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Knack, Brent Andrew (2011) Cell adhesion factors in cnidarians. PhD thesis, James Cook University.
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Cell Adhesion Factors in Cnidarians
Thesis Submitted by
Brent Andrew KNACK BSc (Hons)
in August, 2011
for the degree of Doctor of Philosophy
in the School of Pharmacy and Molecular Biology
James Cook University
i
Statement of Sources I declare that this thesis is my own work and has not been submitted in any form for another
degree or diploma at any university or other institution of tertiary education. Information
derived from the published or unpublished work of others has been acknowledged in the
text and a list of references is given.
I declare that the electronic copy of this thesis provided to the James Cook University
Library is, within the limits of the technology available, an accurate copy of the print thesis
submitted.
6th August 2011 Brent Andrew Knack
ii
Statement of Access I, the undersigned, author of this work, understand that James Cook University will make
this thesis available for use within the University Library and, via the Australian Digital
Theses Network, for use elsewhere.
I understand that, as an unpublished work, a thesis has significant protection under the
Copyright Act and I do not wish to place any further restriction on access to this work.
6th August 2011 Brent Andrew Knack
iii
Contribution of Others Nature of Assistance Contribution Names
Core research funding Australian Research
Council Centre of
Excellence for Coral Reef
Studies
Stipend James Cook University,
Graduate Research School
Travel Assistance Faculty of Medicine,
Health and Molecular
Sciences Graduate
Research Scheme
Financial Support
Accommodation Network for Genes and
Evironment in
Development
Database programming
(See page iv)
Dr. Wayne Mallett, JCU
High Performance
Computing
Collaboration and
Technical assistance with
Drosophila Cell Culture
Dr. Tom Bunch, University
of Arizona Data Collection & Support
Collaboration on
Expression of Wnt
signalling component
Chuya Shinzato and
Svetlana Ukolova
iv
Collaborator contributions to development of JCUSMART:
The concepts and programming behind JCUSMART grew from a relatively simplistic installation
of the annotation programs into a large database with a comprehensive search capability. I manaed
and directed the entire development process. I accessed support from JCU High Performance
Computing (HPC) through personal contacts. The contribution of the HPC team members was
primarily programming support. I actively contributed to the design and testing of all system
components developed, as well as working closely with computing staff to correct errors.
During the initial phases of development Dr. Wayne Mallet (System administrator for the JCU
High Performance Computing Department) and myself worked closely on the design of the
computational pipeline, the handling of character conflicts, and the design of the data storage
system. Decisions on what analyses and results were required in order to be informative, how
character conflicts were resolved, and how data should be linked were primarily my responsibility
as Dr. Mallett had very limited knowledge of biology and bioinformatics.
Dr. Mallet was responsible for programming the scripts that initiate analyses, pre-process input
data, and parse key results into the database. As the system administrator, Dr. Mallett was ideally
positioned to install and troubleshoot programs on the computing cluster. Dr. Mallett’s knowledge
of Perl and PostgreSQL allowed the development of a stable analysis platform, which was
compatible with the JCU HPC system.
Throughout the development process, I tested analysis initialisation; data capture & storage; and
database queries. This was a necessary approach, as each of these steps requires interpretation of
results, which was beyond the knowledge of the HPC staff involved. I then directed the changes in
consultation with Dr. Mallett.
During the development process I actively improved my own programming ability by working
with Dr. Mallet to understand Perl and Prostgres databases in more detail. I also worked with
Wade Tattersall (an undergraduate programmer working on the HPC ARCHER Project) and other
personal contacts to understand Python programming which has a PostgreSQL component
allowing greater control of queries and results. With this knowledge I was able to create more
complex database queries and return more specific results than was previously possible. The
current version of the command line interface was primarily programmed by me, using the
programming skills I developed whilst conducting my PhD.
v
Wade Tattersall assisted me to write the initial scripts for commencing BLAST analyses, parsing
BLAST output for relevant data, and entering the results into the JCUSMART database. I took
over the modification and testing of these scripts and further refined these independently.
The web-server interface was programmed and implemented by Wayne Mallet early in the
development of the JCUSMART system. It was designed to give basic reports on up to ~300
sequences. The size of the datasets being analysed quickly outgrew the web-server functionality.
The web-server interface was not progressed beyond its initial incarnation, as HPC staff could not
spare time to update, nor could I take on writing HTML code. The more flexible & capable
command line interface was adopted as it required less external support.
Collaborator contribution to Figure 6.2: Maximum likelihood phylogenetic analysis of
representative β-Integrins
I selected protein sequences for analysis and collected sequences from NCBI protein database.
Integrin phylogenetic analyses are based on extensively edited protein sequence alignments.
Editing of the full length protein sequence alignment (output of ClustalW) was primarily
conducted by Prof. David Miller. Phylogenetic Analysis conducted using MolPhy was performed
by Prof. Miller. I was responsible for editing of raw analysis output into the published figure
presented in Knack et al, 2008 and Figure 6.2.
Every reasonable effort has been made to gain permission and acknowledge the owners of
copyright material. I would be pleased to hear from any copyright owner who has been omitted or
incorrectly acknowledged.
6th August 2011 Brent Andrew Knack
vi
Acknowledgements The greatest thanks must go to the girl who will very soon become my wife, Ally. In my
work, as in the rest of my life you have been my one source of unwavering support and my
inspiration for perserverance. Your faith in me has been nothing short of amazing, even at
the hardest times and with the distance between us, you kept me going. No words can
explain your true contribution to this work.
Secondly I would like to acknowledge the contribution my supervisor, Prof. David Miller, to
faciliating the initiation of this project. I would also like to thank Dr. Eldon Ball for helping
me to appreciate some of the finer aspects of Acropora development and Dr. David Hayward
for preparing and mailing EST clones as soon as he was able. Thanks must also go to Dr. Tom
Bunch, whose hospitality and patience made every aspect of my time in Arizona worthwhile.
Thanks must also go to Dr. Akira Iguchi for the words he shared during my Honours degree,
“Step by step we will do”. This is a phrase that I have reflected upon many times throughout
the past few years.
My fellow lab members, with whom I have shared the highlights and disappointments of my
work, you have made this experience truly enriched. Your friendship and advice will
continue to be invaluable to me as will the colourful language of 5 foreign tongues. I wish
you all the best in what I am confident will be a long and successful life beyond your studies.
Finally, I must thank my parents who supported and encouraged me to embark on on this
study and helped me to remain focused. Without you, I would not have had the ability to
succeed in my studies.
vii
Table of Contents
Statement of Sources.................................................................................................... i
Statement of Access......................................................................................................ii
Contribution of Others.................................................................................................iii
Acknowledgements .....................................................................................................vi
Abstract xv
Chapter 1: Introduction............................................................................................. 1
1.1 Cell adhesion molecules in development and evolution................................................................1 1.2 Acropora millepora -‐ a model organism for evolution and development ...............................6 1.3 Project objectives ......................................................................................................................................... 10
Chapter 2: Methods .................................................................................................12
2.1 Standard PCR Protocol............................................................................................................................... 12 2.2 Amplification of Gene Fragments from A.millepora cDNA libraries....................................... 12 2.3 Recovery of cDNA from Expressed Sequence Tag libraries ...................................................... 13 2.4 In situ Hybridisation ................................................................................................................................... 13 2.4.1 Riboprobe Synthesis ...................................................................................................................................... 13 2.4.2 Fixation of coral developmental stages ................................................................................................ 13 2.4.3 Hybridisation.................................................................................................................................................... 14 2.5 Cloning of pHS S2 cell expression constructs .................................................................................. 15 2.5.1 Generation of native Acropora integrin expression constructs ................................................. 15 2.5.2 PCR amplification from template pBSII constructs......................................................................... 16 2.5.3 Generation of chimeric Acropora integrin expression constructs ............................................ 17 2.5.4 Generation of chimeric mutant expression construct .................................................................... 19 2.5.5 Insertion of HA and MYC tags to ItgβCN1 and AmItgβ2 ............................................................... 20 2.6 Maintenance of Drosophila S2 cells in culture ................................................................................ 23 2.7 Transfection of Drosophila S2 Cells in culture ................................................................................ 23 2.8 Cell spreading assays.................................................................................................................................. 24 2.9 Antibody staining and flow cytometry of integrin expressing cells....................................... 25
viii
Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of
large protein sequence datasets ...........................................................26
3.1 Introduction.................................................................................................................................................... 26 3.2 Methods (development of JCUSMART)............................................................................................... 31 3.3 Results (Testing and low intensity applications)........................................................................... 34 3.4 Discussion........................................................................................................................................................ 34 3.5 Conclusions..................................................................................................................................................... 37
Chapter 4: Diversity of cell adhesion molecules in cnidarians ...................................38
4.1 Introduction.................................................................................................................................................... 38 4.2 Methods............................................................................................................................................................ 41 4.3 Results ............................................................................................................................................................. 42 4.3.1 Cadherins ........................................................................................................................................................... 42 4.3.2 Integrins ............................................................................................................................................................. 43 4.3.3 Lectins ............................................................................................................................................................... 45 4.3.4 Adhesion LRR.................................................................................................................................................... 48 4.3.5 Class B adhesion G-protein coupled receptors................................................................................... 49 4.3.6 Immunoglobulin superfamily.................................................................................................................... 50 4.3.7 Extra-cellular matrix .................................................................................................................................... 51 4.3.8 Novel cnidarian sequences ......................................................................................................................... 52 4.4 Discussion........................................................................................................................................................ 54 4.4.1 The ancestral adhesion repertoire.......................................................................................................... 54 4.4.2 Novel cnidarian sequences ......................................................................................................................... 60 4.4.3 Differences between cnidarian adhesion systems ............................................................................ 61 4.4.4 Interesting absences...................................................................................................................................... 62 4.5 Concluding comments................................................................................................................................ 63
Chapter 5: Developmental roles of cadherins from Acropora millepora....................65
5.1 Introduction.................................................................................................................................................... 65 5.2 Methods............................................................................................................................................................ 71 5.2.1 Sequence identification................................................................................................................................ 71 5.2.2 Cadherin phylogenetics................................................................................................................................ 71 5.2.3 Isolation of riboprobe template cDNA .................................................................................................. 71 5.3 Results ............................................................................................................................................................. 73 5.3.1 Identification of catenin binding cadherins and planar cell polarity components from
Acropora millepora ...................................................................................................................... 73
ix
5.3.2 Phylogenetic analysis of catenin binding cadherins ....................................................................... 75 5.3.3 In situ hybridisation of catenin binding cadherins and planar cell polarity pathway
components ...................................................................................................................................... 77 5.4 Discussion........................................................................................................................................................ 80 5.4.1 Cadherins involved in epithelial cohesion and migration evolved early in metazoan
evolution............................................................................................................................................ 80 5.4.2 Planar cell polarity but not Am_ACadherin is implicated in gastrulation of Acropora
millepora ........................................................................................................................................... 82 5.4.3 Am_ACadherin and planar cell polarity are implicated in development of the Acropora
larval oral pore............................................................................................................................... 84 5.5 Conclusion ....................................................................................................................................................... 86
Chapter 6: Integrins of Acropora millepora ..............................................................87
6.1 Introduction.................................................................................................................................................... 87 6.2 Methods............................................................................................................................................................ 91 6.2.1 Phylogenetic analyses................................................................................................................................... 91 6.2.2 Preliminary ligand binding assay ........................................................................................................... 91 6.2.3 Optimisation of cell spreading conditions ........................................................................................... 92 6.2.4 Analysis of integrin surface expression................................................................................................. 93 6.3 Results ............................................................................................................................................................. 94 6.3.1 Integrin identification and phylogenetic analysis ........................................................................... 94 6.3.2 Ligand Binding of coral integrins ........................................................................................................... 97 6.4 Discussion......................................................................................................................................................102 6.4.1 Novel Coral integrins may interact with 2 distinct ligand types ............................................ 102 6.4.2 Integrins containing AmItgα1 interact with RGD tripeptide ligands................................... 103 6.5 Conclusions...................................................................................................................................................105
Chapter 7: General Discussion................................................................................106
7.1 Modes of gastrulation in cnidarians...................................................................................................106 7.2 Genetic determinants of cnidarian gastrulation...........................................................................110 7.3 Acropora millepora exhibits an inside-‐out mode of gastrulation..........................................113 7.3.1 The mobile presumptive ectoderm and un-coupling of β-catenin from mesendoderm
development.................................................................................................................................. 113 7.3.2 Maintaining stability in the presumptive endoderm ................................................................... 115 7.3.3 Co-ordinating expansion of the presumptive ectoderm ............................................................. 116
Chapter 8: General Conclusions..............................................................................120
x
Reference List ...........................................................................................................123
Appendix A: Supplementary Material .......................................................................135
Chapter 4 ...........................................................................................................................................................135 Chapter 5 ...........................................................................................................................................................150 Chapter 6 ...........................................................................................................................................................170
Appendix B: JCUSMART Survey of the Cnidarian Adhesome......................................177
Cadherins ...........................................................................................................................................................177 Integrins ...........................................................................................................................................................179 Lectins ...........................................................................................................................................................186 LRR Adhesion ..........................................................................................................................................................192 Class B GPCR 193 Immunoglobulin .....................................................................................................................................................198 Extracellular Matrix ..............................................................................................................................................201 Planar Cell Polarity Signalling ..........................................................................................................................213
xi
List of Figures
Chapter 1: Introduction............................................................................................. 1
Figure 1.1 Evolution of metazoan cellular junctions................................................................................3
Figure 1.2 Lifecycle of Acropora millepora ...................................................................................................8
Figure 1.3 Differing gastrulation strategies of Acropora millepora and Nematostella
vectensis ..............................................................................................................................................8
Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of
large protein sequence datasets ...........................................................26
Figure 3.1 Upward approach to identifying genes of interest in a custom dataset ................ 28
Figure 3.2 Downward approach to identifying genes of interest in a custom dataset using
JCUSMART ...................................................................................................................................... 30
Figure 3.3 Computational Workflow of JCUSMART pipeline............................................................. 33
Chapter 4: Diversity of cell adhesion molecules in cnidarians ..................................38
Figure 4.1 Maximum likelihood analysis of Talin proteins from representative
metazoans ....................................................................................................................................... 44
Figure 4.2 Maximum Likelihood analysis of metazoan haemolytic lectins ................................ 47
Figure 4.3 Domain structure of selected cnidarian innovations ...................................................... 53
Chapter 5: Developmental roles of cadherins from Acropora millepora....................65
Figure 5.1 Generalised protein architecture of catenin binding cadherins................................. 67
Figure 5.2 Milestones in cadherin evolution ........................................................................................... 67
Figure 5.3 Protein conservation and architecture of Am_ACadherin ........................................... 74
Figure 5.4 Maximum likelihood analysis of catenin binding Cadherin cytoplasmic
domains............................................................................................................................................ 76
Figure 5.5 Spatial mRNA expression patterns of Am_ACadherin and PCP components in
embryo’s of Acropora millepora ............................................................................................ 78
Figure 5.6 Spatial mRNA expression patterns of Am_ACadherin and PCP & Wnt components
in larvae of Acropora millepora ............................................................................................. 79
Chapter 6: Integrins of Acropora millepora ..............................................................87
Figure 6.1 Maximum likelihood analysis of representative α-‐Integrins ...................................... 95
Figure 6.2 Maximum likelihood analysis of representative β-‐Integrins....................................... 96
xii
Chapter 7: General Discussion ...............................................................................106
Figure 7.1 Modes of Gastrulation in cnidarians and bilaterians ....................................................107
Figure 7.2 Phylogeny of cnidarian classes ...............................................................................................108
Figure 7.3 Differing gastrulation strategies of Acropora millepora and Nematostella
vectensis .........................................................................................................................................109
Appendix A: Supplementary Material .......................................................................135
Chapter 4
Supplementary Figure 4.1 JCUSMART search terms used for identification of adhesion
genes ............................................................................................................135
Supplementary Figure 4.2 Multiple sequence alignment of Talin proteins used for
maximum likelihood analysis ...........................................................139
Supplementary Figure 4.3 Multiple sequence alignment of meatzoan haemolytic lectins
used for maximum likelihood analysis .........................................141
Supplementary Figure 4.4 Spatial mRNA expression pattern of haemolytic lectin
A036-‐E7 in Acropora millepora larva, polyp & adult ..............142
Supplementary Figure 4.5 Boxshade alignment of full length Am_LRIG3 protein with
representative metazoan LRIG3 orthologues .............................143
Supplementary Figure 4.6 Boxshade alignment of full length Am_PTPRD protein with
representative metazoan PTPRD orthologues ...........................146
Chapter 5
Supplementary Figure 5.1 Aligned nucleic acid and protein sequence of Am_ACadherin 150
Supplementary Figure 5.2 Boxshade alignment of representative Dachsous C-‐terminal
domains .......................................................................................................157
Supplementary Figure 5.3 Multiple sequence alignment of cadherin cytoplasmic domains
used for maximum likelihood analysis .........................................158
Supplementary Figure 5.4 Boxshade alignment of AmFlamingo protein with
representative metazoan Flamingo orthologues ......................160
Supplementary Figure 5.5 Boxshade alignment of full length Am_Van Gogh Like protein
with representative metazoan Van Gogh orthologues............166
Supplementary Figure 5.6 Boxshade alignment of full length Am_Dishevelled protein with
representative metazoan Dishevelled orthologues...................168
xiii
Chapter 6
Supplementary Figure 6.1 Multiple sequence alignment of α-‐Integrin proteins used for
maximum likelihood analysis ............................................................170
Supplementary Figure 6.1 Multiple sequence alignment of β-‐Integrin proteins used for
maximum likelihood analysis ............................................................173
xiv
List of Tables
Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of
large protein sequence datasets ...........................................................26
Table 3.1 Programs used by JCUSMART in the annotation of protein sequences .................. 32
Table 3.2 Conditions under which sequences may not be identifiable using JCUSMART... 36
Table 3.3 Contribution of JCUSMART to publication........................................................................... 37
Chapter 4: Diversity of cell adhesion molecules in cnidarians ..................................38
Table 4.1 Datasets used to explore the adhesome of basal metazoans....................................... 41
Table 4.2 Distribution of cadherin family proteins in basal metazoans ..................................... 42
Table 4.3 Distribution of integrin and associated proteins in basal metazoans ..................... 43
Table 4.4 Distribution of lectin family proteins in basal metazoans ............................................ 45
Table 4.5 Distribution of adhesion -‐LRR proteins in basal metazoans........................................ 48
Table 4.6 Distribution of class B G-‐Protein Coupled Receptors in basal metazoans............. 49
Table 4.7 Distribution of immunoglobulin superfamily proteins in basal metazoans ......... 50
Table 4.8 Distribution of extracellular matrix proteins in basal metazoans............................. 51
Table 4.9 Presence and absence of selected bilaterian adhesion genes with
developmental and immunological significance............................................................ 55
Chapter 5: Developmental roles of cadherins from Acropora millepora ...................65
Table 5.1 Distribution of catenin binding cadherins and planar cell polarity components
in cnidarians .................................................................................................................................. 75
Chapter 6: Integrins of Acropora millepora ..............................................................87
Table 6.1 Concentrations of Mg2+ (MgCl2) and Ca2+ (CaCl2) added to Robb’s Saline for
optimisation of cell spreading conditions......................................................................... 92
Table 6.2 Percentage of cells expressing detectable α and β-‐Integrins on the cell surface
following transient transfection..........................................................................................100
Table 6.3 Percentage of cells expressing epitope tagged β-‐Integrins on the cell surface
following transient transfection..........................................................................................101
Chapter 7: General Discussion ...............................................................................106
Table 7.1 Function of adhesion protein families with demonstrated involvement in
bilaterian gastrulation.............................................................................................................112
xv
Abstract Cell adhesion is central to metazoan evolution and development, facilitating multicellularity,
intercellular communication and co-‐ordinated cell movements. Morphological studies
indicate that cellular and developmental processes involving dynamic changes in adhesive
state, such as cell migration and gastrulation, were established early in metazoan evolution.
However, much of our current understanding surrounding the involvement of cell adhesion
molecules in these processes has been obtained from comparative analyses of bilaterian
development. Cnidarians present an exciting opportunity to investigate cell adhesion and
developmental processes in the simplest extant animals to possess a tissue layer level of
organisation. As the nearest out-‐group to the Bilateria, cnidarians are ideally positioned for
comparative studies between diploblastic and triploblastic development whilst being highly
informative regarding ancestral gene function and protein family evolution.
The diversity of adhesion proteins in cnidarians has remained largely unexplored, with no
single analysis offering an overview of the cnidarian adhesion complement or “adhesome”.
To better understand the complexity of the cnidarian adhesome, sequence data from four
model cnidarians (Acropora millepora – coral, Nematostella vectensis – Sea Anemone, Hyrda
magnipapillata – Hydra, Clytia hemispherica – Hydrozoan Jelly-‐fish) was annotated and all
potential adhesion proteins categorised according to similarity to described protein families.
The cnidarian adhesome shows overall similarity with that of invertebrate bilaterians,
containing a substantial array of recognisable cadherins, integrins, lectins and extracellular
matrix proteins comparable to Drosophila and sea urchin. Such conservation suggests that
most recognised adhesion proteins involved in bilaterian development and innate immunity
were already established in the ureumetazoan ancestor. All four species also demonstrated
an expanded set of lectin domain containing putative pattern recognition receptors which
may act in opsonisation of microbial pathogens. In contrast to this, each species lacked the
large immunoglobulin complement observed in deuterostomes suggesting the predominate
mechanism of microbial recognition may be facilitated by lectins rather than
immunoglobulins.
Catenin binding cadherins and components of the planar cell polarity (PCP) pathway were
among the developmentally significant proteins conserved between cnidarians and
bilaterians. Expression of cadherins, such as E-Cadherin and N-Cadherin, which bind
cytoplasmic β-catenin, strongly influence the ability of cells to undergo epithelial to
mesenchymal transtion, a central event in developmental processes such as gastrulation.
The assymetrical distribution of planar cell polarity cadherins and other PCP proteins at
xvi
both the cell and tissue levels, also affects tissue morphology by co-‐ordinating cellular
structures and providing positional cues during morphogenesis. To identify the potential for
conserved catenin binding cadherins and PCP proteins to participate in cnidarian
development, patterns of mRNA expression were assessed in embryos and larvae of the
coral Acropora millepora. Surprisingly, the only known catenin binding Cadherin from coral
and the first identified outside the Bilateria, Am_ACadherin, does not appear to participate in
gastrulation, which is inconsistent with bilaterian modes of gastrulation. Planar cell polarity,
however may be active during Acropora gastrulation, with AmVan Gogh (AmVangl) and
AmDachsous (AmDs) expressed assymetrically during gastrulation. Am_ACadherin was
instead implicated in oral pore development following gastrulation as indicated by a highly
restricted pattern of expression in the oral ectoderm. In situ mRNA hybridisation also
strongly suggests the involvement of non-‐canonical Wnt/PCP in oral pore development,
however none of the PCP adhesion proteins exhibited oral pore restricted patterns of
expression during larval stages. These results suggest both Cadherin-‐catenin signalling and
PCP are signficant during cnidarian development and may facilitate co-‐ordinated tissue
mobility such as involution of the oral ectoderm.
Survey of the cnidarian adhesome also identified α and β integrins, which, like cadherins,
play signifcant roles in the early development of bilaterians. The expression patterns for 3 (1
α-‐Integrin & 2 β-‐Integrins) of the 5 (3 α-‐Integrins & 2 β-‐Integrins) integrins identified in
Acropora have previously been reported, showing restiction to the presumptive endoderm
throughout gastrulation. The ligand binding properties of basal integrins have not been
reported, obscuring their developmental function. To identify the ligand binding properties
of AmItgα1-ItgβCN1 and AmItgα1-AmItgβ2 integrin heterodimers, transgenic cell spreading
assays were performed on a number of Drosophila ligands. These experiments suggested
coral AmItgα1 containing integrins may bind to arginine-‐glycine-‐aspartate (RGD) sequence
containing proteins. RGD specific integrins are abundant throughout the Bilateria, and along
with Laminin binding integrins, have been suggested to be present in the Urbilaterian
ancestor (the last common ancestor of the Bilateria). Phylogenetic analysis of α-‐Integrins
including the more recently identified AmItgα3 (Acropora) and NvItgα2 (Nematostella)
suggest that these proteins may bind to a second distinct ligand type. The ligand diversity of
cnidarian integrins may therefore be comparable with basal bilaterians.
xvii
Invovlement of cadherins and integrins in gastrulation appears to be conserved between
cnidarians and bilaterians as suggested by mRNA expression during gastrulation of
Acropora millepora. The mode of gastrulation exhibited by Acropora millepora is among the
more peculiar strategies of gastric cavity formation, where by a flat cellular bilayer folds to
produce a spherical bilayer at the end of gastrulation. Expression patterns of the coral
integrins and cadherins, along with protein localisation data for β-catenin, allow the
description of a new model for gastrulation in Acropora. These data suggest the presumptive
ectoderm of the coral embryo is mobile, with expansion of this tissue layer co-‐ordinated by
planar cell polarity. Stability in the presumptive endoderm is suggested to be mediated by
integrin ligand binding, providing the basis for mesoglea (the connective tissue layer that
separates cnidarian ectoderm and endoderm) development. This model is inside-‐out with
respect to most modes of gastrulation which rely upon co-‐ordinated migration of the
presumptive endoderm and represents a unique arrangement of conserved adhesion
proteins and cellular signalling processes.
Chapter One Introduction
1
Chapter 1: Introduction
1.1 Cell adhesion molecules in development and
evolution Cell adhesion and changes in adhesive state are essential for the normal development and
function of metazoans. Developmental processes including fertilisation, gastrulation, and
neurogenesis along with immunity and allorecognition are all dependant on co-‐ordinated
changes in the adhesive capability of cells (Halbleib & Nelson 2006; Kinashi 2005). The
ability of a cell to adhere to other cells, to maintain contact with extracellular matrix, or to
actively migrate to another location, is governed by the expression of a broad group of cell
surface proteins collectively known as cell adhesion molecules. Although this name may
suggest a simple function in making cells “stick”, cell adhesion molecules have far more
important roles than simply securing cellular structures.
Each biological process involving adhesion, such as gastrulation, requires multiple cells to
have structural organisation and behave in a co-‐ordinated manner (Gumbiner 2005). Cell
adhesion molecules perform two fundamental cellular functions, cohesion and intracellular
signalling, which together contribute to tissue organisation and co-‐ordination in
multicellular organisms. The cohesive function is a consequence of extracellular domain
(ecto-‐domain) structure and determines ligand specificity, whereas the cytoplasmic domain
facilitates cell signalling and cytoskeletal attachment by interacting with cytoplasmic
proteins. Unlike most cell surface receptors, which only transduce signals from the
environment into the cell, a number of cell adhesion molecule families frequently respond to
internal cellular stimuli, changing affinity, expression level or signalling ability through
intrinsic structural response, transcriptional regulation and protein regulatory mechanisms
(eg. phosphorylation, domain cleavage, and endocytosis). The range of cytoplasmic
interactions, multiplicity of fast and slow regulatory mechanisms and the ability to
specifically determine cellular cohesion, make adhesion molecules particularly versatile
morphogenic effectors, capable of influencing the structure, organisation and gene
expression profile of both cells and tissues.
Chapter One Introduction
2
The influence of cell adhesion molecules on cellular organisation and co-‐ordination is
common to all multicellular organisms. Whereas unicellular organisms are thought to
primarily utilise adhesive processes for anchoring to their environment, the maintenance of
multicellular form immediately dictates an added role for adhesion in generating continual
cell-‐cell contact (Abedin & Nicole King 2010). The link between multicellularity and
increased adhesive capability is well established (Abedin & King, 2010; King, 2004; Nichols
et al, 2006)and none more evident than in the most complex incarnation of multicellularity,
the Metazoa. An overview of the metazoan tree of life is presented in Figure 1.1A.
Enrichment of cell adhesion genes is among the features that distinguish metazoans from
other eukaryotes, along with enrichment of signal transduction and cell differentiation genes
(Rokas 2008). Even the most morphologically simple metazoan, Trichoplax adhaerens
(essentially a flat epithelial bilayer sandwiching multinucleated fibre cells), contains a
complement of adhesion genes, which is beyond that of the closest unicellular relatives of
metazoans, the Choanoflagellata (King et al., 2008; Srivastava et al., 2008). Expansion of
genetic adhesive capability during metazoan evolution is reflected in the diversity of cellular
junctions observed in different animal lineages. Figure 1.1B&C illustrates the expansion of
adhesive junction types from simple anchorage junctions in choanoflagellates to the
communication, barrier and dynamic cohesive junctions of vertebrates. Although this
increased complexity of adhesive systems was a significant step in the transition to
multicellularity, most of what is known about the function of cell adhesion molecules
originates from studies of bilaterians.
Investigation of adhesion mechanisms within bilaterian animals has demonstrated that
many adhesion and associated signalling mechanisms are conserved from flies to
vertebrates. This is particularly evident in systems affecting early morphogenesis such as
Cadherin-‐β-‐catenin, integrin signalling and planar cell polarity (Klein & Mlodzik, 2004;
Nichols et al., 2006). Despite widely variegated gastrulation strategies in flies, fish, frogs and
mammals, these conserved adhesion systems maintain influence over the same tissue level
mechanisms during the formation of three germ layers (See Chapter 7). Through
conservation of ligand specificity and intracellular signalling properties, adhesion proteins
with similar structure in each taxon govern tissue morphogenic mechanisms such as tissue
flexibility, migration and directional division. Whilst this level of functional conservation has
clearly been demonstrated in higher animals, the morphogenesis of basal phyla, cnidarians
and sponges, is strikingly different.
3
Figure 1.1 Evolution of metazoan cellular junctions. A Phylogeny of the Metazoa (adapted from Technau et al., 2005). A wide range of evidence suggests a common (monophyletic) origin for metazoans from a unicellular ancestor. Cnidarians are basal among animals with a tissue layer level of organisation (Eumetazoa) and are informative about the last common ancestor of cnidarians and bilaterians. B & C Distribution of adhesive cell junctions in metazoans (adapted from Abedin and King, 2010). The diversity of cell junctions expanded multiple times during animal evolution. The presence (grey) or absence (white) of junction types is evidenced by genetic surveys (Gene), electron microscopy (Morph), and experimental (Exp) data. Although experimental data is lacking for non-‐bilaterians, the presence of genes that function in adherens and septate junctions distinguish metazoans from their unicellular ancestors and subsequent evolution of intercellular communication in the eumetazoan ancestor may have been critical to tissue layer formation. Enrichment of communicating, barrier and cohesive junctions is a feature of chordate evolution and particularly evident in vertebrates. Some caveats apply to the data presented: 1 α and β integrins are absent in choanoflagellates but present in C.owczarzaki, a model unicellular organism used to infer the genetic content of Opisthokonts, which are basal to holozoans. 2 Only some sponges exhibit basal lamina like structures and ECM comprised of Collagen IV, a major component of basal lamina.
Chapter One Introduction
4
The methods of gastrulation observed in basal animals are even more diverse than in
bilaterians. This is particularly clear in cnidarians where all strategies present in higher
animals are represented as well as a number of unusual gastrulation methods (Byrum and
Martindale, 2004). Despite broad resemblances to bilaterian gastrulation, formation of the
gastric cavity in cnidarians and the gastrulation-‐like events of sponge embryonic
development result in only two embryonic germ layers. These animals are therefore
referred to as diploblasts. Furthermore, larvae and primary polyps of cnidarians exhibit
radial symmetry, only possessing distinction of oral and aboral structures and not a dorsal-‐
ventral axis or bilateral symmetry, characteristic of higher animals. Given the great impact of
adhesive systems on morphogenesis, these clear developmental differences between
bilaterians and basal phyla are suggestive of changes in adhesion system specificity,
regulation and downstream signalling events.
Evolution of other milestone developmental processes associated with differential adhesion
is also believed to have originated in sponges and cnidarians. The Porifera (sponges)
represent the most basal metazoan phylum to exhibit continual cell-‐cell contact during
development and show specificity in cohesion that allows sorting of dissociated cells
(Burger et al., 1975) . They are also the most basal animal to establish cell layers during
embryonic development, although they lack a single dedicated gastric cavity, which is
defining of true gastrulation. The novelties found in cnidarians are even more significant to
animal evolution. Cnidarians do develop a single dedicated gastric cavity, and possess a
primitive nervous system, which is proposed to facilitate sensory responses to the
environment. They also have added complexity of life cycles that include a medusa jellyfish
stage, which commonly have dedicated defensive structures in the form of
cnidoctye/nematocyte (stinging cell) covered tentacles. The development of comparable
dedicated tissues in higher animals has been shown to require cell adhesion molecules to
direct tissue development.
Despite the multitude of novelties that arose before the Bilateria and the significant
differences between cnidarian development and that of higher animals, very little
information exists about the function of adhesion in cnidarians and sponges. Simply
considering differences among bilaterians provides a highly limited view of the evolution of
adhesion molecules and adhesion-‐signalling systems. This is due to both the large amount of
time between cnidarian and bilaterian divergence and the genetically derived nature of
some common laboratory model organisms such as Drosophila melanogaster. Knowledge of
adhesion in basal animals is limited to sporadic identifications of differentially regulated
Chapter One Introduction
5
adhesion molecules during coral bleaching and metamorphosis, investigation of a single
coral lectin (Millectin), and limited surveys of two complete genomes.
The complete genomes of only 1 sponge (Amphimedon queenslandica) and 1 cnidarian (the
starlet sea anemone, Nematostella vectensis) are currently published (Putnam et al., 2007;
Srivastava et al., 2010). To date, the Nematostella genome has been the only representative
of non-‐bilaterian animals regularly included in comparative analyses of cell adhesion
molecules. Surveys of the available genomes have been targeted towards identification of
genes or conserved functional protein domains associated with developmentally important
cell adhesion molecules. These targeted investigations fail to report many novel or expanded
components of the adhesion complement and are instead focused on highlighting the
ancestral nature of components from a minimal set of developmental adhesion systems.
However, the great diversity of cnidarian developmental processes and near stochastic
patterns of gene loss within the Cnidaria (Forêt et al. 2010; Miller et al. 2007) indicates that
investigation of a single cnidarian genus is often insufficient to accurately infer ancestral
relationships. It is important to be mindful of this when information regarding the cnidarian
adhesion complement is inferred from a single species. Additionally, detailed evolutionary
relationships have been explored for only three families of adhesion genes (integrins,
cadherins and G-‐Protein Coupled Receptors) leaving the evolutionary features of most
adhesion families enigmatic.
Understanding the origins of developmentally significant cell adhesion molecules is
important, although sequence based analyses provide no information as to the function of
these basal cognates. The specificity, regulation, and spatial and temporal expression of
cnidarian adhesion molecules are expected to be substantially different to that of higher
animals and are more informative of ancestral function than identifying the presence or
absence of a homologue. However, no investigation into these aspects of cnidarian adhesion
systems has previously been performed for any family of cell adhesion molecule.
Chapter One Introduction
6
The overall lack of information regarding the ancestral function of cell adhesion molecules
and the evolution of adhesion molecule families is best addressed by investigation of
cnidarian adhesion systems. As the most basal organisms with a tissue layer level of
organisation, cnidarians are ideally positioned to be informative about ancestral
developmental processes while illuminating aspects of molecular evolution. Understanding
the adhesion complement or “adhesome” of more than a single representative species, and
performing investigations into the specificity and expression patterns of key developmental
cell adhesion molecules from cnidarians is the most efficient way to fill critical gaps in our
knowledge of diploblastic development and evolution.
1.2 Acropora millepora - a model organism for
evolution and development Cnidarians are a large and successful phylum of predominantly marine animals, which
diverged from bilaterians approximately 500-‐600 million years ago and have expanded to
over 9000 described species. They are characterised by the presence of cnidocytes (stinging
cells) that are used for defence and prey capture. The Cnidaria are divided into four classes:
Anthozoa, Scyphozoa, Cubozoa and Hydrozoa, each with defining life cycle features.
Acropora are a genus of reef building scleractinian corals abundant throughout the Indo-‐
Pacific region. Phylogenetic analyses have revealed that along with Nematostella (sea
anemones), Acropora are members of the basal class of cnidarians, the Anthozoa (see Miller
& Ball, 2000 for review). Investigations into anthozoan genomes have revealed that despite
a simple, radially symmetrical body plan, these basal animals possess surprisingly
sophisticated genetics, containing a similar gene number and signalling complement to
vertebrates (Miller & Ball, 2000). The sophisticated genetic complement and basal position
of Acropora both within the Cnidaria and among the Eumetazoa (“True metazoans” –
Animals with a tissue layer level of organisation), positions this genus ideally for
investigations of evolution and development.
Acropora millepora is among the best described species of cnidarian in terms of both
genetics and development, and exhibits several features that are likely to involve differential
expression of cell adhesion molecules, such as nervous system development and
metamorphosis (Ball et al., 2004; Putnam et al., 2007). The gastrulation strategy of Acropora
millepora is particularly peculiar, involving the early formation of a flat cellular bilayer and
folding of the presumptive ectoderm around the presumptive endoderm (Figure
1.2)(Hayward et al., 2004). This is in contrast to that of Nematostella vectenisis, which has a
typical blastula stage and gastrulation occurs via invagination (Hayward et al., 2004) (Figure
Chapter One Introduction
7
1.3). Whereas movements similar to those seen in Nematostella gastrulation have been
highly investigated in bilaterian models, the factors facilitating tissue re-‐arrangements
during coral gastrulation are obscured.
Despite the gross morphological differences, Nematostella and Acropora are closely related
on an evolutionary timescale (both are Sub-‐Class Hexcorallia) and commonly have a similar
complement of many signalling pathway components (Lee et al., 2007). The biological
differences and genetic similarity of these two cnidarians provide a unique opportunity for
comparative studies of cell adhesion in closely related animals, whilst elucidating
mechanisms of an unusual developmental strategy.
In addition to the developmental and evolutionary implications of studying cell adhesion in
Acropora, there are coral specific processes reported to involve cell adhesive changes.
Acropora live in obligatory symbiosis with unicellular algae called Zooxanthellae. The
uptake and maintenance of symbionts in the gastroderm of adult Acropora millepora
requires expression of Millectin, a cell adhesion factor of the lectin family (Kvennefors et al.,
2008; Kvennefors et al., 2010). Whether other adhesion factors play a role in the recognition,
uptake and translocation of algal symbionts from ectodermal cells to the gastrodermis
remains unresolved.
The wide spread loss of Zooxanthellae from coral tissue is known as coral bleaching and is
also suggested to involve changes in cell adhesion. Although bleaching involves both
apoptotic and necrotic processes, the sloughing of gastrodermal tissue has also been
reported and is assumed to be due to a change in cellular cohesive state (Kvennefors et al.,
2008; Kvennefors et al., 2010). Separate experiments have found changes in the expression
level of undescribed adhesion molecules (eg. Lectins and cadherins) during thermal stress
(Seneca et al., unpublished; (Gates et al., 1992), which further implicates adhesion molecules
as central players in coral bleaching. The precise adhesion genes that are responsible for
sloughing of zooxanthellae containing gastrodermal cells are equivocal and could be
resolved through direct analysis of adhesion protein function, although such an undertaking
is not straightforward in sensitive marine invertebrates like coral.
Chapter One Introduction
8
Figure 1.2 Lifecycle of Acropora millepora. Electron micrographs of each major stage in the embryonic development of Acropora millepora (Image courtesy of E.Ball, Unpublished). The anthozoan lifecycle lacks the medusa stage observed in other cnidarian classes and asexual reproduction in coral results in colonial expansion rather than budding of a separate individual. Sexual reproduction occurs annually when mature Acropora colonies release sperm and ova into the water column where fertilisation and embryonic development takes place. Developmental times from fertilisation are temperature dependant and times indicated are within normal ranges for embryos collected in late September at Magnetic Island, Queensland.
Figure 1.3 Differing gastrulation strategies of two representative cnidarians, Acropora millepora and Nematostella vectensis. Gastrulation in Acropora (A; Hayward et al., 2004) is preceded by formation of a flat cellular bilayer, which reduces in circumference and thickens at the onset of gastrulation. The edges then begin to fold upward producing a concavity on the side of the presumptive endoderm. As the concavity deepens (gastrula) the blastopore becomes apparent and eventually closes to make a sphere. By contrast, Nematostella gastrulation (B; Lee et al., 2007) is preceded by blastula (a hollow spherical monolayer of cells) formation and occurs by involution of presumptive endoderm from one side of the blastula. Unlike Acropora, the blastopore does not close and becomes the larval oral pore.
Chapter One Introduction
9
There are a number of limitations to using Acropora millepora as a model organism.
Maintaining coral in a marine aquarium is a difficult task due to fastidious temperature,
light, pH and nutritional requirements and those kept in large reef aquariums do not
reproduce sexually in a predictable manner, which makes them unsuitable for
developmental studies. Sexual reproduction of Acropora millepora on the reef flat is an
annual event and occurs by broadcast spawning of sperm and ovum bundles. The annual
reproductive cycle and generation times of 5-‐10 years have inhibited the development of
gene knockdown, protein over expression and cell tracking methods in this model.
Application of these techniques, which have become the standard tools of developmental
research and functional analyses, has been further constrained by the fragility of coral
embryonic stages and requirement of a high salt and high pH (pH 7.8 – 8.0) environment.
The study of adhesion in coral morphogenesis has therefore required some exploration and
extension of the techniques established in other model metazoans.
Chapter One Introduction
10
1.3 Project objectives Developmental roles of adhesion molecules are poorly comprehended in basal metazoans,
which provide a fundamental view of ancestral gene function and the evolutionary history of
gene families. The objective of this project is to address the deficit in understanding of
ancestral adhesion gene complement and function using a combination of bioinformatic and
molecular techniques. Acropora millepora is the primary model for these investigations,
which focus on a number of specific aspects of cnidarian adhesion systems.
A representative view of adhesion genes in cnidarians has not yet been achieved from
previous studies exploring the genome of Nematostella vectensis. Through the investigation
of four large cnidarian datasets (genome, transcriptome and expressed-‐sequence tag
projects), the first component of this project aims to establish an overview of the cnidarian
adhesome and provide insight into the evolution of adhesion genes originating in the
eumetazoan ancestor. Accurate annotation of coding sequences is central to identifying the
gamut of adhesion proteins within cnidarians and to discerning their evolutionary
relationships. The datasets analysed from Acropora millepora, Nematostella vectensis, Hydra
magnipapillata and Clytia hemispherica contain over 100,000 predicted peptides that must
be considered for adhesive potential. No single, publically available tool is able to provide
the level of annotation required and permit sufficient data exploration for gene discovery.
Therefore, a secondary aim of the above survey component was to assemble a tool capable
of high throughput annotation and exploration of protein sequences which would allow
analysis of private data for genes with specific protein features (eg. conserved domains or
signal peptides). Development of such software has potential to contribute to projects
beyond simply identifying adhesion related genes and is applicable to all experiments
requiring discrimination of uncharacterised sequences.
The second aim of this project focuses on elucidating the functional roles of specific families
of adhesion proteins found during the survey described above. Members of the Cadherin and
integrin families are of particular interest due to their critical function in cell sorting,
directed cell migration and neuronal pathfinding in bilaterians. The diversity and
developmental impact of these proteins has not been functionally assessed in cnidarians,
although the expression patterns for some of the coral integrins during development of
Acropora millepora have been previously reported (Knack et al. 2008). This work on coral
integrins will be extended as a part of the second project component by determining the
ligand specificities of 3 Acropora integrins using transgenic cell spreading assays. Whereas
Chapter One Introduction
11
some preliminary data on cnidarian integrins has been published, the expression and
function of Cadherin mediated cell signalling systems from cnidarians are recondite. Chapter
5 explores the phylogenetic relationships between cnidarian catenin binding cadherins,
which have been demonstrated to be central to bilaterian gastrulation mechanisms. The
expression of coral cadherins and planar cell polarity components are also investigated in
Chapter 5, revealing potential roles in both embryonic and larval development.
Chapter Two Methods
12
Chapter 2: Methods
2.1 Standard PCR Protocol The standard protocol used to perform amplification from plasmids is as follows:
H20 to 20.0ul Buffer (10x) 2.0µl dNTPs (2mM ea) 1.5µl MgCl2 (25mM) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Taq Polymerase 0.6µl Template (variable)
2.2 Amplification of Gene Fragments from A.millepora
cDNA libraries Gene targets were amplified by PCR from an equal mix of cDNA libraries
representing embryonic, larval and adult tissues of A.millepora. cDNA libraries were
generated by Dr. David Hayward (Australian National University) with RNA isolated
from multiple individuals at adult, planula and prawnchip stages of development.
PCRs used to amplify partial sequences of target genes (Chapters 5 and 6) were
performed using the standard PCR protocol (Chapter 2.1) with the following
alterations:
• Primer concentration 2.5µl Primer (20µM ea)
• MgCl2 concentration 2.5µl MgCl2 (25mM)
• Template volume 0.5µl
• Final volume 25µl
1 95oC 2min 2 95oC 30sec 3 52-‐56oC 30sec 4 72oC 1min/Kb 5 Repeat steps
2-‐4 25x
6 72oC 2x extension
time 7 4oC Hold
Chapter Two Methods
13
2.3 Recovery of cDNA from Expressed Sequence Tag
libraries cDNA isolated from EST libraries were donated by Eldon Ball and David Hayward
(Australian National University, Canberra) and shipped to James Cook University on
Filter Paper. Recovery of cDNA was performed by placing the filter paper in a 1.5ml
microfuge tube and adding 50µl of ddH20 before overnight incubation at 4oC. 5µl of
resuspended cDNA was then used to transform competent E.coli NM522.
2.4 In situ Hybridisation 2.4.1 Riboprobe Synthesis
Anti-‐sense riboprobes were synthesised from partial or full-‐length cDNA cloned in
pGEM-‐T or pBluescript II KS-‐ vectors. Vectors were linearised by restriction
digestion to produce a 5’ end, 5’ overhang. Reverse Transcription was performed
using T7 or SP6 RNA polymerase (Promega) in the presence of digoxygenin (DIG)
labelled UTP. A sample of the resulting RNA was visualised by electrophoresis
through a 1% agarose gel. Labelled RNA was purified by ethanol precipitation and
resuspension in RNase-‐free water before being hydrolysed using carbonate buffer to
produce a theoretical fragment length of 250bp. Finally, riboprobes were purified by
ethanol precipitation and resuspended in resuspension buffer (50% formamide,
50% TE, 0.1% Tween-‐20).
2.4.2 Fixation of coral developmental stages
Developmentally staged embryos and larvae from Acropora millepora were fixed for
10-‐12minutes in 4% (v/v) formaldehyde in HEPES buffered Millipore Filtered Sea
Water pH8.0 (0.2µm filter; HEPES-‐MPFSW). Embryos and larvae were then washed
repeatedly in MPFSW before dehydration through a methanol/H20 series (20%,
50%, 70%, 90%, 100%x2) and stored in absolute methanol at -‐20°C.
Chapter Two Methods
14
2.4.3 Hybridisation
In situ hybridisation was carried out by methods similar to that previously described
(Hayward et al., 2001). Staged embryos were removed from storage and allowed to
come to room temperature before rehydration to 50% through a methanol series
(90%, 70%, 50%), followed by washing with PBS then PBT (once each). Overnight
incubation (4°C) in RIPA was performed to remove the large amount of lipid present
in the coral embryos & larvae. Animals were then dehydrated through an ethanol
series (25%, 50%, 70%, 90%, 100% x2) and treated with Xylene for 3-‐4hrs. Xylene
was removed by multiple washes with absolute ethanol followed by rehydration
through ethanol series and washing with PBT.
Prehybridisation was performed by replacing PBT with hybridisation solution and
incubating at hybridisation temperature (50°C-‐57°C) for 2hrs. Riboprobe was added
at a final dilution of 1/125 and allowed to incubate for 72hrs at hybridisation
temperature. Unbound probe was removed by extensive washing in several volumes
of hybridisation wash solution at 55°C-‐60°C. Hybridisation wash solution was
gradually replaced by PBT before addition of Alkaline Phosphatase conjugated anti-‐
DIG antibodies (Roche) at a 1:1600 dilution by PBT. The animals were incubated in
antibody suspension for 2 hours with gentle agitation and washed with PBT.
Detection was performed using NBT/BCIP (Vector Laboratories) diluted in PBT as
per the manufacturer’s instructions. Colour development was stopped by washing
with PBT and background colour was removed with absolute ethanol where
necessary.
Chapter Two Methods
15
2.5 Cloning of pHS S2 cell expression constructs Expression constructs were generated from pBluescript II KS-‐ plasmids containing complete
coding sequence of AmItgα1 (EU239371), ItgβCN1 (AF005356) or AmItgβ2 (EU239372) as
described in Knack et al. 2008. For each integrin subunit, both native and chimeric genes
were cloned into the pHS vector, which has previously been described for the expression of
integrins in Schneider’s line 2 (S2) cells ( Bunch & Brower, 1992; Jannuzi et al., 2002; Bunch
et al., 2004). Chimeric genes were then used as a template to incorporate integrin
heterodimer activating mutations by overlap extension PCR. Primer sequences and
amplification conditions follow.
2.5.1 Generation of native Acropora integrin expression
constructs
Native constructs included complete coding sequence amplified using the below
primers and conditions. PCR products of the expected size (~3Kb) were cleaned
using a PCR cleanup kit (MoBio) before direct restriction digestion (NEB restriction
enzymes) of PCR products and pHS vector. PCR clean up was again performed prior
to ligation of 200ng insert at 6:1 ratio insert:vector using T4 ligase (Promega).
Transformation was carried out by heat shock of 50µl competent NM522 cells. DNA
was isolated from overnight cultures of selected colonies (Axygen Miniprep kit) and
sequenced (Macrogen).
Note: Ext (Brown) is random sequence which exhibits very low specificity with
sequences in the Acropora transcriptome and pHS vector. This sequence was
included in primers to improve the specificity of restriction digests used during
cloning. 5’ and 3’ Ext are not complementary to each other or internal primer
sequences to minimise primer dimer formation.
Chapter Two Methods
16
Ext HndIII-‐Alpha1 5' Tm =60oC 5’-TGTCAACACGCGCTGTACGAAAGCTTATGCTCTTCACTTCAATAACTTG-3’ Ext EcoRI-‐Alpha1 3' Tm=58oC 5’-TGTCAACACGCGCTGTACGAGAATTCCTACAGGGCTGTCGTTTCT-3’ Ext KpnI-‐BetaCN1 5' Tm=60oC 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ Ext SacI-‐BetaCN1 3' Tm=58oC 5’-TGTCAACACGGGCTTCAGCAGAGCTCACTACTGTCTTCCACCAGC-3’ Ext HindIII-‐Beta2 5' Tm=58oC 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ Ext SacI-‐Beta2 3' Tm=58oC 5’-TGTCTTCACGCGCTACGGCAGAGCTCTTATTTCCCACCATACGTAGG-3’ 2.5.2 PCR amplification from template pBSII constructs
H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.6µl pBSII Template 50ng
1 95oC 2min 2 95oC 45sec 3 55oC 30sec 4 72oC 7min 5 Repeat steps
2-‐4 30x
6 72oC 8min 7 4oC Hold
Chapter Two Methods
17
2.5.3 Generation of chimeric Acropora integrin expression
constructs
Chimeric constructs included 5’ sequence from Acropora integrins coding the
extracellular region of the integrin protein and 3’ coding sequence from Drosophila
malanogaster integrin-‐αPS2C or integrin-‐βPS. 5’ and 3’ sequences were amplified
using the conditions and primers described in PCR1. Chimeras were generated from
PCR1 products using overlap extension PCR (PCR2) before direct digestion, cleanup
and ligation to pHS vector. Transformation was carried out by heat shock of 50µl
competent NM522 cells. DNA was isolated from overnight cultures of selected
colonies (Axygen Miniprep kit) and sequenced (Macrogen). Chimeric genes are
referred to as AmItgα1-‐ItgαPS2, AmItgβ1-‐PS and AmItgβ2-‐PS.
5’ Fragment Primers Ext HndIII-‐Alpha1 5' Tm =60oC 5’-TGTCAACACGCGCTGTACGAAAGCTTATGCTCTTCACTTCAATAACTTG-3’ Alpha1Δ 3' Tm=57oC 5’-CCACCACGGCGTCTTCT-3’ Ext KpnI-‐BetaCN1 5' Tm=60oC 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ BetaCN1Δ 3' Tm=60oC 5’-GGGAGCCTCTGTCGGGC-3’ Ext HindIII-‐Beta2 5' Tm=58oC 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ Beta2Δ 3' Tm=57oC 5’-TTCAGCTTCCTTTGGGCA-3’ 3’ Fragment Primers Ext EcoRI-‐AlphaPS2 3' 5’-TGTCAACACGCGCTGTACGAGAATTCCTACAGGTGCTCGTCGCC-3’ Alpha1-‐AlphaPS2 adaptor 5’-GAAGAAGACGCCGTGGTGGGTCGTCGTACTGGCCGC-3’ Ext SacI-‐BetaPS 3' 5’-TGTCAACACGCGCTGTACGAGAGCTCCTATTTGCCCGCATACATG-3’ BetaCN1-‐BetaPS adapt 5’-TGCCCGACAGAGGCTCCCATGTTGGGCATCGTTATGG-3’ Beta2-‐BetaPS adapt 5’-GTTTGCCCAAAGGAAGCTGAAATGTTGGGCATCGTTATGG-3’
Chapter Two Methods
18
PCR1 – Amplification of 5’ and 3’ Sequences H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.5µl pBSII Template 50ng PCR1- 5’ Amplification Cycle 3’ Amplification Cycles
1 95oC 2min 1 95oC 2min 2 95oC 45sec 2 95oC 45sec 3 59oC 30sec 3 59oC 30sec 4 72oC 6min 4 72oC 2min 5 Repeat
steps 2-‐4 30x
5 Repeat steps 2-‐4
30x
6 72oC 4min 6 72oC 4min 7 4oC Hold 7 4oC Hold
PCR2 – Overlap Extension PCR H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl PCR1 5’ product 50ng PCR1 3’ product 50ng Pfu Polymerase 0.5µl pBSII Template 50ng
1 95oC 2min 2 95oC 45sec 3 60oC 30sec 4 72oC 6min 5 Repeat
steps 2-‐4 10x
7 4oC Hold Add Outer
primers
8 95oC 45sec 9 59oC 30sec 10 72oC 7min 11 4oC Hold
Chapter Two Methods
19
2.5.4 Generation of chimeric mutant expression construct
A single amino acid mutation was introduced into the chimeric expression vector
pHSItgβ2-‐βPS pHS by overlap extension PCR. 5’ and 3’ sequences were amplified
using the conditions and primers described in PCR1. Chimeras were generated from
PCR1 products using overlap extension PCR (PCR2) before direct digestion, cleanup
and ligation to pHS vector. Transformation was carried out by heat shock of 50µl
competent NM522 cells. DNA was isolated from overnight cultures of selected
colonies (Axygen Miniprep kit) and sequenced (Macrogen). Chimeric AmItgβ2
containing an activating mutation is referred to as AmItgβ2L>D-‐PS.
5’ Fragment Primers
Ext HindIII-‐Beta2 5' 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ AmItgb2L>D S-‐ 5’1065-GGCTTGTCTGATAAGTTGGTCAAGGTTAGATGAGTCCTCTCTCAACG-1019 3’
3’ Fragment Primers Ext SacI-‐BetaPS 3' 5’-TGTCAACACGCGCTGTACGAGAGCTCCTATTTGCCCGCATACATG-3’ AmItgb2L>D S+ 5’1019-CGTTGAGAGAGGACTCATCTAACCTTGACCAACTTATCAGACAAGCC-1065 3’
PCR1 – Amplification of 5’ and 3’ sequences
H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.5µl pBSII Template 50ng
1 95oC 2min 2 95oC 45sec 3 60oC 30sec 4 72oC 6min 5 Repeat
steps 2-‐4 10x
7 4oC Hold
Chapter Two Methods
20
PCR2 – Overlap Extension PCR H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl PCR1 5’ product 50ng PCR1 3’ product 50ng Pfu Polymerase 0.5µl pBSII Template 50ng
1 95oC 2min 2 95oC 45sec 3 60oC 30sec 4 72oC 6min 5 Repeat
steps 2-‐4 10x
7 4oC Hold Add outer
primers
8 95oC 45sec 9 59oC 30sec 10 72oC 7min 11 4oC Hold
2.5.5 Insertion of HA and MYC tags to ItgβCN1 and AmItgβ2
HA and MYC epitope tags were cloned into expression constructs for ItgβCN1 and
AmItgβ2 respectively. Epitope tag sequences were inserted into the loop encoding
region between bases 258 and 259 of ItgβCN1 and bases 248 and 249 of AmItgβ2.
This loop region was extended using sequence from the corresponding loop in the
Drosophila integrin ItgβPS. Sequences 5’ and 3’ of the insertion site were amplified
in separate PCRs from the coral template using the following primer combinations
before restriction digestion.
Template 5’ Sequence 3’ Sequence
5’ Primer 3’ Primer 5’ Primer 3’ Primer
ItgβCN1 Ext-‐KpnI βCN1
5’
ItgβCN1
insert tag 5’ S-‐
βCN1 insert tag
3’ S+
βCN1 NgoMIV
3’ S-‐
AmItgβ2 Ext-‐HindIII β2
5’
AmItgβ2
insert tag 5’ S-‐
AmItgβ2 insert
tag 3’ S+
AmItgβ2 NcoI
3’S-‐
Chapter Two Methods
21
Sense and anti-‐sense oligonucleotides encoding the HA and MYC epitopes flanked by
SacI and BamHI restriction sites were annealed by immersing a microfuge tube
containing equal portions of sense and antisense oligonucleotides in boiling water
for 5 minutes before allowing to cool slowly to room temperature. Three way
ligation was performed to combine the 5’ and 3’ coral sequences with the HA or MYC
tag insert. The resulting DNA containing the epitope tag corresponded
approximately 1/3 of original coral template in the 5’ end. This sequence was
inserted into the ItgβCN1 or AmItgβ2 expression vector by restriction digestion and
ligation. Success of the cloning process was determined by restriction digestion and
sequencing. Only 2 vectors, ItgβCN1-‐HA and AmItgβ2-‐MYC, were completed in the
timeframe required to perform cell spreading experiments. HA and MYC tagged
ItgβPS were kindly donated by Dr. Tom Bunch (University of Arizona) for
experimental control.
5’ and 3’ Fragment Primers
coral adaptor sequence SacI restriction site BamHI restriction site Ext KpnI-‐BetaCN1 5' 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ ItgbCN1 insert tag 5’S-‐ 5’-ctcgacgagctcgaactcgacgagtaggagctgctgccggacatggcggagccgccgccaccagccgccagttcagcTTTTGCACCTACATTACTGT-3’ ItgbCN1 insert tag 3’S+ 5’-gatctgggatccagttccgccagcggatacgaagagtactctgccggcgaaattGTGCAAGTTCAGCCAAAG-3’ Ext HindIII-‐Beta2 5' 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ AmItgb2 insert tag 5’S-‐ 5’-
ctcgacgagctcgaactcgacgagtaggagctgctgccggacatggcggagccgccgccaccagc
cgccagttcagcTGTATCAAGTGGCTTGTTC-3’
AmItgb2 insert tag 3’S+ 5’-gatctgggatccagttccgccagcggatacgaagagtactctgccggcgaaattAATGTGAAAGTGAAACCACA-3’
Chapter Two Methods
22
HA and MYC epitope tag oligonucleotides
SacI restriction site BamHI restriction site HA or MYC Tag sequence Myc loop middle S+ 5’-cgtcgagcttctactcgcagagctcctcggagcagaagctgatctccgaagaggatctgg-3’ Myc loop middle S-‐ 5’-gatcccagatcctcttcggagatcagcttctgctccgaggagctctgcgagtagaagctcgacgagct-3’ HA1 loop middle S+ 5’-cgtcgagcttctactcgcagagctcctcgTACCCGTACGATGTGCCGGATTACGCCg-3’ HA1 loop middle S-‐ 5’-gatccGGCGTAATCCGGCACATCGTACGGGTAcgaggagctctgcgagtagaagctcgacgagct-3’
Chapter Two Methods
23
2.6 Maintenance of Drosophila S2 cells in culture All experiments were performed using Drosophila S2/M3 cell line (Schneider’s line 2 cells
adapted for growth in M3 medium (Schneider 1972); referred to as S2 Cells). S2 cells were
maintained at a density of 2x106 – 10x106 cells/ml in Shields and Sang M3 medium (M3;
Sigma) supplemented with 12.5% heat inactivated foetal calf serum (FCS), 100U/mL
Penicillin and 100μg/mL Streptomycin (PS). Cells were cultured under controlled
temperature conditions at room temperature (25OC) with an air gas phase. Transformed cell
lines were maintained in M3 + FCS + PS with 2x10-‐7 Molar Methotrexate (MTX).
2.7 Transfection of Drosophila S2 Cells in culture Unless otherwise stated, transfections were performed in 6 well tissue culture plates
(2ml/well) 1 day after resuspension of cells at 3x106 cells/ml in M3 +FCS +PS medium to
allow cells to reach the exponential growth phase and recover from stress. Immediately
prior to transfection, DNA mix was prepared by adding serum free M3 medium and the
following components to achieve a final volume of 100µl:
• Transient transfection only -‐ 1µl 250ng/µl GFP expression plasmid (under
Actin promotor)
• Transient transfection only -‐ 2µl 250ng/µl Mys (ItgβPS) RNAi
• Equal parts of:
o 250ng/µl pH8CO (methotrexate resistance; Rebay et al., 1991)
o 250ng/µl α integrin pHS expression construct
o 250ng/µl β integrin pHS expression construct
Negative controls contained empty pHSMCS and pH8CO vectors only. 100µl of diluted
Cellfectin (Invitrogen) was also prepared immediately prior to transfection by addition of
10µl Cellfectin to 90µl serum free M3 medium. The diluted Cellfectin and DNA mix were
gently combined before incubation at room temperature for 15minutes. During incubation,
growth medium (M3 + FCS +PS) on the cells to be transfected was replaced with 2ml serum
free M3 medium. After incubation and working no more than 2 wells at a time, M3 (serum
free) medium was removed from the cells, 800µl M3 (serum free) medium was added to the
200µl Cellfectin-‐DNA mix before being gently added to cells. Cells were incubated in the
transfection mix for 5hrs at room temperature. After incubation, the transfection mix was
replaced with 2ml M3+FCS+PS and incubated overnight. Following this, medium was
replaced with supplemented M3 medium containing MTX.
Chapter Two Methods
24
2.8 Cell spreading assays Potential ligands for coral integrins (Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow,
PacI, Drosophila Trimeric Laminin, Sucrose fractionated Drosophila Laminin) were diluted
in Phosphate Buffered Saline (PBS) and 200µl added to the wells of a 96well tissue culture
plate. The plate was sealed with parafilm and incubated overnight at 4OC. Prior to assay,
liquid was removed from the wells by decanting and replaced with 200µl of spreading
medium (optimal: Robb’s Saline + 1mM Mg2+ + 0.2mM Ca2+; Alternatives: BES-‐Tyrodes (1mM
Mg2+, 10µM Ca2+, 1% BSA), PBS (1mM Mg2+, 10µM Ca2+, 1% BSA), serum free M3 medium (no
additional cations)).
Cells at 3 days post-‐transfection were counted and collected by centrifugation at 1000g for 2
minutes. Cells were resuspended at 4x105cells/ml in spreading medium (Optimal: Robb’s
Saline with 1mM Mg2+ and 0.2mM Ca2+). Spreading medium was removed from the wells and
replaced with 100µl of cells in spreading medium (4x104cells/well). Attachment and
spreading was allowed to proceed for 2hrs before fixation with 2% paraformaldehyde (PFA)
by PBS. Images were captured on a Nikon X5000 attached to a Zeiss Axiovision 200
microscope.
Chapter Two Methods
25
2.9 Antibody staining and flow cytometry of integrin
expressing cells Duplicate cultures of S2 cells were sampled 2 days after transfection with coral and/or fly
integrin subunits. Samples of 0.5x106 -‐ 1x106 cells were resuspended in 50µl of primary
antibody diluted as follows:
• Rabbit anti-‐AmItgα1 1:500 dilution (1:100 and 1:1000 also trialled)
• Rabbit anti-‐ItgβCN1 1:500 dilution (1:100 and 1:1000 also trialled)
• Rabbit anti-‐AmItgβ2 1:500 dilution (1:100 and 1:1000 also trialled)
• Mouse anti-‐ItgαPS1 1:500 dilution
• Mouse anti-‐ItgαPS2C 1:1000 dilution
• Rabbit anti-‐HA-‐488 1:1000 dilution
• Rabbit anti-‐MYC 1:1000 dilution
Samples were incubated in primary antibody for 20-‐25minutes before washing with 500µl
M3 serum free medium and resuspension in 50µl secondary antibody diluted as follows:
• Goat anti-‐Rabbit 546 1:1000 dilution – Coral integrins
• Goat anti-‐mouse-‐546 1:1000 dilution – Fly integrins
Samples were incubated in secondary antibody for 20-‐25minutes before washing with
500µl serum free M3 medium and fixation in 500µl 2% PFA by PBS. Surface antigen
detection was then performed by flow cytometry. To sort antigen expressing cells for cell
spreading assays (Section 6.3.2), cells were washed 2x in 500µl Robb’s Saline after
incubation with secondary antibody and maintained on ice prior to cell sorting.
Chapter Three JCUSMART
26
Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of large protein sequence datasets
3.1 Introduction The traditional view of many evolutionary and developmental biologists has been that the
simple morphology of basal invertebrates such as cnidarians is a reflection of low genetic
complexity. The genomic complexity of cnidarians has often been underestimated through
the reasonable expectation that simple morphology is correlated with low complexity.
However, studies in the early 2000’s examined this expectation and demonstrated specific
examples where aspects of cnidarian genetics were considerably more complex than
expected (Schmitt & Brower, 2001; reviewed in Ball et al., 2004; Martindale et al., 2002).
Compelling evidence for broad genetic complexity within basal metazoans arose from
analysis of the moderately sized expressed sequence tag (EST) projects of Acropora
millepora and Nematostella vectensis. Sequence analysis of these EST data showed that
Acropora possesses a higher proportion of sequences with greater similarity to humans than
traditional invertebrate models (ie. Drosophila and Caenorhabditis) (Kortschak et al., 2003).
Both Acropora and Nematostella datasets contain a significantly higher than expected
representation of signalling pathways previously assumed to be vertebrate specific, and
exhibit a degree of ancestral gene maintenance not observed in bilaterians (Technau et al.
2005). Evidence for ancestral complexity within the Cnidaria was consolidated with the
publication of Nematostella genome, which supported the above findings and revealed that
the number of coding genes is more similar to that of Deuterostomes than Ecydosozoans
(Ball et al., 2004; Martindale et al., 2002; Schmitt & Brower, 2001). The demonstrated lack of
correlation between genetic and phenotypic complexity has lead to a new appreciation for
the complexity that arose prior to bilaterian evolution.
The dramatic change in understanding of cnidarian genetics and its implications for
metzoan evolution exemplifies the importance of sequence analysis as a research tool. The
power of sequence analysis to yield results capable of making a significant impact on any
field of study is heavily reliant on both the size and quality of the sequence data available.
Development of high throughput sequencing technologies including Illumina and 454
sequencing has made generation of high quality, large scale datasets (such as transcriptome
Chapter Three JCUSMART
27
and genome sequencing) feasible for a much wider range of non-‐traditional model
organisms. Some publically available examples available at the Ensembl genome browser
(http://www.ensembl.org/index.html) include Dolphin, Alpaca, Wallaby, Turkey and Shrew.
Thee hundred and twenty three (323) whole genome projects are currently (July 2011) in
the public domain (available through Ensembl) and the decreasing cost of sequencing is
stimulating the occurrence of private large scale datasets. Despite the increasing ease of
generating data, the ability to navigate large datasets in order to explore genetic
relationships and genetic novelties remains out of reach for most individual molecular
biologists and requires the assistance of a dedicated bioinformatician. As a result, the huge
potential of large scale sequencing for furthering our understanding of genetic diversity is
underutilised.
Increasing the accessibility of large datasets for analysis by a greater proportion of the
scientific community hinges on accurate annotation of sequence data, principally of coding
sequences and predicted peptides. This is particularly important in organisms such as
cnidarians where current data indicates a significant proportion of the detectable coding
sequences are dissimilar to all sequences described in the primary public databases
(Swissprot and NCBI). However, annotation of these datasets by molecular biologists is often
limited by the procedural aspects of operating commonly accepted annotation software,
including installation on an appropriate computing environment, operation of the
computing environment and the annotation tools using a command line interface, and
management of the output data.
Two of the most powerful and common annotation tools are BLAST, to explore pairwise
similarity, and HMMER to explore conservation of functional domains within protein
sequences. Like most common annotation tools, these can be utilised for individual
sequences by virtue of online servers accessible through a graphical interface. These public
servers carry heavy limitations to compute time and the number of sequences that can be
processed and are therefore not suitable for large scale processing.
Limitations to the capacity of graphical and easy to manage annotation tools have
contributed to the development of an upward or “gene by gene” approach to annotation and
large dataset exploration (Figure 3.1). The “gene by gene” approach starts with a small
number or narrow range of sequences to be annotated and often yields a narrow range of
results. For example, in the case of identifying a single or small number unknown sequences,
Chapter Three JCUSMART
28
BLAST can be performed online against public databases and similarity to reported
sequences can be identified. For this application, the “gene by gene” approach is ideal.
However, this approach is less suitable for identifying sequences of interest from a pool of
unknowns (eg. a transcriptome or genome sequence dataset). This task requires use of a
known homologue of the sequence of interest, which is used to query a custom BLAST
database and may require some verification of results, such as BLAST of the best hits for
each gene of interest against NCBI non-‐redundant (nr) or SwissProt databases. The analysis
process quickly becomes time consuming as the number of sequences increases.
Figure 3.1 Upward approach to identifying genes of interest in a custom dataset. The process undertaken to analyse the data is shown in A, whilst the impact on the size of the gene’s of interest group is displayed in B As the identification process proceeds, the number of genes that need to be considered increases with each step. Steps marked with “*” have potential to introduce identification / annotation error to the system, through false matches which may not be easily detectable if only BLAST is considered.
A B
Chapter Three JCUSMART
29
The time required to processes data by the gene by gene approach can reduce the number of
annotative tools that are used, which can have adverse affects on the accuracy of the final
annotation. Often the only tool employed by molecular biologists in sequence identification
is BLAST. As with all bioinformatics programs, BLAST has implicit limitations. When
considering large proteins with common or repeated domains, for example those of the
Cadherin family, which can contain more than 30 Cadherin domains, the BLAST analysis fails
to accurately distinguish between members of the family, even if repeat masking is applied.
In these cases, and cases where the query sequence is potentially highly derived as expected
in cnidarian data, the protein architecture (domain structure, presence of signal peptides
and transmembrane regions etc) must also be considered. The Simple Modular Architecture
Research Tool (SMART) (Letunic et al. 2009; Schultz et al. 1998) served from Heidelberg
University (http://smart.embl.de) is one online tool that allows for more accurate peptide
annotation through identification of protein architecture, however the same limits to input
size as with other online serves apply.
In order to more effectively explore large datasets from potentially divergent organisms, an
approach that asks “What genes are present?” must be used instead of one that asks “Is this
specific gene present?”. This more permissive, downward approach (Figure 3.2) is better
suited to surveying large or multiple datasets for whole families of genes. Using a downward
approach, genes of interest are identified by searching pre-‐assigned annotations for specific
combinations of features that are consistent with the genes of interest. Through control of
search terms, this approach allows greater flexibility and control over the scope of results
obtained.
For public genome project data, the level of exploration offered by a downward approach is
primarily accessed through genome browsers (eg. JGI genomes and Ensembl), which usually
offer results for BLAST against SwissProt, HMMER analysis against Pfam and SMART, and
organisation of sequences into Eukaryotic Cluster of Orthologous Groups (KOG) or Kyoto
Encyclopaedia of Genes and Genomes (KEGG) pathways. Using a genome browser to survey
public genomes for genes with specific features often begins with exploring the contents of
the appropriate KOG and/or KEGG annotations. The results can then be narrowed on the
basis of protein architecture (structural features of a protein including signal peptides,
conserved domains and transmembrane regions) or BLAST annotations. Unfortunately,
accurate mapping of private data to the KOG and KEGG hierarchies is not always possible
and is particularly complicated for lower organisms such as cnidarians due to small dataset
size and the strong vertebrate bias in publically available genomes on which KOG and KEGG
Chapter Three JCUSMART
30
are based. Instead exploration of large private datasets must be based on the remaining
annotative tools, protein architecture and BLAST. Fortuitously, software to perform these
analyses are freely available for academic purposes and can be installed on multiple
computing platforms.
Figure 3.2 Downward approach to identifying genes of interest in a custom dataset using JCUSMART. The process undertaken to analyse the data is shown in A, whilst the impact on the size of the gene’s of interest group is displayed in B. As the process proceeds, the number of sequences to consider is reduced in a manner reflecting the stringency of search criteria. Error is not introduced into the system, however some sequences may not be identifiable due to the nature of the custom dataset. These sequences are more easily detected using the downward approach rather than falsely annotated.
In order for large scale sequencing data to be effectively utilised by a greater proportion of
molecular biologists, a single access point for data annotation and exploration using a
downward approach must be available. Currently, no single package is publically available
to perform both of these functions. To address this deficit, I have developed a tool named
JCUSMART that aims to act as a single platform for automated annotation of large protein
datasets and for exploration of the resulting annotations using a downward approach.
Adoption of this tool will facilitate exploration of large datasets by a larger number of
molecular biologists who have only a basic background in bioinformatic techniques.
A B
Chapter Three JCUSMART
31
3.2 Methods (development of JCUSMART) In order to provide a more accessible single platform for employing a downward approach
to large scale data exploration I developed a system named JCUSMART that allows
assessment of both BLAST results and protein architecture. JCUSMART was designed to
address both analytical and user interface requirements.
The analytical requirements of JCUSMART were:
• To perform timely analysis of large private datasets by BLAST against public
datasets and determination of protein architecture
• To extract key information from raw results for storage and display
• To store key information in a searchable format
Analytical requirements were addressed using a computational pipeline, which was capable
of thin deployment over a local computing cluster thereby reducing analysis time. Outputs
from annotative programs (Table 3.1) were parsed for key information, which was passed
into a PostgreSQL database. The computational workflow is detailed in Figure 3.3. BLAST
annotation was not included in the automated analysis in order to give greater control over
the BLAST parameters. A separate script was developed to enter BLAST data into the
database.
To make this tool accessible the user interface was required to:
• Be presented for use in a single, intuitive, graphical environment
• Have simple operation for initiation of analyses and exploration of results
• Present results clearly
JCUSMART is accessible through either a web interface
(https://kanga.hpc.jcu.edu.au/jcusmart/index.html) or command line interface. Both offer
the ability to launch new JCUSMART analyses and search the database for sequences with
common annotations. Both interfaces have simple operation and display results in an easily
interpreted table.
Chapter Three JCUSMART
32
Program Function Origin Link HMMER2: HMMPFAM
Predict Conserved Domains using HMMs. PFAM and SMART databases utilised
Howard Hughes Medical Institute and Dept. of Genetics, Washington University
http://hmmer.janelia.org/
TMHMM Predict Transmembrane Helicies
Center for biological sequence analysis – Technical University of Denmark (CBS-‐DTU)
http://www.cbs.dtu.dk/services/TMHMM (Krogh et al., 2001)
SignalP Predict signal peptides
CBS-‐DTU http://www.cbs.dtu.dk/services/SignalP (Bendtsen et al., 2004)
TargetP Predict cell
membrane/ mitchondreal/ Chloroplastic signal peptides
CBS-‐DTU http://www.cbs.dtu.dk/services/TargetP (Krogh et al., 2001)
SEG Identify low complexity segments
CBS-‐DTU (Wootton, 1994)
NCOILS Predict coiled-‐coil regions
CBS-‐DTU (Lupas et al., 1991)
Prospero Identify internal repeats
Welcome Trust Centre for Huan Genetics
http://www.well.ox.ac.uk/ariadne/intro.shtml (Emanuelsson et al., 2000)
Netphos Predict Phosphorylation sites
CBS-‐DTU http://www.cbs.dtu.dk/services/NetPhos (Blom et al., 1999)
Table 3.1 Programs used by JCUSMART in the annotation of protein sequences, showing the information gathered and the program origin. Conserved Functional domains were detected using HMMPFAM in conjunction with PFAM and SMART Hidden Markov Model Libraries. TMHMM was used to predict the presence of transmembrane helices. Membrane targeting signal sequences were identified using SignalP and TargetP. Low complexity segments and coiled-‐coil predictions were predicted by SEG and NCOILS respectively. Prospero and Netphos were used to identify internal repeats and putative phosphorylation sites. All programs are freely available for academic use. No citation is available for HMMER2 as per the program manual.
Chapter Three JCUSMART
33
Figure 3.3 Computational Workflow of JCUSMART pipeline. JCUSMART accepts protein sequences in FASTA format. The input file is parsed for characters that raise errors in the analysis programs, removing or replacing characters where required (this does not affect the protein sequence). JCUSMART then loads the cleaned input file to each program for analysis. Each program runs on a separate node of the James Cook University High Performance Computing Cluster (HMMPFAM on 2 nodes) and is assigned the maximum number of CPUs allowed by the program. Raw output files are parsed for results informative to most molecular biologists, which are then entered into the JCUSMART database. The JCUSMART graphic user interface and command line interface allow text based searching of database results for identification of sequences containing specific combinations of domains and protein architectural features (eg. Singal peptide, specific combinations of domains, transmembrane helix, and phosphorylation sites). Refining search terms allows narrowing of the number of sequences of interest. BLAST results are also stored in the JCUSMART database, however this analysis is run independently. Results of BLAST can also be searched for key words allowing identification of a greater number of potential sequences of interest (dependant on similarity limits imposed on BLAST analysis), whilst providing enough architectural information to accurately assign orthology even in cases where BLAST alone is insufficient.
Chapter Three JCUSMART
34
3.3 Results (Testing and low intensity applications) Initial testing and development was centred around the sequences of the three integrins
from Acropora millepora which had previously been characterised in terms of protein
architecture (Knack et al., 2008). For each annotation program, a comparison was made
between the known protein architecture, the raw program output and the results in the
database. These results were consistent for the three sequences used. The same comparison
was made for a 10 sequence analysis and also found to be 100% consistent, demonstrating
annotations were not lost or re-‐assigned before entry into the database. Analysis of 100 and
1000 sequence datasets were performed in order to check for errors in the database.
SignalP, HMMER, TMHMM and TargetP programs produce a database entry for every
sequence analysed. Errors could therefore be identified by comparing the number of input
sequences to the number of entries for these programs. Re-‐testing using these datasets was
performed after each adjustment to the system. Testing and development of database
queries were also perform using these initial datasets.
3.4 Discussion The increasing ease of generating large amounts of sequence data is broadening the scope of
what can be achieved through sequence analysis. However, a large proportion of molecular
biologists who could benefit from exploring large private datasets for proteins containing
specific features of interest are not utilising the data for their maximum benefit. In many
cases this is simply due to inexperience with the procedural aspects of bioinformatic
analysis on large datasets. JCUSMART provides a single tool for running basic automated
annotation of large datasets and exploring the results using a downward approach.
Currently the JCUSMART pipeline can analyse protein data from a whole genome containing
30,000 sequences in less than 3 days. After analysis, multiple large datasets that may consist
of over 200,000 sequences in total can then be searched for specified features in a matter of
minutes. Common queries used to explore data from one or more datasets in the JCUSMART
database include:
• Searching for proteins with specified conserved domains
• Searching for proteins with specified architectural features (eg. signal peptide or
number of transmembrane domains)
• Searching for sequences that have BLAST hits with descriptions matching specified
key words using Boolean operators (AND, OR, NOT)
Chapter Three JCUSMART
35
For many of the data types (eg. conserved domains and BLAST hits) E-‐value cutoffs can be
specified, allowing control over the specificity of results returned by a query, and any chosen
fields can be retrieved using a list of sequence names generated using JCUSMART or any
external method. The rapid cross dataset search times and ability to control search
stringency makes this tool ideal for comparative studies and identification of novel or
divergent sequences of interest from a large pool of data. A number of limitations currently
exist in JCUSMART. The current web based interface lacks features such as downloadable
results file and fetching sequences from the database in FastA format. These issues have
been resolved in the command line interface. The web interface could also be improved by a
graphical display of results with links to external information (eg. conserved domain
descriptions), which would make interpretation of results more straightforward than the
current tabular format.
JCUSMART is primarily designed to facilitate identification of sequences of interest from a
pool of unknown sequences. It is not designed to identify orthologous sequences or group
sequences automatically. This is due to the difficulties of automating the integration of
BLAST (which is potentially misleading for divergent or basal animals) and protein
architecture information to reach an accurate result. Protein grouping is better performed
manually using the JCUSMART information as it allows the user to consider their prior
knowledge of the proteins of interest. Protein identification therefore involves refining
search criteria to suit a specific question. Some sequences may not be identifiable using
JCUSMART for a number of reasons (Table 3.2). It should be noted that the inability to
identify a homologue of a particular gene of interest is not a definitive indication of its
absence from an organism, it may simply be absent from the dataset.
Chapter Three JCUSMART
36
Reason for Absence Description
Fragmentation of genes
into multiple models
This is an inherent limitation of the contiguous sequence assembly
process (unrelated to JCUSMART). In the case of genome sequencing
this is aided by increased sequence coverage and EST support.
Incomplete models may
not represent the gene
sufficiently to be
identified using the
search criteria
Gene models and therefore predicted peptides, may represent
regions of a gene that are specific to a particular biological function
or to a specific gene. These sequences cannot be resolved without
laboratory investigation (ie. cloning and sequencing).
Incomplete models more
closely resemble other
sequences
Limitations in BLAST and HMMER can produce misleading results.
Often in this case the results of BLAST against NCBI nr will not agree
with the domain structure of the predicted peptide.
Inappropriate search
criteria
Narrowing search criteria to focus on a diagnostic region of the gene
of interest can sometimes correct this. Search criteria involving
keywords commonly require refinement.
Table 3.2 Conditions under which sequences may not be identifiable using the JCUSMART method. Both errors in gene or protein prediction, such as fragmentation of genes into multiple models or truncated gene models, and errors in the search approach (ie. inappropriate search criteria) can result in unidentifiable sequences.
Currently the JCUSMART database holds annotations for a total of 112,868 sequences
(~35Gb) from five basal animal datasets:
• Nematostella vectensis genome filtered models – 27273 sequences
• Hydra magnipapillata EST project protein predictions – 19845 sequences
• Acropora millepora Transcriptome protein predictions – 48 335 sequences
• Clytia hemispherica EST project protein predictions – 8219 sequences
• Monosiga brevicolis Genome predicted peptides – 9196 sequences
The application of JCUSMART in identification of specific gene families across several genera
has already made a significant contribution to a number of research projects. The projects
and the application of JCUSMART is outlined in Table 3.3. One further documented
application for JCUSMART has been in the identification of adhesion genes throughout the
Cnidaria. This survey of the cnidarian adhesion complement is presented in Chapter 4 and
was the model use case for JCUSMART.
Chapter Three JCUSMART
37
Research Contribution using JCUSMART Foret et al (2010), New tricks
with old genes: the genetic
bases of novel cnidarian traits.
Trends in Genetics, 26 (4), 154-‐
158
Identification of CEL-III like lectins in Clytia hemispherica but
not in Nematostella vectensis or Hydra magnipapilata. This
distribution of CEL-III demonstrates the stochastic nature of
gene loss in cnidarians
Detection of Selenium related
proteins in Acropora millepora
(In preparation)
Identification of GPx (1), TR (2), Other Seleno-‐proteins (8),
selenium binding proteins(2), other related factors (1) from
Acropora millepora EST data. These sequences formed the
basis of protein characterisation and coral stress experiments
to meet PhD project requirements.
Detection of caspase and NOD
related genes in Acropora
millepora, Nematostella
vectensis, Hydra magnipapillata,
Clytia hemispherica
Identification of NOD and caspase proteins from each of 4
cnidarian datasets prior to cloning and laboratory
investigation used to meet PhD project requirements.
Table 3.3 JCUSMART has already contributed to published and unpublished work though the identification of genes of interest and sequence annotation. The project and the contribution of JCUSMART are outlined in the table.
3.5 Conclusions Through the use of a distributed computational pipeline and a relational PostgreSQL
database, JCUSMART delivers accurate and highly searchable automated protein annotation
in a timely manner. Having already demonstrated its usefulness in a number of projects,
JCUSMART provides an efficient tool for employing a downward approach to identifying
sequences of interest, and facilitates the exploration of large protein datasets by molecular
biologists with limited bioinformatic knowledge.
Acknowledgements Initial development and implementation of pre-‐processing, job dispatch and data storage
systems were performed in conjunction with Dr. Wayne Mallet of the James Cook University
High Performance Computing Department. Testing and development of BLAST dispatch,
BLAST data storage and command line query scripts were performed with the assistance of
Wade Tattersall of the JCU ARCHER project. Thanks also to Russell Sim and David Lang for
guidance on programming and query design.
Chapter Four Diversity of Adhesion Molecules in cnidarians
38
Chapter 4: Diversity of cell adhesion molecules in cnidarians
4.1 Introduction Cellular contacts with the extracellular matrix (ECM) and surrounding cells play a critical
role in the development and survival of animals, as demonstrated by the multitude of severe
and often lethal phenotypes resulting from misregulation of cell cohesion and cell migration
(Hogg & Bates, 2000). A combination of genome analyses and functional studies in
representative bilaterians has led to an appreciation that cell adhesion molecules, the
proteins facilitating cellular cohesion and migration, form a complex network capable of
regulating diverse biological processes in both protostomes and deuterostomes (Klein &
Mlodzik, 2004; Schambony, Kunz, & Gradl, 2004; Tan et al., 2001). The importance of cell
adhesion molecules to fundamental processes such as morphogenesis and immunity has
been described in detail. Examples include integrins, which function in leukocyte trafficking
and formation of the gastric cavity (Kinashi, 2005; Serini, Valdembri, & Bussolino, 2006;
Yang et al., 1999), cadherins, which influence cell polarity and directed cell movements
(Kimura-‐yoshida et al., 2005; Rodríguez, 2004), and immunoglobulins, which are the
predominant immune mediators in bilaterians, facilitating vertebrate adaptive immunity.
Comparative analyses have also identified that many families of adhesion genes, such as
integrins, catenin binding cadherins, and several components of the basement membrane
are conserved throughout the Bilateria, playing analogous roles in a variety of model
organisms. However, the complement and function of cell adhesion molecules from outside
the Bilateria is poorly investigated.
Biological processes governed by cell adhesion in bilaterian animals are also observed in
lower metazoans such as cnidarians and sponges. Cnidarians, the most basal phylum to
contain a tissue layer level of organisation (Technau et al., 2005), undergo a broad variety of
processes that centre around adhesion molecule expression and dynamic changes in
adhesive state including complex morphogenic cell re-‐arrangements during gastrulation and
metamorphosis (Grasso et al., 2008; Hayward et al., 2004), allorecognition between
individuals and colonies (Nicotra et al., 2009), innate immunity (Miller et al., 2007), nervous
system development (de Jong et al., 2006) and the uptake/loss of zooxanthellae in symbiotic
cnidarians such as coral (Gates et al., 1992; Kvennefors et al., 2008, 2010). This diverse
phylum is also positioned at the base of the Eumetazoa (‘true animals’), owing to possession
of a nervous system and dedicated tissues. Furthermore, gastrulation results in development
of only two germ layers rather than three as observed in bilateral animals. These attributes
Chapter Four Diversity of Adhesion Molecules in cnidarians
39
make cnidarians a significant comparator to bilaterian systems and particularly informative
for studies of evolution and development, allowing the genetics of the last common
eumetazoan ancestor to be inferred. Despite the importance of cnidarians to understanding
the fundamental roles of adhesion molecules in tissue development and the molecular
evolution of adhesion genes, the diversity and function of adhesion genes in cnidarians are
largely unexplored.
Investigations encompassing aspects of cnidarian adhesion have previously been focused on
elucidating the distribution of a single gene family (defined by the presence of specific
functional domains) throughout the whole of the Metazoa. The evolution of only 3 families of
adhesion molecules (integrins, cadherins and G-‐Protein Coupled Receptors) has been
investigated in this manner with each analysis suggesting several family members
originated prior to the eumetazoan ancestor (Hulpiau & van Roy, 2011; Nordström et al.,
2009; Sebé-‐pedrós et al., 2010). No common evolutionary trend could be determined from
these proteins families, as each family has a broad variety of functions and therefore
selective pressures, however, expansion of protein families is generally evident at the base
of the bilaterian and vertebrate lineages. Although these surveys provide perspective on the
origin of specific adhesion families, they provide a limited view of the cnidarian adhesion
gene complement or ‘adhesome’ as a whole. Such targeted studies also have a restricted
capacity to consider the importance of novel or structurally distinct family members in a
biological context, which is central to understanding the significance of ancestral adhesion
molecules.
In order to establish an accurate overview of the cnidarian adhesome, the present analysis
considered 7 families of adhesion molecules with described roles in bilaterian development
or immunity. In addition to investigation of the integrin, cadherin and immunoglobulin
families mentioned above, members of the lectin, class B G-‐protein coupled receptors
(GPCR), Adhesion leucine rich repeat (LRR) and extracellular matrix families were also
identified. Analysis of these families attempted to identify all possible members with
relevance to cell adhesion processes, although additional members of these families may be
detectable using less stringent criteria.
Chapter Four Diversity of Adhesion Molecules in cnidarians
40
Previous studies into the evolution of adhesion molecules often considered only a single
representative, the sea anemone Nematostella vectensis, as representative of cnidarians as
whole. However, the genetic diversity and near stochastic nature of gene loss observed in
the Cnidaria suggest the use of a single species may not be suitable to gain insight into the
evolution of adhesion genes originating in the eumetazoan ancestor. To overcome this
limitation the present analysis examines EST, transcriptome and genomic data from 4 model
cnidarians, Acropora millepora, Nematostella vectensis, Hydra magnipapillata and Clytia
hemispherica. This selection of cnidarians includes representatives from 2 taxonomic
classes, Hydrozoa (Hydra and Clytia) and Anthozoa (basal among cnidarians; Acropora and
Nematostella), and 2 habitats, marine (Acropora and Clytia) and estuarine (Hydra and
Nematostella). Developmental strategies also vary between the 4 species, with Hydra and
Nematostella developing directly from larvae to adult, whilst Acropora and Clytia (which
also exhibits a medusa stage) undergoing metamorphosis to a sedentary polyp. The variety
of the taxonomic classes, habitats and developmental strategies are typical of cnidarians and
allows this analysis to more accurately elucidate conservation, novelty, and differences in
cnidarian adhesion systems, whilst developing a strong basis for inferring the adhesion
repertoire of the common metazoan ancestor.
Chapter Four Diversity of Adhesion Molecules in cnidarians
41
4.2 Methods The adhesion complement of four cnidarians (Acropora millepora, Nematostella vectensis,
Hydra magnipapillata and Clytia hemispherica) and one choanoflagellate (Monosiga
brevicolis) was assessed using the automated annotation and data exploration capacities of
JCUSMART (described in Chapter 3). Predicted peptides with potential cell adhesion
capabilities were identified from 5 large sequencing datasets (Table 4.1) by searching
JCUSMART annotations with the terms presented in Supplementary Figure 4.1. These
searches focused on the discovery of peptides associated with 7 broad families of cell
adhesion molecules (cadherins, integrins, lectins, Adhesion-‐Leucine Rich Repeat (Adhesion-‐
LRR), Adhesion G-‐protein coupled receptors (Adhesion-‐GPCRs), immunoglobulin
superfamily, and extracellular matrix (ECM)), which are central to developmental,
immunological and defensive processes in metazoans. Many of the predicted peptides could
be further categorised into sub-‐families according to the presence of key architectural
features used in the classification of bilaterian proteins. Orthology of a prediction to any
previously described sequence was assigned only after manual consideration of both the
predicted protein architecture and results of BLASTp against the NCBI non-‐redundant (nr)
database. As detailed in chapter 3, genes were more easily detected in anthozoans, owing to
the larger size of the available data and longer predicted peptides. Therefore the absence of
some genes from this analysis (particularly from Clytia) may reflect limitations of the
predicted peptide collection rather than gene losses.
Animal Data type Number of sequences
Reference
Acropora millepora Transcriptome (2010) 48 335 Miller et al., unpublished
Nematostella vectensis Genomic predicted peptides
(version 1)
27 273 Putnam et al., 2007
Hydra magnipapillata Unigenes (EST contigs) 19 845 Bosch et al.,
unpublished
Clytia hemispherica Unigenes (EST contigs) 8 219 Houliston et al., 2010
Monosiga brevicolis Genome predicted peptides
(version 1)
9 196 King et al., 2008
TOTAL 112 868
Table 4.1 Predicted peptide datasets used to assess the adhesion complement of four cnidarians with diverse lifecycles and developmental features. Each dataset was annotated and explored using JCUSMART as described in Chapter 3. The Number of Sequences presented in the table represents an unadjusted total of the sequences analysed and does not take into account occasions where models were later combined to provide complete coding sequences.
Chapter Four Diversity of Adhesion Molecules in cnidarians
42
4.3 Results 4.3.1 Cadherins
Hydra magnipapillata
Clytia hemispherica
Nematostella vectensis
Acropora millepora
Monosiga brevicolis
Catenin binding 1 -‐ 4 1 -‐ Flamingo -‐ -‐ 1 1 -‐ Calsyntenin -‐ -‐ 1 2 -‐ FAT -‐ -‐ 1 -‐ -‐ FAT-Like -‐ -‐ 2 -‐ -‐ Dachsous like -‐ -‐ 10 1 6 Unique -‐ -‐ 2 -‐ 4
Table 4.2 Distribution of Cadherin family proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
A modest number of predicted peptides from each cnidarian contained extracellular
Cadherin (EC) domains (maximum of 56 distinct sequences in the complete dataset
available, the Nematostella genome) and very few peptides contained other conserved
domains. Although the number of cadherins in each animal was limited there was a
surprising richness of cadherins with recognised developmental and signalling roles
including orthologues of Type-‐III Catenin Binding cadherins (Figure 5.3), Flamingo (CELSR)
(Supplementary Figure 5.4), FAT and FAT-like (in Nematostella), Dachsous (Supplementary
Figure 5.2), Cadherin23 and Calsyntenin as well as a limited number of Protocadherins.
The number of genes in each sub-‐family is comparable between anthozoans, however, large
cadherins are difficult to detect and may obscure understanding of the complete hydrozoan
Cadherin complement. It is expected that representatives of the developmentally significant
cadherins (above) are in fact present in each cnidarian, supporting previous suggestions by
Hulpiau and van Roy (2011) that their origin predates the cnidarian divergence. Monosiga
peptides containing EC domains are unlike those of the Metazoa, consisting of novel
combinations of domains or chains of EC domains up to 58 repeats long.
Investigation of mRNA expression patterns during coral development and discussion of
putative protein function of some members of the Cadherin superfamily are presented in
Chapter 5.
Chapter Four Diversity of Adhesion Molecules in cnidarians
43
4.3.2 Integrins Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
integrin Alpha 1 1 2 3 -‐ integrin Beta 2 1 4 2 -‐ integrin Linked Kinase
1 1 1 1 -‐
ILK associated Ser/Thr Kinase
1 -‐ 1 -‐ 1
Talin 2 2 1 2 1 PINCH 1 -‐ 1 1 -‐ Parvin 1 -‐ 1 1 -‐ Paxillin 1 1 1 1 1 FAK -‐ 1 1 1 -‐ c-Src 1 -‐ -‐ 1 -‐
Table 4.3 Distribution of integrin and integrin associated proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
All components of the integrin signalling system reported in vertebrates were identified in
the cnidarians except Nematostella c-Src, Clytia PINCH & Parvin, and Hydra Focal adhesion
Kinase, although these too are expected to be present. Cytoplasmic signalling proteins of the
integrin pathway such as Talin, integrin Linked Kinase (ILK), PINCH and α-‐Parvin are
represented by a single peptide prediction in each cnidarian, however the number of α and β
integrin models is variable. As previously reported in Knack et al (2008), Acropora contains
only 2 integrin β-‐subunits (confirmed here by exhaustive transcriptome searches) compared
to Nematostella 4, Hydra 2 and Clytia 1. The distribution of α-‐Integrins is similarly fluctuant
between cnidarians, with Acropora possessing 3, Nematostella 2, Clytia 1, and Hydra 1. This
distribution combined with phylogenetic analyses (Figures 6.1 & 6.2) suggests that the
eumetazoan ancestor possessed a complement of at least 2 β and 1 α integrins and
expansion in each cnidarian is the result of independent duplications. The heterodimer
combinations and ligand binding properties of the cnidarian integrins are unknown, but may
hold clues as to their biological function and relationship with bilaterian integrins.
Evolutionary relationships and ligand binding properties of Acropora integrins are
presented in Chapter 6. Patterns of mRNA expression for Acropora integrins have previously
been reported by Knack et al (2008).
Chapter Four Diversity of Adhesion Molecules in cnidarians
44
Figure 4.1 Maximum likelihood analysis of Talin proteins from representative metazoans. Clytia Talin1 and Clytia Talin2 are closely associated, grouping together with Nematostella with strong bootstrap support (91%). Cnidarian sequences form an independent clade basal to the bilaterian and vertebrate clade, suggesting that duplication in Clytia is genus specific and does not support an ancestral origin of the duplication observed in vertebrates. Phylogenetic analysis is based on alignment of Talin proteins in the region of overlap between Clytia talin1 and Clytia talin2 (Supplementary Figure 4.2). Branch numbers represent percentage bootstrap support based on 100 bootstrap replicates.
In contrast to the complete conservation of integrin signalling components in basal
metazoans, the complement of Monosiga lacks critical components. Protein domains
resembling the α-‐integrin repeat domain were detected however no complete α or β
integrins could be identified. ILK, PINCH and α-‐Parvin were also absent, however Talin was
found to be present, which suggests an alternative function for FERM domain proteins,
which are enriched in choanoflagellates (Nicole King et al., 2008). Phylogenetic analysis of
Talin genes (Figure 4.1) places Monosiga and cnidarian Talin genes independent of
metazoan counterparts showing that vertebrate Talin1 and Talin2 arose from a lineage
specific independent duplication.
Chapter Four Diversity of Adhesion Molecules in cnidarians
45
4.3.3 Lectins Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
C-‐Type Soluble
12 4 42 3
C-‐Type Secreted 3 5 13 6 C-‐Type Transmembrane
4 2 10 2
Galectin 19 4 42 2 Fucolectin 1 -‐ 25 21 -‐ Mannose Binding (Legume Like)
2 -‐ 2 3 2
Haemolytic -‐ 2 -‐ 3 -‐ Table 4.4 Distribution of lectin family proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
lectin families known to be important to metazoan processes including cell-‐cell adhesion
and immunity were represented in cnidarians with unexpected diversity. The most
expansive lectin family of higher animals, the C-‐type lectins, were also particularly well
represented in all four of the cnidarians investigated and comprised the greatest proportion
of the lectins identified from each species. Galactose binding lectins were the next most
abundant being found in similar numbers to C-‐type lectins. Despite the number of C-‐type
lectins (42 from Nematostella), there was only a limited selection of predicted peptides that
are likely to be secreted (13 -‐ Nematostella) as determined by the presence of a signal
peptide. Even fewer models (9 -‐ Nematostella) contained a transmembrane helix and could
be considered potential cell surface antigens. The C-‐type lectin family did however contain a
surprising diversity of associated domains, which more closely resembles vertebrates than
protostome invertebrates (Wood-‐Charlson & Weis, 2009). One model of C-type lectin 16A
(CLEC16A), which is highly expressed in circulating immune cells and associated with a
range of auto-‐immune diseases in man, was found in each cnidarian. The CLEC16A models
were the only predicted lectins to demonstrate significant homology to known lectins (based
on BLAST) and have not previously been reported outside the Bilateria.
lectins containing a collagen domain, followed by a lectin domain were conserved
throughout the cnidarians. This structure is consistent with the Collectin family (Figure 4.3),
which has only been reported once in lower animals during a microarray analysis of
Acropora metamorphosis (Grasso et al., 2008). Unlike the bilaterian proteins, which consist
of Collagen -‐ C-‐Type lectin combination, 14 of the Collectins from cnidarians contain a
galactose binding domain (Gal_lectin), and only 2 (1 Nematostella and 1 Clytia) have a C-‐
Chapter Four Diversity of Adhesion Molecules in cnidarians
46
type lectin domain. Nematostella, Acropora and Clytia each contain three models with
predicted signal peptides and none of the sequences have transmembrane helices,
suggesting they are secreted.
Fuctose binding lectins (Fucolectins/F-‐type lectins) are also not reported in pre-‐bilaterian
animals and have limited abundance in basal deuterostomes such as the purple sea urchin
(Strongylocentrotus purpuratus – 2), yet a surprising number were identified in the
Nematostella genome (26) and Acropora transcriptome (23). More modest numbers were
identified in Hydra and Clytia, which contained 2 and 1 predicted peptides respectively,
suggesting an anthozoan specific expansion of the Fucolectin repertoire.
Acropora and Clytia each contain 2 members of the haemolytic lectin family, which has a
restricted distribution in metazoans being first reported in the sea cucumber, Cucumeria
echinata (Hatakeyama et al., 1994) then in Acropora millepora (Grasso et al., 2008).
Exhaustive searches of Hydra and Nemtatostella data failed to identify homologues of this
family suggesting selective gene losses have occurred (Forêt et al., 2010). Maximum
likelihood analysis (Figure 4.2) shows the Acropora haemolytic lectins clade independently
of the Clytia sequences, suggesting lineage specific duplications occurred from a single
ancestral gene. In situ hybridisation of coral haemolytic lectins demonstrate expression in
cells thought to be nematoblasts, the precursors to nematocysts (“stinging” cells)
(Supplementary Figure 4.4).
The lectin complement of Monosiga is much smaller than that of the cnidarians with only 15
peptides matching the search criteria. The majority of these (9) contain C-‐type lectin
domains and no representatives of the collectin, fucolectin or haemolytic lectin families are
present.
Chapter Four Diversity of Adhesion Molecules in cnidarians
47
Figure 4.2 Maximum likelihood analysis of metazoan haemolytic lectins shows sequences from Clytia and Acropora form separate clades with 100% bootstrap support, suggesting duplication of the ancestral haemolytic lectin occurred independently in each genus. The limited distribution of CEL-III like haemolytic lectins implies a number of independent gene losses occurred in the Cnidaria and elsewhere during bilaterian evolution (Foret et al., 2010). Phylogenetic analysis is based on alignment of haemolytic lectins shown in Supplementary Figure 4.3. Branch numbers represent percentage bootstrap support based on 100 bootstrap replicates. Acropora sequences are labelled according to their EST of origin (Grasso et al., 2008) and Clytia sequences correspond to Contigs IL0ABA2YE22RM1 (1) and IL0ABA8YL09RM1 (2).
Chapter Four Diversity of Adhesion Molecules in cnidarians
48
4.3.4 Adhesion LRR Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
LRR-‐Dsl -‐ -‐ 1 -‐ -‐ LRR-‐EGF -‐ 2 4 1 -‐ LRR-‐FN3 -‐ -‐ 1 -‐ 1 LRR-‐IG -‐ -‐ 2 4 -‐ LRR-‐LDLa -‐ -‐ -‐ -‐ 12 LRR-‐Sushi -‐ -‐ 1 -‐ 1 LRR-‐VWA -‐ 1 -‐ -‐ -‐ Scribble -‐ -‐ 1 1 -‐
Table 4.5 Distribution of proteins containing a leucine rich repeats (LRR) and an adhesion domain in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
Overall the adhesion-‐LRR complement of cnidarians is much smaller and shows less
diversity than that of bilaterians. More than 230 Leucine Rich Repeat containing proteins
were identified across the 5 species investigated and only 26 of them had potential adhesive
capability based on the presence of one or more adhesion domains. LRR containing models
proposed to have adhesive functions were sorted into dsl, EGF, FN3, IG, LDLa, SUSHI, and
VWA groups based on domain content and none of these groups were identified in Hydra.
Cnidarians appear to lack the LRR and LDLa domain combination, characteristic of the
relAxin hormone receptor family (LGR 7 and LGR 8) consistent with previous suggestions
that these proteins arose early in the vertebrate lineage (Wilkinson et al., 2007). This
domain combination was however dominant in Monosiga, which contained 12 LRR-‐LDLa
proteins compared to 1 LRR-‐Sushi and 1 LRR-‐FN3.
The number of models that are likely orthologues of known bilaterian proteins was limited
to 4. Each of the anthozoans contained 1 model of Leucine-Rich Repeats and Immunoglobulin-
Like Domains 3 (LRIG3) and 1 of Scribble. LRIG3 is suggested to play a role in tumour
suppression and neural tube closure (Abraira et al., 2010; Zhao et al., 2008), whilst Scribble
is crucial for signalling from the Cadherin FAT-1 and may be a cross-‐over point between
Planar Cell Polarity and Hippo signalling (Abraira et al., 2010; Zhao et al., 2008).
Chapter Four Diversity of Adhesion Molecules in cnidarians
49
4.3.5 Class B adhesion G-protein coupled receptors Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
7tm_2 Only 4 4 28 23 1 GPS-‐7tm_2 1 2 13 8 3 VLGR1 -‐ -‐ 1 1 -‐ CLECT-‐GPS-‐7tm_2
-‐ -‐ 3 -‐ 1
NvX Group -‐ -‐ 6 -‐ -‐ GPCR125 -‐ -‐ 1 1 -‐ Novel -‐ -‐ 3 2 1
Table 4.6 Distribution of Class B Adhesion G-‐Protein Coupled Receptors in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
The Class B G-‐Protein Coupled Receptors (characterised by a 7tm_2 domain) comprise the
most interesting group of GPCRs due to their roles in developmental signalling processes.
Like the Adhesion-‐LRR family, proteins containing a 7tm_2 domain were rare in all the
organisms investigated and a restricted distribution among cnidarians was observed for 3
protein subgroups. The majority of 7tm_2 proteins from each species have no detectable
extracellular domains and adhesion domains are extremely rare, consistent with reports
that adhesion-‐GPCRs expanded after the divergence of vertebrates (Nordström et al., 2009).
Orthologues of Frizzled, Smoothened and Very Large G-protein Couple Receptor1 (VLGR1)
were identified in each cnidarian but absent from Monosiga. C-‐type lectin and 7tm_2 domain
proteins were only identified in Nematostella as were proteins containing a Somatomedin B
(SO) domain. SO-‐7tm_2 proteins have previously been reported from Nematostella
(Nordström et al., 2009) and the present investigation confirms that these proteins are not
shared with other cnidarians. The third group with a restricted distribution, the GPCR125
orthologues, were found only in the anthozoans. Novel structures were also found in 6 genes
(3 Nematostella, 2 Acropora, 1 Monosiga).
Chapter Four Diversity of Adhesion Molecules in cnidarians
50
4.3.6 Immunoglobulin superfamily Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
DCC 1 -‐ 1 -‐ -‐ DS-‐CAM 1 1 9 -‐ -‐ F11/Contactin 1 -‐ 1 -‐ -‐ L1-‐like -‐ -‐ 2 1 -‐ N-‐CAM -‐ -‐ 3 4 -‐ IgLON 2 2 13 2 Ig and FN3 Containing
4 1 23 20 -‐
Ig and Adhesion
2 9 11 -‐ -‐
COLIG -‐ -‐ 5 9 -‐ TIR (Toll Pathway)
4 -‐ 12 10 2
RPTP -‐ -‐ 2 1 -‐ MALT-1 -‐ 1 2 2 -‐
Table 4.7 Distribution of immunoglobulin Superfamily proteins in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
The abundance of immunoglobulins containing any other associated domain is much smaller
in cnidarians than higher animals, with the largest representation consisting of 87 individual
predicted peptides (Nematostella). The majority of sequences found in Nematostella (55)
were associated with Fibronectin Type3 (FN3) domains and could be further categorised
into 6 sub-‐groups (N-‐CAM, L1-‐like, Contactin, DS-‐CAM, Repeat Protein Tyrosine Phosphatase
(RPTP), and Other Ig and FN3 containing), whilst most of the remaining sequences were
specified as part of the DCC, or Toll like families. Interestingly no Toll Like Receptors (TLR)
possessing the canonical structure known from higher animals were identified during this
survey. One LRR containing Toll receptor (NvTLR-1) has previously been identified in
Nematostella (Miller et al., 2007) and remains the only example from a cnidarian despite
exhaustive searches.
A number of models unrelated to the above sub-‐groups were also identified including
members of the FGF receptor family, Bystin proteins, and DCLK2/3 proteins. Mucosa
Associated Lymphoid Tissue 1 (MALT1) was also found in Nematostella, Clytia and Acropora,
and although these are not adhesion related genes they have previously only been reported
in vertebrates, C.elegans and Dictostellium. Partial putative models of extremely large
adhesion molecules were also identified in cnidarians including Titin, Roundabout and
VEGFR, however genes similar to those functioning in the adaptive immunity of vertebrates,
such as I-CAM and V-CAM were not identified in either cnidarians or choanoflagellates.
Chapter Four Diversity of Adhesion Molecules in cnidarians
51
One novel group of proteins, consisting of a Collagen domain followed by 2-‐5 Ig domains was
identified in Nematostella and Acropora, but are absent from the hydrozoan data. The
structure (Figure 4.3) is somewhat reminiscent of collectins, however the function has not
been described.
4.3.7 Extra-cellular matrix Hydra
magnipapillata Clytia
hemispherica Nematostella vectensis
Acropora millepora
Monosiga brevicolis
Collagen4 2 1 6 -‐ Fibrillar Collagen 12 12 10 2 Collagen 6 4 12 1 Minicollagen 20 2 3 -‐ Fibrinogen Domain Containing
2 44 92 3
Fibrillin 4 5 15 -‐ FN2 Domain Containing
1 2 11 -‐
FN3 Domain Containing
-‐ -‐ 1 2
Laminin Alpha 2 -‐ 9 1 Laminin Beta -‐ -‐ 1 -‐ Laminin Gamma 4 -‐ 4 1 Thrombospondin 4 6 3 -‐ Rhamnospondin -‐ 5 6 10 -‐ HSPG2 1 1 1 -‐ SPOCK 1 1 1 -‐
Table 4.8 Distribution of Extracellular matrix proteins in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.
The major components of the invertebrate extra-‐cellular matrix (ECM) such as type 4
Collagen, Laminins, Fibrillin and Heparin Sulphate Proteoglycan 2 (HSPG2) were identified
throughout the Cnidaria and a similar number of proteins in each family were detected
across Nematostella, Hydra, Cyltia and Acropora. All of these classes of ECM proteins occur
in the Bilateria with the exception of the mini-‐collagens, a family of proteins containing
simplified collagen like domains which were identified as a structural component of the
nematocyst cell wall in Hydra. Their existence in Acropora has been acknowledged in the
literature (Srivastava et al., 2010) however the relative number of mini-‐collagen domain
proteins in Nematostella (2), and Clytia (2) has not been reported. The laminin complement
of Nematostella is similar to that of vertebrates consisting of 3 or more alpha chains, 3 or
more gamma chains and only 1 beta chain. Alpha and Gamma chains are also predicted in
Hydra, whereas no laminins were detected in Clytia.
Chapter Four Diversity of Adhesion Molecules in cnidarians
52
A few of the large multidomain matrix proteins and proteoglycans were also represented
including Usherin, Sidekick, Nidogen and HSPG2, Titan, Polydom, SPARC and SPOCK, Slit and
notch. A surprising discovery was the presence in Nematostella of a model similar to
periostin, a matrix protein involved in regulation of bone mass in response to load in
vertebrate models. Fibronectin, Vitronectin, Tenascin’s and von Willebrand Factor were
absent from cnidarians further supporting their designation as vertebrate specific genes.
4.3.8 Novel cnidarian sequences Rhamnospondins are a family of thrombospondin and lectin domain containing proteins,
which have immune functions in Hydractina. This survey has identified homologues of
Rhamnospondins in the wider cnidarian community but not in Monosiga suggesting this
family originated in cnidarians. Similarly, minicollagen proteins, previously known from
Hydra, were found in each of the cnidarians, although this is perhaps unsurprising given
their role in cnidocyte cell wall formation. A novel protein family consisting of a signal
peptide followed by a Collagen domain and 2-‐4 Ig domains (Figure 4.3) was also found in
Nematostella and Acropora. The only other novel cnidarian adhesion family are GPCR
proteins containing a Somatomedin-‐B domain in extracellular region. This family are
described as the NvX GPCR family and through this analysis have been confirmed as
Nematostella specific. A few examples of novel protein architectures not shared between the
cnidarians were also identified in the adhesion GPCR family, although these protein
predictions need to be experimentally confirmed.
Chapter Four Diversity of Adhesion Molecules in cnidarians
53
Figure 4.3 Domain structure of selected cnidarian innovations. Collectin like proteins, containing a Galactose binding lectin domain rather than the C-‐type lectin domain of Collectins, were identified as novel sequences in the Cnidaria. A Family of proteins, which I named COLIG’s, containing a Collagen domain followed by 2-‐4 immungoglobulin domains was also identified for the first time in this analysis. COLIG’s are restricted to the anthozoan data and appear to be absent from hydrozoans, however it is unclear whether this is an anthozoan innovation or an example of hydrozoan gene loss. Rhamnospondins, which are characterised by a series of thrombospondin domains followed by a rhamnose binding lectin domain, also appear to be an innovation of the Cnidaria or ureumetazoan ancestor. Each of these families hold a structure similar to pattern recognition receptors of bilaterian humoral immune systems. The presence of a predicted signal peptide suggests these proteins are secreted, which may allow them to function in opsonisation of bacteria or other invasive micro-‐organisms.
Chapter Four Diversity of Adhesion Molecules in cnidarians
54
4.4 Discussion 4.4.1 The ancestral adhesion repertoire Cnidarian adhesion genes or gene families possessing features of known bilaterian proteins
are likely to have been retained from the Ureumetazoan ancestor (the last common ancestor
of cnidarians and bilaterians). The presence and absence of known gene families is
summarised in Table 4.9. Genes from each of the 7 adhesion families (cadherins, integrins,
lectins, Adhesion-‐LRR, Adhesion-‐GPCRs, immunoglobulin superfamily, and ECM) were
identified in all 4 cnidarians investigated and many showed homology to described
bilaterian protein architectures, although clear orthologues of higher animal proteins were
rarely apparent. Comparison of the cnidarian cell adhesion molecule complement with the
published bilaterian adhesion gene repertoire, has revealed a number of significant
functional and evolutionary aspects of cell adhesion.
The cnidarian extracellular matrix, cadherin, and immunoglobulin families all showed
characteristics more consistent with invertebrates than chordates. The extracellular matrix
proteins were remarkably well conserved with all major components of invertebrate ECM
(eg. Type-‐IV and fibrillar collagens, laminins, fibrilin, fibulin and Thrombospondin) present
in each cnidarian. The most surprising aspect of the cnidarian ECM was the diversity of
proteins associated with human disorders, which were identified primarily from the
Nematostella genome. These proteins include Titin, Usherin, Heparin Sulphate Proteoglycan 2
(HSPG2), MEGF, Polydom, and SPOCK. Although the precise roles of these proteins in
cnidarian development are unclear, their presence suggests the capacity to produce complex
cell-‐ECM interactions that facilitate cell migration, cilia formation and regulate matrix
assembly, is an ancestral trait.
Chapter Four Diversity of Adhesion Molecules in cnidarians
55
Family Genes present Genes absent Novel Genes Genes not previously Identified outside Bilateria
cadherins • Type-‐III Cadherin (catenin binding)
• FAT • FAT-‐Like • CELSR/Flamingo • Dachsous • Protocadherins
• Type I/II/IV Cadherin (Catenin Binding)
• Desmocolins • Desmogliens • CDH26 • 7D family
• 2 unique -‐ Nematostella
• Type-‐III Cadherin
• Dachsous • FAT • FAT-‐Like
integrins • α-‐Integrin • β-‐Integrin • integrin Linked Kinase (ILK)
• ILK associated phosphatase
• PINCH • Parvin • Talin • Kindlin • ADAM • ADAM_TS • GON family
-‐ -‐ -‐
lectins • C-‐type • Galactose Binding • Fucolectins • Haemolytic lectins
• Selectins • Collectin-‐Like • Rhamnospondins
• Collectins • Fucolectins
Immunoglobulin • N-‐CAM • L1-‐like • F11/Contactin • IgLON • RPTP • DCC • DS-‐CAM • Ig-‐FN3 • TIR containing • NvTLR-1 • MALT-‐1
• Canonical Toll Receptors
• Adaptive Immune Components
• COLIGs • RPTP • MALT-1
Adhesion LRR • LRIG3 • Scribble • LRR-‐EGF • LRR-‐dsl • LRR-‐IG • LRR-‐Sushi
-‐ -‐ • LRIG family • Scribble
Class B GPCRs • CLECT-‐7tm_2 • GPCR125 • VLGR
• Group II • Group VI • Group VII • Group VIII • Methuselah family
• Somatomedin B-‐GPCRs
• 2 Unique Acropora
• 3 Unique Nematostella
-‐
Chapter Four Diversity of Adhesion Molecules in cnidarians
56
Extracellular Matrix
• Fibrillar Collagens • Collagen 4 family • Contactin • Collagen Triple Helix containing
• Fibrinogen containing
• Fibrillin • Polydom • Fibulin • Thrombospondins • Periostin • Titan • Perlecan/HSPG2 • MEGF • Netrin • Fibropellin • Notch • Nidogen • Slit • Agrin • Usherin • Fras1 • Rabconnectin • Sortilin • Sorting nexin • Sidekick • SPOCK
• Fibronectin • Vitronectin • Osteonectin • Von Willebrand Factor
• Tenascin
• Minicollagens -‐
Table 4.9 Presence and absence of selected bilaterian adhesion genes with developmental and immunological roles. The present analysis has identified a number gene families previously thought to be bilaterian restricted including Type-‐II cadherins, FAT cadhrins and Fucolectins. The abundance of known bilaterian adhesion genes demonstrates that many of the adhesion systems recognised from higher animals were already established in the last common eumetazoan ancestor. Further diversity in mammalian systems is therefore likely to be the result of lineage specific expansions occurring after the cnidarian divergence. Genes involved in vertebrate sight and hearing are also present in cnidarians and are likely to have been sequestered from other biological functions, which are yet to be elucidated in lower animals. Gene families absent from the cnidarian data are largely associated with evolution of closed circulatory systems and circulating cellular immune systems. Hormone receptors associated with long range cellular signalling are also absent from cnidarians.
Chapter Four Diversity of Adhesion Molecules in cnidarians
57
cadherins that influence the early embryogenesis of higher animals have only recently been
reported in one cnidarian, Nematostella vectensis (Fung et al., 2008; Hulpiau & van Roy,
2009; Whittaker et al., 2006), and no expression or functional data has been published from
this model. Conservation of Type-‐III Cadherin, Flamingo, and Dachsous among the
cnidarians (as well as FAT and FAT-‐like protein in Nematostella) combined with their
absence from choanoflagellates suggest the critical roles these proteins play in β-‐catenin
signalling and planar cell polarity originated prior to the cnidarian divergence (See Chapter
5 for discussion). Reports from the genome of Amphimedon queenslandica (Porifera) have
also yielded a sequence for a Type-‐III Cadherin, but do not indicate the distribution of other
cadherins in sponges (Srivastava et al., 2010). The presence of these cadherins in both
cnidarians and sponges implies that Cadherin based morphogenic systems date back to the
metazoan common ancestor.
Despite the richness of morphologically crucial cadherins in cnidarians, the structure and
low total number of Cadherin proteins is most consistent with the invertebrate complement.
Type-‐III cadherins possess a characteristic domain structure and are not found in chordates
(with few exceptions). Using the JCUSMART method, the total number of genes with 3 or
more Cadherin domains in Nematostella (the most complete dataset used here) was found
to be 20 -‐ 4 more than found by Hulpiau et al (2011). In either case, the number of cnidarian
cadherins is similar to that of fly (17) and sea urchin (14), compared to over 110 sequences
in mouse and human (Fung et al., 2008; Hulpiau & van Roy, 2009; Whittaker et al., 2006),
supporting previous suggestions that diversification of cadherins occurred specifically in
chordates (Hulpiau & van Roy, 2011).
Immunoglobulin proteins lacked the complexity observed in higher animals. Whereas the
sea urchin genome yielded over 20 adhesion-‐LRR proteins with a wide variety of associated
extracellular domains, only 13 sequences were found across the 4 cnidarians (Nematostella -‐
9, Acropora -‐1, Clytia -‐3). The range of associated domains was also limited with only
EGF(7), Ig(3), FN3(1), Sushi (1), VWA(1), represented. The immunoglobulin family
contained representatives of all Ig-‐FN3 sub-‐families and the IgLON family, however, the total
number of immunoglobulin proteins falls far short of what is observed in sea urchin (~400;
Whittaker et al., 2006). Such an explosive expansion of the immunoglobulin superfamily in
deuterostomes most likely facilitated the occurrence of adaptive immunity, the components
of which are absent from cnidarians. Both the adhesion-‐LRR and immunoglobulin families
have recognised roles in innate immunity, serving largely as pattern recognition receptors
(PRRs) in other model organisms (Pancer & Cooper, 2006). This function is apparently
Chapter Four Diversity of Adhesion Molecules in cnidarians
58
conserved in cnidarians, with a member of the IgLON family implicated in allorecognition of
Hydractinia (Hydrozoa) colonies (Nicotra et al., 2009). The relative simplicity of these
adhesion families, as identified during this survey, suggest cnidarian pattern recognition
mediated by immunoglobulin and adhesion-‐LRR proteins is primitive when compared to
bilaterian systems.
The predominant PRR system of cnidarians may instead centre around the lectin family.
Cnidarian lectins were found to be surprisingly diverse and include a variety of C-‐type and
galactose binding lectins (Gal-‐lectins) with a range of associated domains. This level of
complexity in cnidarian C-‐type lectins more closely resembles the vertebrate lectin diversity
than that of model protostomes (Cambi et al., 2005; Dam & Brewer, 2010), which is
consistent with previous reports of genetic simplification among Ecdysozoans (eg.
Drosophila and Caenorhabditis) (Lin et al., 2000; Wood-‐Charlson & Weis, 2009). Both C-‐type
and Gal-‐lectins have been suggested to play roles in pattern recognition (Cambi et al., 2005;
Dam & Brewer, 2010; Weis et al., 1998). Pattern recognition is particularly important to
cnidarians that exist in obligatory symbiosis with intracellular algae (Zooxanthellae) and
lectins have been shown to facilitate the uptake of symbionts into host cells through specific
interaction with glycoproteins of the algal cell wall (Lin et al., 2000; Wood-‐Charlson & Weis,
2009). The most prominent example of this interaction comes from the only characterised
PRR lectin from a coral, Millectin, a C-‐type lectin isolated from Acropora millepora,
(Kvennefors et al., 2008; Kvennefors et al., 2010).
The lectin family also yielded some of the most interesting and unexpected cnidarian
innovations, all of which have implications for innate immunity. The first unexpected find
was the presence of fucolectins (F-‐type/Fucose binding), secreted innate immune effectors
first identified in eels (Honda et al., 2000) that have not previously been described in lower
animals. The anthozoans, Nematostella and Acropora, each contain an expanded set of more
than 20 (26 and 23 respectively) fucolectins compared to between 2 and 8 representatives
in bilaterians, suggesting that fucolectins form a central part of the anthozoan immune
system. Other unexpected finds included orthologues of CLEC16A (a disease associated
protein highly expressed in mammalian B cells and dendritic cells) and CEL-III (haemolytic
activity), further adding to the secreted innate immune repertoire of cnidarians.
Chapter Four Diversity of Adhesion Molecules in cnidarians
59
Comparison of the cnidarian adhesome with that of Monosiga was consistent with previous
indications that adhesion systems sustaining unicellular life are considerably simpler than
what is required in multicellular animals (Abedin & Nicole King, 2010). This was particularly
evident in the ECM, which contained limited examples of fibrillar collagens, a limited set of
laminin α and γ chains, and lacked all components of functional basement membranes (eg.
Type-‐IV collagens and laminin-β). Immunoglobulin domains were almost completely absent
from Monosiga, as were adhesion GPCR proteins, and whilst many of the domains known
from other adhesion families (eg. Cadherins and integrins) were identifiable in low
abundance (eg. Cadherin & integrin α domains), the overall protein architecture was unlike
that of animal adhesion molecules.
In contrast to systems such as innate immunity, characterised by genes absent in pre-‐
metazoans and likely to have originated in the ureumetazoan or urmetazoan ancestor,
integrin signalling is suggested to have originated well before the evolution of
multicellularity despite its absence from Monosiga. All components of the integrin signalling
complex were identified in cnidarians and only the terminal signalling molecules (α and β
integrins) varied in number. This finding is consistent with investigations into the pre-‐
metazoan ancestry of integrins, suggesting the integrin heterodimer, ILK, Talin, PINCH and
α-‐Parvin originated in the Unikont ancestor with canonical integrin signalling (involving FAK
and c-Src) appearing in the Opsithokont ancestor (Sebé-‐pedrós et al., 2010) (Although the
later is based on data from a single species). The function of integrin signalling components
in the primitive model organisms investigated by Sebé-‐Pedrós et al (2010), and therefore
the pre-‐metazoan ancestral function, is likely to be distinct from that of the metazoan
integrin system. Instead, these proteins are likely to have been co-‐opted from other
biological processes into morphogenic roles concurrent with the evolution of animal
multicellularity.
Chapter Four Diversity of Adhesion Molecules in cnidarians
60
4.4.2 Novel cnidarian sequences Novelty in the cnidarian adhesome is primarily centred around expansion of the immune
system with most phylum specific protein families exhibiting strong similarity to secreted
pattern recognition proteins. Models containing an N-‐terminal collagen domain followed by
a C-‐terminal Gal_lectin domain were identified in each of the cnidarians, an architecture
which closely resembles that of Collectins (Collagen – C-‐type lectin). Vertebrate Collectins
function in humeral immunity through opsonisation, neutralisation, agglutination,
phagocytosis and activation of the complement system in addition to modulating apoptotic
cell clearance (Gupta & Surolia, 2007; van de Wetering et al, 2004). Each of these immune
responses are expected to also occur in cnidarians as apoptotic processes and components
of the complement system (eg. C3, MASP, Ficolins (also identified by the present analysis),
and Factor B) have both been identified (Ainsworth et al., 2007; Kimuraet al., 2009; Lasi et
al., 2010). However, the range of proteins able to elicit these responses awaits clarification.
Two other taxon specific families with a similar general architecture, consisting of a
conserved homophilic matrix protein domain (TSP1 or Collagen) in conjunction with an
antigen recognition domain (lectin or Immunoglobulin) were also identified. The
Rhamnospondin family (TSP1 – Rhamnose binding lectin) have previously been identified in
Hydractinia, where they are suggested to mediate bacterial defence in the oral region
(Schwarz et al., 2007). The present analysis detected Rhamnospondins in the wider
cnidarian community, suggesting they are not a hydrozoan innovation. Rhamnospondins
have also been reported from the tunicate Botyllus schlosseri (Oren et al, 2007), however
their identification in this urochordate is unconvincing. The final family of novel proteins
with the matrix-‐recognition architecture, which have here been given the name “COLIGs”, is
previously undescribed anywhere in the Metazoa, consisting of a collagen domain followed
by 2 Ig domains. This structure again suggests a role in resisting colonisation by micro-‐
organisms.
Whereas the Collectin-‐like proteins, Rhamnospondins and COLIGs provide secreted defence
by opsonisation, the minicollagens, which are also unique to cnidarians, are reported to be
critical to physical defence. Minicollagens are demonstrated components of the cnidocyte
cell wall, which is a defining defensive structure in all cnidarians. It is therefore unsurprising
that minicollagen proteins were identified in both anthozoans and hydrozoans.
Chapter Four Diversity of Adhesion Molecules in cnidarians
61
4.4.3 Differences between cnidarian adhesion systems Although many of the major components of bilaterian adhesion could be identified in all of
the cnidarians, a number of proteins with adhesion domains had a restricted distribution.
These proteins showed no bias towards any one biological process or adhesion family. The
Anthozoans were found to each possess a single LRR & Ig domain containing protein, which
showed clear orthology to vertebrate LRIG3. The LRIG family was thought to be vertebrate
specific and LRIG3 has been experimentally determined to influence neural crest formation
and complex tissue morphogenesis (eg. inner ear morphogenesis), possibly through
modulation of the FGF and Wnt pathways (Abraira et al., 2010; Zhao et al., 2008).
Orthologues of XPTP-D, a Xenopus receptor Protein Tyrosine Phosphatase involved in
retinal axon extension and neurite outgrowth (Johnson & Holt, 2000; Johnson et al., 2001),
were also restricted to Anthozoans. The prospect of ancient roles for these genes in
embryogenesis and neurogenesis of anthozoans is exciting and warrants further
investigation, however a reason for their absence from hydrozoans cannot yet be suggested.
The genome of Nematostella vectensis revealed a number of proteins that are not found in
other cnidarians such as NvX group and C-‐type lectin domain containing (Group V) GPCRs.
Extensive targeted searches of the available cnidarian data have confirmed that these
families are species specific innovations. Nematostella also possesses the only known
canonical Toll receptor in cnidarians (Miller et al., 2007). The canonical structure of the Toll
receptor is expected to be ancestral and has apparently been lost from other model
cnidarians.
The most peculiar distribution among cnidarians is that of the haemolytic lectins, which
bind to target cells via the lectin domain and cause osmotic rupture by pore formation.
Haemolytic lectins were identified in only 1 of the anthozoans (Acropora; Grasso et al.,
2008) and 1 of the hydrozoans (Clytia), with the 2 sequences in each species resulting from
independent duplications (Figure 4.2). These cnidarian lectins are orthologous to the CEL-III
haemolytic lectin from sea cucumber (Cucumaria echinata) and together make up the entire
known metazoan complement of haemolytic lectins. The distribution of haemolytic lectins in
echinoderms and cnidarians suggest these genes have been lost in a number of independent
lineages, with parallel losses occurring in anthozoans and hydrozoans.
Chapter Four Diversity of Adhesion Molecules in cnidarians
62
The remarkable distribution of haemolytic lectins, along with the restriction of canonical
Toll receptors, highlights both the ubiquity and stochastic nature of gene loss in cnidarians
and throughout the Metazoa (Forêt et al., 2010). Furthermore, cnidarian haemolytic lectins
are retained only in species that inhabit a marine environment, suggesting estuarine and
marine cnidarians have distinct defensive strategies and that adaptation to an estuarine
habitat has impacted on gene loss in Nematostella and Hydra.
4.4.4 Interesting absences The cnidarian adhesome lacks a number of genes present in bilaterians, particularly those
recognised to be chordate specific expansions such as some classes of cadherins and
integrins. The missing Cadherin family members include Type I and II cadherins (eg. E-
Cadherin and N-Cadherin) as well as the recently evolved desmocolins and desmoglians. The
absence of β-catenin binding Type I and II cadherins was predictable due to a demonstrated
degree of functional analogy with invertebrate Type-‐III cadherins, which are considered the
ancestral ‘classical’ Cadherin (Hulpiau & van Roy, 2009; 2011). Other predictable absences
include proteins associated with adaptive immunity and matrix proteins such as Fibronectin
and Von Willebrand Factor, which function in maintenance of vascular systems, a structure
lacking from cnidarians.
integrins with VWA domains involved in binding of collagens and those functioning in
leukocyte trafficking were also absent from all the cnidarians investigated. Cnidarian
integrins are not directly related to mammalian types and have unknown binding
capabilities, although the ability to bind collagen is unlikely (See Chapter 6). Reelin, a cell
surface receptor, which functions in positioning neuroblastic cells, is another significant
absence from the cnidarian adhesome. This exclusion may at first seem surprising given the
presence of other large neural adhesion proteins in Nematostella such as Slit, Netrin, and
Roundabout, however, Reelin binds to a soluble ligand, acting in the neurocrine system.
Hormone receptors are completely absent from cnidarians, however they are prevalent in
basal deuterostomes such as sea urchin.
Chapter Four Diversity of Adhesion Molecules in cnidarians
63
4.5 Concluding comments The overall composition of the 7 adhesion families making up the cnidarian adhesome is
remarkably similar to adhesion systems of bilaterian invertebrates, with the exception of the
lectin family, which has undergone significant simplification in Ecdysozoans. This degree of
conservation suggests that most of the recognised bilaterian adhesion components affecting
developmental, innate immune and defensive processes were already established in the
ureumetazoan ancestor. Determining which of these adhesion systems are linked to the
origin of metazoan multicellularity and the earliest forms of morphogenesis will require
similar surveys of the recently released sponge genome (Amphimedon queenslandica). Such
studies could also clarify the influence of adhesion proteins on the evolution of dedicated
tissues, which are a eumetazoan feature absent from sponges.
The abundance of secreted innate immune proteins observed in all 4 species investigated
here, highlights the importance of pattern recognition to cnidarian survival. The aqueous
environment inhabited by cnidarians could easily facilitate colonisation by pathogenic
micro-‐organisms, which may go some way towards explaining the development of a
predominantly secretory cnidarian immune system in preference to forms of cell-‐contact
mediated immunity. The secretion of mucus as a general stress response in cnidarians may
also increase the efficacy of these immune mediators in resisting colonisation by reducing
loss of secreted proteins to the water column. Alternatively, a proportion of the pattern
recognition receptors may be associated with nematocysts (‘stinging’ cells) that form the
primary mechanism for cnidarian defence and prey capture. In situ RNA hybridisation of
both haemolytic lectins and Collectin-‐like proteins in Acropora polyps demonstrate
expression in putative nematoblasts, the precursors to nematocysts (Grasso et al., 2008;
Supplementary Figure 4.4). Further investigation of pattern recognition protein expression
is required to determine whether the secreted innate immune proteins of cnidarians
constitute a form of humoral immunity or if the innovation in cnidarian pattern recognition
is primarily associated with cnidarian specific structures.
Whether the cnidarian pattern recognition complement represents a phylum specific
expansion or the higher animal repertoire is a simplification of the ancestral state is unclear.
However, surveys of other lower metazoan phyla will resolve these issues as complete data
becomes available for a broader range of basal animals. The evolutionary and functional
impacts of cnidarian innate immunity are therefore an area of interest for future
investigations and may also contribute to our knowledge of symbiont uptake and coral
bleaching mechanisms, which will aid in reef conservation efforts.
Chapter Four Diversity of Adhesion Molecules in cnidarians
64
Many of the cell adhesion molecules directing morphogenic cell movements during
gastrulation and neural development were identified in each of the cnidarians. The effect of
these proteins in the development of diploblastic animals however has not been explored,
with most studies instead focusing on the expression and regulation of transcription factors
expressed in tissue restricted patterns. The insight into cnidarian cell adhesion presented
here establishes a strong foundation for future analyses aimed at understanding the
expression and function of genes that ultimately determine tissue differentiation and
morphogenesis in lower animals.
Chapter Five Developmental roles for cadherins from Acropora millepora
65
Chapter 5: Developmental roles of cadherins from Acropora millepora
5.1 Introduction Developmental processes in metazoans require regulated adhesion and communication
between cells in order to properly co-‐ordinate morphological change and ensure correct
functional associations are established. Members of the Cadherin family are critical to these
processes and whilst their primary capability may appear to be adhesion between cells
achieved through calcium dependant homophilic interactions, cadherins have a far more
influential part to play than simply mediating cell-‐cell contact (Hulpiau & van Roy, 2009;
Magie & Martindale, 2008; Nollet et al., 2000; Yagi & Takeichi, 2000). Differential regulation
and a variety of cytoplasmic connections allow cadherins to take an active role in regulating
the cytoskeleton, cell shape and tissue polarisation as well as playing roles in cell sorting and
the intracellular signalling that determines morphogenesis (Gumbiner, 2005; Halbleib &
Nelson, 2006).
The earliest recognised cadherins were isolated because of their clear roles in gastrulation
and are now considered “classical” or Type-‐I cadherins (Yoshida and Takeichi, 1982; Kemler
1992). Members of this group, which include E-Cadherin (Cdh1) and N-Cadherin (Cdh2), have
5 extracellular Cadherin domains, followed by a transmembrane helix and a characteristic
cytoplasmic region. There are now more than 15 recognisable groups of cadherins based on
the arrangement of conserved domains and motifs along the protein (referred to herein as
the protein architecture). Phylogenetic analysis of first true Cadherin repeat in the
ectodomain has identified 6 major clades and revealed substantially more familial
associations (Hulpiau & Frans van Roy, 2009; Nollet et al., 2000; F van Roy & Berx, 2008).
Although there are many sub-‐families of cadherins, some genes stand out as particularly
significant for their functions in development, such as Type-‐I cadherins, CELSR
(Flamingo;Fmi), Dachsous (Ds) and FAT (Ft) cadherins (Halbleib & Nelson, 2006). Like Type-‐
I cadherins, those from the Type-‐III and Type-‐IV groups have been shown in different
animals to be critical to the cellular re-‐arrangements of germ layer formation (Miller &
Mcclay, 1997; Oda et al., 1998; Choi and Gumbiner, 1989; Hatta et al., 1987; Takeichi et al
1986). Each of these 3 families, which vary in overall protein architecture (Figure 5.1), share
a common feature -‐ the ability to make functional connections to the cytoskeleton through
binding cytoplasmic catenins (Yagi & Takeichi, 2000). The Cadherin cytoplasmic domain
(CCD) is a defining feature of these groups and contains 2 highly conserved motifs, the juxta-‐
Chapter Five Developmental roles for cadherins from Acropora millepora
66
membrane domain (JMD), responsible for binding δ-catenin (p120-catenin), and the catenin
binding domain (CBD), responsible for binding β-catenin (van Roy & Berx, 2008). The ability
of these proteins to influence the cytoplasmic pool of β-catenin sets them apart and conveys
the influence over morphogenic events for which these cadherins are known (Gumbiner,
2005).
Two functions of catenin binding cadherins are particularly well conserved in the Bilateria,
cell cohesion and cell migration. In vertebrates, expression of E-Cadherin is detectable at
adherens junctions even before gastrulation has occurred. Like in adult tissues, the role of E-
Cadherin at this stage of development is to maintain stable cellular contacts and limit the
movement of cells within the cell layer. As gastrulation commences, cells fated to become
mesoderm and endoderm begin to express N-Cadherin. As these cells begin to migrate by
ingression or involution, they cease to express E-Cadherin at the cell surface (Takeichi,
1988). This occurs by both endocytosis of membrane associated E-Cadherin and
transcriptional inactivation (Le et al., 1999; Hatta et al., 1987). The loss of epithelial
character associated with the migratory or invasive phenotype has been demonstrated to be
a direct result of N-Cadherin expression. Moreover, co-‐expression of N-Cadherin and E-
Cadherin produces cells that have the same invasiveness as those expressing N-Cadherin
alone (Nieman et al., 1999).
In protostomes, Type-‐IV Cadherin (DE-Cadherin/Shotgun in Drosophila) is functionally
analogous to vertebrate E-Cadherin, showing a similar protein localisation and undergoing
the same down-‐regulation during mesoderm development (Oda et al., 1998). Like in
vertebrates, DN-Cadherin is up-‐regulated in migrating mesodermal cells, although the
protein architecture of DN-Cadherin (a Type-‐III Cadherin) is significantly different from that
of DE-Cadherin (type-‐IV) and vertebrate N-Cadherin (type-‐I). In sea urchin, an invertebrate
deuterostome, no functional analogue of N-Cadherin has been described. Instead, the
flexibility of cell junctions that allows invagination of presumptive mesendoderm has been
suggested to be facilitated by de-‐coupling of LvG-Cadherin from the cytoskeleton. Protein
localisation studies indicated that whilst LvG-Cadherin expression at the cell surface is
maintained, cell surface expression of α-catenin and β-catenin is greatly reduced compared
to the surrounding ectoderm (Miller & Mcclay, 1997). Together, these observations suggest
both protostomes and deuterostomes require at least 1 catenin binding cadherin that
facilitates tissue stability and in most cases a 2nd to promote a migratory or invasive
phenotype.
Chapter Five Developmental roles for cadherins from Acropora millepora
67
Figure 5.1 Generalised protein architecture of catenin binding cadherins. Catenin binding cadherins possess distinct ectodomain structures allowing classification as Type-‐I/II, Type-‐III or Type-‐IV. Type-‐I & Type-‐II cadherins (classical cadherins) contain 5 Cadherin domains (ectodomain repeats; EC) followed by a transmembrane domain and Cadherin cytoplasmic domain. The highly conserved Cadherin cytoplasmic domain (CCD) is responsible for functional connections to the cytoskeleton through interaction with δ-‐catenin via the JMD motif, and β-catenin via the CBD motif. Unlike Type-‐I & Type-‐II cadherins, which are chordate specific, Type-‐III & Type-‐IV cadherins possess longer extracellular domains consisting of 8 or more EC repeats and a membrane proximal “Primitive Classic Cadherin Domain” (PCCD). The PCCD consists of a single “non-‐chordate” domain, followed by alternating EGF-‐like and Laminin G (LamG) domains. Cadherins of Type-‐IV structure have 8 EC domains and their PCCD contains only 1 EGF-‐like and 1 LamG domain. By contrast, the number of EC domains in Type-‐III cadherins varies, with some proteins containing as many as 30 EC domains. The Type-‐III EC domains are also preceded by N-‐Terminal EC-‐like domains, which hold a similar structure but are not capable of homophilic binding. The PCCD of Type-‐III cadherins contains a 3 EGF-‐like domains and 2 LamG domains, more than other types of catenin binding cadherins.
Figure 5.2 Milestones in cadherin evolution (adapted from Hulpiau and Van Roy, 2009). The proposed appearance of features in the Cadherin domain structure throughout evolution are marked in blue with losses marked in red. Genomic surveys suggest early cadherins contained a wide variety of associated domains (eg. Nhh, vWA and IgCAM domains) and simplification of these associations occurred in the bilaterian lineage. Catenin binding cadherins have been suggested to be a bilaterian invention, first occurring as Type-‐III cadherins in the Urbilateria (common ancestor of bilaterians). From the Type-‐III structure, these cadherins are believed to have diversified through a serries of domain simplifications into Type-‐IV and Type-‐I/II cadherins in protostomes and chordates respectively.
Chapter Five Developmental roles for cadherins from Acropora millepora
68
In addition to roles in making cytoskeletal connections at the cell membrane, β-catenin is an
important cytoplasmic signalling molecule. If not active at the cell membrane, ‘free’
cytoplasmic β-catenin is rapidly degraded following phosphorylation by the GSK3β complex,
consisting of Glycogen Synthase Kinase-3β (GSK3β), Adenomatous Polyposis Coli (APC) and
Axis Inhibition Protein (Axin). Alternatively, β-catenin can function as an activating co-‐factor
to T-‐Cell Factor (TCF/LEF) transcription factors, which lay downstream of the canonical Wnt
Pathway. Canonical Wnt signalling through the Frizzled group of cell surface receptors
inhibits GSK3β mediated degradation of β-‐catenin, thereby stabilising the cytoplasmic pool
and effecting its nuclear translocation (Nelson & Nusse, 2004; Schambony et al., 2004). In
the nucleus, TCF/LEF is activated by replacement of the repressing co-‐factor, Groucho, with
the activating co-‐factor β-‐catenin (Chen & Courey, 2000; Range et al., 2005). Through this
mechanism, β-‐catenin is critical to canonical Wnt signalling, which is commonly regarded as
one of the most important signalling cascades in bilaterian development, governing diverse
processes such as axis specification and limb development.
Canonical Wnt signalling is recognised to activate epithelial to mesechymal transitions
(EMT), which is a critical step during gastrulation of bilaterians (Chuai & Weijer, 2009).
Among the main features of a full EMT is the loss of epithelial adhesions (adherens
junctions) and increase in cell motility (Shook & Keller, 2003). It is therefore perhaps
unsurprising that Canonical Wnt has a reciprocal inhibitory relationship with the expression
and function of CCD containing cadherins, as both compete for β-catenin activity to facilitate
opposite adhesive states (Vincan & Barker, 2008; Wang et al., 2010). Canonical Wnt
signalling has been demonstrated to stabilise and up-‐regulate the E-Cadherin repressor,
Snail, which is required for EMT (Yook et al., 2006; Katoh and Katoh, 2006; Yook et al.,
2005). Furthermore β-catenin has been suggested to directly repress transcription of E-
Cadherin (van Roy & Berx, 2008). Conversely, E-Cadherin has been demonstrated to inhibit
the translocation of β-catenin to the nucleus in over-‐expression experiments (Logan et al.,
1999; Wikramanayake et al., 2003), thereby acting in a positive feedback loop to maintain
cellular adhesions and highlighting the importance of β-catenin regulation in determining a
cell’s adhesive state.
The developmental roles of CELSR, Ds and Ft cadherins are also influenced by Wnt signalling,
although here signalling through the non-‐canonical pathway is upstream of their expression
(Strutt, 2003). These cadherins are all components of systems that determine the
polarisation of cells within an epithelial cell sheet or planar cell polarity (PCP). Two main
systems of planar cell polarity have been described, the Fmi-Fz (‘core’) pathway and the Ds-
Ft pathway. In most well described examples, these systems function within the same tissue
Chapter Five Developmental roles for cadherins from Acropora millepora
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and both are required for correct morphogenesis (for review see Fanto and McNeill, 2004),
however regulatory mechanisms between these systems remain enigmatic. In each system,
cell polarity is effected by the asymmetrical localisation of membrane bound components to
opposing ends of the cell.
Interactions between the membrane bound components have been shown to occur through
direct binding of opposing extracellular domains. The Fmi-‐Fz system has opposing
localisation of Fz-‐Dgo-‐Dsh-‐Fmi and Vangl-‐Pk-‐Fmi complexes, whereas the Ds-‐Ft system relies
on opposing localisation of Ds and Ft at the membrane as well as asymmetrical cellular
localisation of four-‐jointed (fj), a golgi protein that modifies the Ds protein (Wu & Mlodzik,
2009). Components of each system are expressed in a graduated pattern across the
epithelium and polarisation of tissues is determined by the relative expression and activity
of each system. The relationship between their relative expression and morphological
events is not simple. Two classical examples are the Drosophila Wing and Eye, where both
the Fmi-‐Fz and Ds-‐Ft systems are expressed. In the Wing, graduated activity of Ds-‐Ft along
the proximal-‐distal axis opposes that of Fmi-‐Fz, whereas in the eye, the activity gradient of
both systems are concurrent (Wu & Mlodzik, 2009). Although there are a number of
unresolved aspects of planar cell polarity function, the influence of appropriate PCP on
morphogenesis has been clearly demonstrated in two distinct processes. Convergent
extension, the process of elongating an epithelial sheet through intercalation of existing
cells, is seen in vertebrate gastrulation and is a critical process for neuralation (Shindo et al.,
2008; Wang et al., 2006). The second process is polarised cell division, whereby
establishment of tissue polarity determines the direction of mitotic spindle alignment
through constraint of the cells shape (Gong et al., 2004).
The involvement of PCP in both convergent extension and polarised cell division has been
shown in Zebrafish, Drosophila and Caenorhabditis, suggesting these are evolutionary
conserved morphogenetic events (Carreira-‐Barbosa et al., 2009; Gong et al., 2004; Seifert &
Mlodzik, 2007). Similarly, the role of β-catenin in both transcriptional and membrane
associated roles has been highly investigated in protostomes and deuterostomes. Very few
investigations however, have described homologues of β-catenin and PCP adhesion systems
in ‘lower’ metazoans.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Consideration of the distribution and arrangement of adhesion domains within Cadherin
proteins from Nematostella and choanoflagellates has revealed the ancestral nature of the
Cadherin extracellular domain and describes a variety of domain associations that have
since been lost in the Bilateria (Abedin & King, 2009). The presence of the catenin binding
Cadherin cytoplasmic domain is reportedly unique to metazoans however, in a number of
basal cognates, the overall architecture of proteins containing this domain has not been
reported. Hulpiau and Van Roy (2009) proposed the model of Cadherin evolutionary
milestones described in Figure 5.2, suggesting that a number of developmentally significant
cadherins, including those that bind β-Catenin are restricted to the Bilateria, despite the
parallels in early development of selected cnidarians and bilaterians.
Genomic surveys of the sea anemone Nematostella vectensis have previously revealed the
existence of Canonical and Non-‐canonical Wnt components including β-‐catenin, the GSK3β
complex, Frizzled and Van Gogh, however no direct analysis of the Cadherin complement of
cnidarians has been made (Kusserow et al., 2005; Putnam et al., 2007). Furthermore, the
expression patterns of the Nematostella Wnt genes and β-catenin have been described and
discussed in terms of their signalling during gastrulation (Lee et al., 2007; Wikramanayake
et al., 2003), however the localisation and putative roles of planar cell polarity genes and
catenin binding cadherins have not been reported outside the Bilateria.
Investigation of cadherin-‐catenin and planar cell polarity signalling components in
cnidarians and sponges will resolve the importance of these systems to metazoan evolution
and reveal their ancestral significance in development. Here, I describe the distribution of
genes significant to Cadherin-‐catenin signalling and planar cell polarity in 4 representative
cnidarians, with different developmental features. The expression of catenin binding
cadherins and a number of planar cell polarity components are investigated by in situ
hybridisation of coral embryos and larvae and the functional and evolutionary implications
of these systems are considered.
Chapter Five Developmental roles for cadherins from Acropora millepora
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5.2 Methods 5.2.1 Sequence identification Sequences for “classical” cadherins and other key components of the Wnt -‐ Planar Cell
Polarity pathway were identified using JCUSMART (See Chapter 3). Sequences from 4 large
datasets representing Acropora millepora, Nematostella vectensis, Hydra magnipapilata and
Clytia hemispherica were assessed for the presence of key architectural features and overall
sequence similarity (BLASTp) to known representatives of target genes already in Genbank.
5.2.2 Cadherin phylogenetics Maximum likelihood analysis was performed on 28 cnidarian and bilaterian cadherins
possessing a classical Cadherin cytoplasmic domain which consists of both a JMD motif and a
CBD motif. Due to the highly repetitive nature of the Cadherin extracellular domain, this
analysis is based on truncated sequences corresponding to 148 positions within the
cytoplasmic region and encompassing both the JMD and CBD motifs. Maximum likelihood
analysis was performed using PhyML3.0 (Guindon & Gascuel, 2003) and supported by 100
bootstrap replicates.
5.2.3 Isolation of riboprobe template cDNA Partial sequences of selected genes identified in the Acropora millepora transcriptome were
amplified from Acropora cDNA libraries and cloned into pGEM-‐T vector (Promega) as
described in Chapter 2.2. Amplified regions correspond to sections of the coding sequence
that exhibit the least homology to other genes and do not encompass internal repeats of
functional protein domains. These strategies were utilised to increase the specificity of the
resulting probe. Although a number of primer combinations were assessed, the sequences of
only the most successful pair for each gene are shown below. Full or partial sequences for
the remaining genes were recovered from the Acropora millepora Expressed Sequence Tag
(EST) project. In situ hybridisation was performed as described in Chapter 2.4.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Am_ACadherin F3 5’-‐GGGCAACATCATCTCAAGC-3’
Am_ACadherin R5 5’-‐ATGCGATTGAGGTTATCGAA-3’
AmFlamingo F4 5’-‐GGTCCACGTACCACCAAG-3’
AmFlamingo R4 5’-‐AGAGAATCAAGGAATATCAAG-3’
Am_Van_Gogh like F3 5’-‐GTGATCGCAATATGGGCTT-3’
Am_Van_Gogh like R3 5’-‐TCTGCTTCTTCATAGAAACG-3’
Am_Wnt16 Forward 5'-‐GTACAGTGGTTGGGGAATGG-‐3'
Am_Wnt16 Reverse 5' -‐ CAGTGCAAGAGCCAGAAACA-‐3'
Am_beta-‐Catenin Unigene: B025-‐E11
Am_Frizzled4 Unigene: C007-‐H4
AmDachsous Unigene: D041-‐C8
Am_WIF Unigene: C014-‐E11
Am_Axin Unigene: D027-‐D10
Am_Dishevelled Unigene: B036-‐C4
Chapter Five Developmental roles for cadherins from Acropora millepora
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5.3 Results 5.3.1 Identification of catenin binding cadherins and planar cell
polarity components from Acropora millepora Two sequences with homology to the catenin-‐binding Cadherin cytoplasmic domain were
identified in the Acropora millepora transcriptome using JCUSMART. One contiguous
sequence (Contig6389 + Contig5439; Sequence presented in Supplementary Figure 5.1) of
8,933bp, which I named Am_ACadherin, showed significant homology to Type-‐III cadherins
in both protein architecture and cytoplasmic domain sequence alignment (Figure 5.3).
Although the 5’ sequence of Am_ACadherin could not be resolved with the available data, the
cytoplasmic region contains both a p120/δ catenin binding juxta-‐membrane domain (JMD)
and β-catenin binding domain (CBD) (Figure 5.3 A). The second sequence (Contig10872),
7,786bp, was identified as an orthologue of Dachsous (Ds) (see Supplementary Figure 5.2 for
cytoplasmic region boxshade alignment), an atypical protoCadherin that participates in
planar cell polarity in bilaterians. AmDachsous (AmDs), the first reported Ds orthologue
outside the Bilateria, contains only a CBD in the cytoplasmic region as is consistent with
bilaterian Dachsous genes.
Two partial sequences (Contig8737 and Contig3665) corresponding to the central and C-‐
terminal regions (3,073bp and 3,898bp respectively) of the core planar cell polarity
Cadherin CELSR/Flamingo (Fmi) were also identified using JCUSMART. The presence of coral
orthologues of core (Fmi) and secondary (Ds) planar cell polarity pathway cadherins
prompted investigation of other components of this pathway. Almost all investigated genes
associated with Cadherin-‐catenin signalling, PCP signalling and Wnt/PCP pathway are
represented throughout the Cnidaria (Table 5.1). The range of genes apparently absent from
Hydra and Clytia is likely due to data limitations rather than gene losses. These signalling
pathways can therefore be considered evenly represented among species with different
developmental mechanisms, suggesting they are of fundamental importance to survival.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Figure 5.3 Protein conservation and architecture of Am_ACadherin
A) Alignment of Am_ACadherin cytoplasmic domain to representative sequences from bilaterians. Shading demonstrates >50% consensus to Am_ACadherin (Back –conservation, Grey – conservative substitution). Am_ACadherin contains both a JMD (Yellow box) and CBD (blue box), which are required for interaction with β-‐catenin and the cytoskeleton.
B) Domain structure of Am_ACadherin. Sequences gained from the 2010 assembly of Acropora millepora show only an N-‐terminal truncated model. The total number of EC repeats is therefore unknown. The assembly of 2 overlapping contigs (Contig 6389 + Contig5394) demonstrates Am_ACadherin contains at least 13 EC domains followed by the canonical structure of PCCD, TMH, CCD consistent with Type-‐III cadherins. This is the first reported type-‐III Cadherin outside of the Bilateria suggesting the developmentally critical function of cadherins in facilitating cell adhesion and influencing β-‐catenin balance originated earlier than previously believed.
(Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum
Chapter Five Developmental roles for cadherins from Acropora millepora
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Gene Acropora Nematostella Hydra Clytia
Type-‐III Cadherin 1 3 -‐ -‐ Beta-‐Catenin 1 1 1 1 Axin 1 1 1 -‐ APC 1 1 1 -‐ GSK3b 1 1 1 1 Dishevelled 1 1 1 1 CELSR/Flamingo 1 1 -‐ -‐ Frizzled 4 4 2 1 Van Gogh/Strabismus 1 1 1 1 Diego -‐ -‐ -‐ -‐ LRP5/6 1 1 -‐ -‐ Dachsous 1 1 -‐ -‐ FAT -‐ 3 -‐ -‐ Four-‐jointed -‐ -‐ -‐ -‐ Wnt16 1 1 1 1 Groucho 1 1 1 1 Inversin 3 (partial) 1 -‐ -‐
Table 5.1. Distribution of catenin binding cadherins and planar cell polarity pathway components in cnidarians. For each protein, the number of homologues that were positively identified by the JCUSMART method are shown for each cnidarian species investigated. This value may vary from previously published values, for example, 2 Frizzled proteins have been reported in Clytia (Momose & Houliston, 2007), however only 1 protein could be confirmed as Frizzled on the basis of EST data used in JCUSMART analysis, despite the presence of 4 protein models with BLAST hits to Frizzled. A “-‐“ indicates no sequences were identified but may not be suggestive of gene loss, due to inherent limitations of the initial data and sequence assembly and analysis. Representatives of all components of the Cadherin-‐catenin (Green) and planar cell polarity pathways (blue) are present in cnidarians with the exception of the cytoplasmic-‐side regulatory proteins, Diego and Four-‐jointed. Genes involved in Wnt/PCP signalling are coloured yellow. Conservation of a single homologue of most genes across cnidarian species with minimal to no expansion of gene repertoire suggests that systems influencing β-catenin distribution and cell polarity are highly constrained despite varying developmental methods in each species. Identifiers for models included in table are presented in Appendx B.
5.3.2 Phylogenetic analysis of catenin binding cadherins Maximum likelihood analysis of catenin binding cadherins (Figure 5.4) shows that
sequences from protostomes and deuterostomes form two distinct major clades. Cadherins
with similar overall protein architecture (ie. each Cadherin type) group together within
these major clades. Consistent with this, Type-‐III cadherins from protostomes and
deuterostomes clade separately from each other and from the cnidarian Cadherin
sequences, which also possess Type-‐III architecture. The position of the cnidarian
sequences, as a third major clade is well supported albeit on a long branch, which most
likely reflects the large amount of time since cnidarian divergence from other metazoans
rather than a lack of evolutionary constraint on the cytoplasmic region of cnidarian
cadherins.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Figure 5.4 Maximum likelihood analysis of Cadherin Cytoplasmic Domains of Type-‐I, Type-‐II, Type-‐III & Type-‐IV cadherins from representative metazoans. Cadherin major clades reflect the taxonomy of each animal as Protostome (Green), Deuterostome (yellow) or Cnidarian (blue). Bilaterian Type-‐III cadherins occur in 2 well supported, lineage specific clades (Deuterostome & Protostome), which reflect their function as epithelial stabilising or invasion promoting as well as reflecting their evolutionary history. The position of Type-‐I & Type-‐II cadherins within the Deuterostome clade, and Type-‐IV cadherins within the Protostome clade is consistent with previous suggestions that these families arose from independent lineage specific diversification of ancestral Type-‐III cadherins. The position of cnidarian Type-‐III cadherins in an independent clade is likely due to the early divergence of the cnidarians from bilaterian evolution and is not informative as to the function of cnidarian or ancestral catenin-‐binding cadherins. Branch numbers are bootstrap values (100 replicates). Values of less than 50 are not marked. The edited alignment of sequences used for maximum likelihood analysis and Genbank accession numbers are presented in Supplementary Figure 5.3. (Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum
Chapter Five Developmental roles for cadherins from Acropora millepora
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5.3.3 In situ hybridisation of catenin binding cadherins and planar
cell polarity pathway components In situ RNA hybridisation of Am_ACadherin shows that detectable levels of expression are
not present until after gastrulation, at the sphere stage (Figure 5.6). Expression in spheres is
restricted to a small region of ectoderm on one side of the larva, likely to correspond to the
site of the future oral pore. Expression in the involuted ectodermal cells of the oral pore is
apparent in pear and planula larvae, however no expression could be detected after
metamorphosis.
Coral orthologues of planar cell polarity (PCP) components exhibit a variety of spatial
expression patterns throughout development. Two components of the Fz-Fmi PCP system,
Am Van Gogh like (AmVangl) and AmDishevelled (AmDsh), along with AmDs of the FAT-Ds
PCP system, are first expressed at the prawnchip stage, prior to gastrulation (Figure 5.5).
Each of these three genes are expressed only in the presumptive ectoderm of the prawnchip,
however, whilst AmDsh is expressed ubiquitously throughout this presumptive germ layer,
AmVangl and AmDs are expressed in a gradient from one side of the embryo. It is not clear
whether these graded expression patterns are overlapping or opposing. In contrast to these
early patterns, only one of the three cloned Acropora Frizzled genes are expressed prior to
gastrulation (AmFz4), however its expression is restricted to the presumptive endoderm
(Ukolova et al., unpublished). AmFmi expression is also not detectable until after
gastrulation.
The expression of PCP related genes in larval stages occurs in two main regions, the aboral
ectoderm and the oral region. AmFmi, AmDsh, AmVangl and AmDs are all expressed
throughout the aboral ectoderm of planula larvae. In the oral region, AmFmi and AmDsh are
expressed in the oral endoderm, whilst genes involved in maintaining non-‐canonical Wnt
signalling (Wnt16, Axin, Wnt inhibitory factor (WIF) and Groucho) are expressed either in a
ring of ectodermal cells surrounding the oral pore (Wnt16, Axin) or in the oral ectoderm
itself (WIF, Groucho) (Figure 5.6). Although its role in canonical or non-‐canonical Wnt
signalling is unclear, AmFz4 is also strongly expressed in the oral ectoderm and potentially
overlaps the area of Wnt16 expression.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Figure 5.5. In situ hybridisation of prawnchip (left) and early donut (right) stage Acropora embryos with genes from the Cadherin-‐catenin and planar cell polarity pathways. Embryos are shown with presumptive blastopore in the centre and the presumptive endoderm visible. Unlike catenin binding cadherins from other species, Am_ACadherin (A & B) is not expressed during gastrulation of Acropora millepora, suggesting this protein is not required to maintain cellular stability during embryonic morphogenesis. Am_β-catenin (C & D) and Am_Dishevelled (E & F) are both expressed in the presumptive ectoderm, which is in contrast to blastula forming animals where embryonic expression of both proteins occurs in the presumptive mesendoderm. Planar cell polarity genes Am_Van_Gogh (G & H) and AmDachsous (I & J) demonstrate a lateral gradient of expression in the presumptive ectoderm during embryonic development (the relative direction of these gradients could not be confirmed), which may indicate an active PCP. However, the apparent absence of AmFlamingo expression (K &l) during this stage would suggest otherwise. Am_Dishevelled and Am_β-catenin insitu hybridisations (C-‐F) were performed by C.Shinzato, Unpublished)
Chapter Five Developmental roles for cadherins from Acropora millepora
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Figure 5.6. In situ hybridisation of Acropora larvae with genes from the Cadherin-‐catenin and planar cell polarity pathways. Larvae are shown with oral pore at top except in B where oral pore is centred. Am_ACadherin is expressed in a restricted pattern at the oral pore commencing at sphere stage (A & B) and is maintained during the pear (C & D) and planula (E) larval stages. This pattern overlaps that of β-catenin (F-‐H), consistent with canonical interactions of β-catenin and CCD containing cadherins and a role in oral pore development. Expression patterns of non-‐canonical Wnt signalling components Wnt16 & Frizzled 4 (M & P) suggest Planar Cell Polarity may also function in the oral pore. This is supported by repressors of canonical Wnt signalling WIF (N) and Axin (O) in the oral pore, however the absence of PCP effector (Dachsous, Van Gogh, Flamingo and Dishevelled) expression from the oral ectoderm is contradictory. In situ hybridisation of Am_β-catenin (F-‐H) performed by E.Ball, Unpublished; In situ hybridisation of Wnt components (M-‐P) performed by S.Ukolova, unpublished; In-‐situ hybridisation of AmGroucho (Q) performed by L.Grasso, Unpublished.
Chapter Five Developmental roles for cadherins from Acropora millepora
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5.4 Discussion 5.4.1 Cadherins involved in epithelial cohesion and migration
evolved early in metazoan evolution cadherins capable of binding cytoplasmic β-catenin have long been recognised to have roles
in embryonic development where their expression correlates to both stable cohesive and
invasive cellular phenotypes. Using JCUSMART, I have identified 4 cnidarian cadherins (3
complete from Nematostella vectensis and 1 5’ truncated from Acropora millepora) with
conserved catenin binding motifs in their cytoplasmic domain. Genomic surveys have
revealed the presence of CCDs in representatives of all metazoan taxa, including a cnidarian
(Nematostella vectensis) and sponge (Amphimedon queenslandica – preliminary sequences)
(Abedin & King, 2009; King et al., 2008; Sakarya et al., 2007). These domains are notably
absent from the genome of Monosiga brevicolis, a representative of the choanoflagellata,
which are considered the closest extant metazoan outgroup (Abedin & King, 2009; King et
al., 2008). The absence of CCDs from choanoflagellates suggests the CCD arose only after the
animal transition to multicellularity in the common metazoan ancestor (King et al., 2008).
Although the CCD is recognised as metazoan specific, the complete coding sequence of CCD
containing proteins from non-‐bilaterian representatives has not previously been
investigated. The protein architecture of each cnidarian sequence is consistent with that of
type-‐III cadherins (Figure 5.1), found primarily in non-‐chordates (with the exception of Hz-‐
cadherins) and have not previously been reported outside the Bilateria. Cadherins of this
type are distinct from typical chordate (type-‐I and II) catenin binding cadherins due to the
presence of a variable number of N-‐terminal Cadherin (EC) repeats followed by a
characteristic combination of NC, CE and LamG domains (the PCCD complex), which may
play a role in translocation to the plasma membrane (Oda & Tsukita, 1999). The sponge CCD
containing protein, first reported by Sakarya et al (2007), is also a Cadherin however is
somewhat divergent in its ecto-‐domain architecture. Following the N-‐terminal EC repeats
(>12), are 12 EGF domains and 1 LamG domain. The domain organisation and the absence of
a PCCD are dissimilar to type-‐III cadherins and all other CCD containing proteins. The
sponge protein has instead been considered a FAT-‐like Cadherin (King et al., 2008) despite
its unknown functional significance. Although this one reported sponge catenin binding
Cadherin does not exhibit an architecture characteristic of any described ‘classic’ Cadherin
types, future analyses of the recently completed A.queenslandica genome (Srivastava et al.,
2010) may reveal a more complex complement of CCD containing cadherins in sponges.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Type-‐III cadherins have previously been suggested to be a bilaterian ‘invention’ due to their
presence in both protostomes and deuterostomes (Hulpiau & van Roy, 2009). The
identification of CCD containing proteins from basal metazoans as cadherins with type-‐III
structure contradicts this notion. Instead, their presence suggests that type-‐III cadherins
arose early in metazoan evolution and diversified in the bilaterian lineage, giving rise to
functionally similar type-‐IV and type-‐I cadherins in protostomes and chordates respectively.
Type-‐III cadherins have subsequently been lost from the majority of chordates with only
three representatives (Hz-‐cadherins) remaining in vertebrates.
Phylogenetic analysis of the catenin binding region (Figure 5.4) was unable to identify any
clear relationship between cnidarian and bilaterian CCD containing cadherins. Protostome
and deuterostome sequences produce 2 discrete clades as is consistent with previously
published analyses (Hulpiau & van Roy, 2009). Type-‐III cadherins also form 2 clades
consistent with the functional distinction between type-‐III cadherins from different
bilaterian lineages. Grouping of cnidarian sequences with either clade would suggest a
common evolutionary history, however the cnidarian sequences form a third discrete clade
and are not clearly associated with either of the bilaterian type-‐III Cadherin groups. The
functional significance of the cnidarian CCD containing cadherins therefore remains unclear
as they are not more closely related to either invasive or stabilising type cadherins. It may be
that cadherins fulfilling both of these roles are present in cnidarians and that the distinction
between invasiveness and stability is more closely associated with differential regulation
than with the interaction between the cytoplasmic domain and catenins. Differential
regulation however, only partially explains this distinction in bilaterians, as expression of E-
Cadherin on N-Cadherin expressing cells does not diminish the invasive phenotype (Nieman
et al., 1999). To further the explanation, a mechanism of matrix-‐metalloprotease mediated
cleavage of the E-Cadherin ectodomain has been proposed (Xian et al., 2005), although the
possibility of similar protein level regulation in cnidarians would not be reflected in the
present analysis.
The apparent tight association of the CCD with cadherins in even the most primitive
metazoan phylum, the sponges, and the high degree of conservation in the catenin binding
motifs of the CCD, indicates the presence of a functional constraint on the Cadherin-‐catenin
relationship. The critical nature of roles for β-catenin and Cadherin described during
gastrulation and normal function of bilaterian animals is the most likely reason for this
constraint. It therefore stands to reason that similar vital roles for the conserved Cadherin-‐
catenin signalling axis in germ layer specification and morphogenesis could be evident
during development of more primitive metazoans such as cnidarians.
Chapter Five Developmental roles for cadherins from Acropora millepora
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5.4.2 Planar cell polarity but not Am_ACadherin is implicated in
gastrulation of Acropora millepora The presence of catenin binding cadherins at adherens junctions of the presumptive
ectoderm prior to gastrulation is conserved throughout the Bilateria. A similar distribution
in the presumptive ectoderm was expected for Am_ACadherin prior to gastrulation of
Acropora millepora. However, in situ hybridisation of Acropora embryos revealed no
detectable expression of Am_ACadherin until after gastrulation was complete (Figures 5.5 &
5.6). In each branch of the Bilateria for which catenin binding cadherins have been
described, a role analogous to that of E-Cadherin in stabilising the structure of the
presumptive endoderm has been demonstrated. The unexpected absence of Am_ACadherin
from gastrulation stages indicates it does not function in tissue stability prior to
gastrulation. Only a single Cadherin capable of binding β-catenin has been identified from
A.millepora, suggesting that the most highly conserved role for the Cadherin-‐catenin axis is
not represented in this species.
The most direct evolutionary interpretation of the absence of cadherins effecting tissue
stability is that the tissue stabilising function arose after the divergence of cnidarians. This
prospect is however highly unlikely. Cnidarians demonstrate a considerable range of
gastrulation processes including invagination, ingression, delamination and epiboly (Byrum
and Martindale, 2004). Such diversity is even reflected among coral and the bi-‐layer folding
exhibited by Acropora is a particularly peculiar mode of gastrulation. In contrast to
Acropora, the gastrulation of Nematostella occurs by invagination following blastula
formation (Kraus & Technau, 2006; Lee et al., 2007; Magie, Daly, & Martindale, 2007)
variations of which are reasonably common in bilaterians. The clear evolutionary
relationship between Nematostella and bilaterian gastrulation suggests that stabilisation of
the presumptive ectoderm by a catenin-‐binding Cadherin is likely to be represented in some
parts of the cnidarian lineage. As such, the tissue stabilising function can still be considered
ancestral. The absence of this function in Acropora is therefore attributable to a genus
specific diversification related to the mode of gastrulation and is not reflective of the
ancestral state.
Simplification of Acropora catenin binding Cadherin function is also supported by the results
of cnidarian genomic surveys. Whereas a single Type-‐III Cadherin was identified in coral, the
genome of Nematostella vectensis has revealed 3 predicted Cadherin genes with Type-‐III
architecture. Two of the Nematostella genes appear to be tandem repeats and may produce
only a single functional protein (based on protein alignment – not shown), however the
third gene resides on a separate scaffold of the genome assembly and is likely to be a distinct
Chapter Five Developmental roles for cadherins from Acropora millepora
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and functional Cadherin. The presence of only a single Cadherin in coral compared to 2 in
Nematostella could indicate that functional simplification is paralleled by gene loss. This
then raises the question of how epithelial associations are maintained when the major
adhesive component of adherens junctions has been lost. When considered along with the
incomplete nature of the Acropora transcriptome, there is a reasonable possibility that
further catenin binding cadherins may be found in the upcoming Acropora millepora genome
(Miller et al, unpublished).
Planar cell polarity has also been implicated in gastrulation, however most studies have
focused on roles during convergent extension processes such as neuralation. In Acropora,
expression of AmDsh, AmVangl and AmDs is restricted to the presumptive ectoderm (Figure
5.5). Unlike AmDsh, expression of AmVangl and AmDs is observed as a gradient across the
embryo. The relative orientation of the gradients could not be confirmed although the
presence of graded expression is consistent with classical descriptions of these genes in
polarised epithelial tissues (Wu & Mlodzik, 2009). These patterns of expression suggest that
planar cell polarity has a role in morphogenesis of the coral ectoderm during gastrulation.
Like most forms of gastrulation, that of Acropora requires progressive changes in cellular
arrangement. The best described methods of achieving the necessary re-‐arrangements are
directed cell movements (eg. convergent extension, integrin mediated migration, ingression,
and involution) and polarised cell division. Directed cell movements are expected to play a
minimal role in Acropora gastrulation with no clear morphological evidence for the common
forms. Despite this, the process by which convergent extension is regulated is worth
considering. Experiments in Xenopus have demonstrated that convergent extension
requires active planar cell polarity controlled by Dsh and that non-‐canonical Wnt is only
permissive in this process and not required (Wallingford et al., 2000). Of more direct
importance is the demonstration that planar cell polarity, through Dishevelled, is
responsible for directing polarised cell division along the AV axis in Zebrafish. Similarly the
expression of AmDsh in the presumptive ectoderm of coral (Figure 5.5) could play roles in
permitting cell movement and directing cell polarity. This would also be consistent with a
lack of catenin binding Cadherin in this tissue. Asymmetric distribution of AmVangl and
AmDs may therefore act in establishing cell polarity and the direction of cell division,
allowing extension of the ectodermal cell sheet to occur in an ordered manner where a
single plane of ectodermal cells is maintained.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Contradictory to this suggestion is the absence of detectable AmFmi and Fz expression in the
ectoderm prior to gastrulation. Frizzled and Fmi are the major cell surface components of
the core (Fz-‐Fmi) planar cell polarity pathway and are expected to be present in polarised
epithelium. Whilst it is likely that multiple Frizzled proteins remain undiscovered in
Acropora, some of which may be expressed in the presumptive ectoderm, the same is less
likely to be true for Flamingo. Genome surveys from a range of representative metazoans
indicate diversification of CELSR occurred only in the vertebrate lineage, so alternatively
expressed coral cognates are not expected. The roles and organisation of non-‐bilaterian
planar cell polarity systems are yet to be defined, however the asymmetrical distribution of
AmDs and AmVangl and their potential involvement in directing cell division are a starting
point for future investigations which would fill a gap in both our knowledge of cnidarian
gastrulation and the evolution of a major metazoan patterning system.
5.4.3 Am_ACadherin and planar cell polarity are implicated in
development of the Acropora larval oral pore. The possibility of developmental roles for Am_ACadherin and components of the planar cell
polarity pathway were investigated by in situ hybridisation of Acropora millepora
developmental stages. Spatial patterns of expression in and around the oral pore (larval
mouth structure) for a number of the genes investigated were consistent with roles in oral
pore development, a process which is poorly described in Acropora. Morphological analyses
have demonstrated that following Acropora gastrulation the blastopore closes completely
(sphere stage) prior to establishment of the oral pore (pear stage). This is in contrast to the
development of other cnidarians, such as Nematostella, where the blastopore does not close
instead becoming the oral pore. Morphological data from Acropora also show that cells of
the oral pore are ciliated and more closely resemble the epithelial organisation of
ectodermal tissue (Ball et al., 2002), suggesting they are of ectodermal origin. The possibility
that oral pore formation occurs through involution of ectodermal tissue is therefore not
unreasonable.
Expression of Am_ACadherin was first detectable in a restricted region of ectoderm in sphere
stages and later expressed specifically in the oral pore, during pear and planula stages
(Figure 5.6). Expression both prior to and following establishment of the larval mouth,
suggests a role in oral pore development. The restricted pattern of larval expression also
coincides with that described for Amβ-catenin, supporting the proposition of an ancestral
Cadherin-‐catenin signalling axis. The association of Amβ-catenin with Am_ACadherin in these
cells is further supported by expression of AmWIF and AmGroucho in the same cell
population. In bilaterian models, WIF has been demonstrated to compete with Wnts for Fz
binding, thereby antagonising the canonical Wnt system and subsequent stabilisation and
Chapter Five Developmental roles for cadherins from Acropora millepora
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nuclear translocation of β-catenin. Groucho, on the other hand, represses the transcriptional
targets of Wnt signalling by binding TCF/LEF transcription factors in the absence of nuclear
β-catenin. Expression of WIF and Groucho together suggests that β-catenin is not
transcriptionally active in the oral pore and is more likely to be associated with Cadherin at
the plasma membrane. Evidence for a Type-‐III Cadherin-‐β-catenin interaction in cells of the
oral pore, which are potentially undergoing involution, is consistent with an
invasive/migratory type function for Am_ACadherin.
Genes up stream of the planar cell polarity pathway were also found to be expressed in the
oral region of Acropora larvae. AmWnt16 is expressed in a restricted pattern in a ring of cells
at the edge of the oral pore. Bilaterian orthologues of Wnt16 have been demonstrated to
activate the planar cell polarity pathway whilst inhibiting canonical Wnt signalling in
bilaterians. These effects constitute non-‐canonical Wnt signalling. The functional
significance of AmWnt16 in coral development has not yet been clearly demonstrated,
however gene expression is abolished by the presence of nuclear beta-‐catenin following
alsterpaullone treatment (Ukolova et al., unpublished), which is consistent with previously
described reciprocal antagonism between the canonical and non-‐canonical Wnt systems
(Bryja et al., 2009; Topol et al., 2003). AmAxin is also expressed in the ectodermal cells
surrounding the mouth of coral larvae. In bilaterians, Axin is a constituent of the GSK3β
complex responsible for the degradation of cytoplasmic β-catenin and is capable of binding
β-catenin directly. Expression coinciding with Wnt16 is consistent with the expected
negative regulation of the canonical Wnt system. The role of Axin as part of the GSK3β
complex outside the Bilateria is however currently under scrutiny due to the absence of the
beta-‐catenin binding motif in Amphimedon, Nematostella and Acropora Axin orthologues
(Srivastava et al., 2010; Ukolova et al unplublished).
The presence of a functional planar cell polarity system at the outer lip of the oral pore may
have roles in orientating cilia appropriately for directing food to the gastric cavity or simply
in defining the structure of the oral pore from surrounding ectodermal tissue. The
expression patterns of planar cell polarity effectors, however, contradict the existence of a
non-‐canonical Wnt mediated planar cell polarity in this area. In situ hybridisation of AmFmi,
AmDsh, AmVangl, and AmDs failed to identify expression of components from either the Fmi-‐
Fz or FAT-Ds PCP systems at the outer lip of the oral pore. The only possible PCP component
expressed in the oral pore was AmFz4. Although AmFz4 expression may overlap that of
AmWnt16, co-‐expression is not necessarily suggestive of a role in PCP signalling. Frizzled
proteins are recognized to hold diverse functions so no conclusion as to the implication of
AmFz4 in the oral pore can be drawn without the support of functional data.
Chapter Five Developmental roles for cadherins from Acropora millepora
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Despite the apparent lack of PCP component expression, the suggested role of Wnt16
expression in oral pore development cannot be ignored. Protein localisation studies are the
only conclusive indicator of planar cell polarity and provide added sensitivity over in situ
hybridisation. These experiments may uncover asymmetric distribution of protein within a
limited cell population thereby re-‐enforcing non-‐canonical Wnt signalling as a factor in oral
pore development. Likewise, protein localisation and further analysis of the morphological
movements during oral pore formation could clarify the role of Am_ACadherin. Further
investigation involving both protein localisation and in vivo functional studies are also
required to clarify the roles of Cadherin-‐catenin signalling and planar cell polarity in
cnidarian gastrulation. Of particular interest is the influence of these cadherins on the
nuclear translocation of β-catenin. It has already been demonstrated in Nematostella that
over expression of the CCD can inhibit gastrulation, however no direct studies into the
function of ‘classic’ cadherins have been undertaken outside the Bilateria.
5.5 Conclusion Investigation into the Cadherin complement of cnidarians has revealed an ancestral origin
for two adhesion systems that are important in bilaterian development. Here I have
described that the conserved Cadherin-‐catenin axis, active during bilaterian development, is
conserved in cnidarians and likely to have originated in the eumetazoan ancestor. Whilst the
function of this system in cnidarians is yet to be tested, early investigations into expression
in Acropora suggest roles in involution rather than the tissue stabilising functions seen in
bilaterians. The possibility that the function of the Cadherin-‐catenin axis is simplified in
Acropora is also evidenced. Components of the planar cell polarity pathway, from signalling
to membrane bound effectors, are also conserved in cnidarians. Expression in both
gastrulation and oral pore development in Acropora implies planar cell polarity is not
limited to previously described functions in bilaterian development and is of broader
morphogenic significance than currently recognised.
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Chapter 6: Integrins of Acropora millepora
6.1 Introduction Cohesion of cells to the extracellular matrix (ECM) is critical to the evolution of animal
multicellularity and morphogenesis of complex metazoan body structures (Abedin & King,
2010). The integrin family of transmembrane cell surface receptors are the primary
mediators of cell-‐ECM interactions in bilaterians (De Arcangelis & Geogres-‐Labouesse, 2000)
and have been implicated in a broad array of biological processes including gastrulation,
neuron outgrowth and leukocyte trafficking. Integrin genes are present in all metazoans
investigated to date, although the number of α and β-‐subunits varies greatly between taxa
(Sebé-‐pedrós et al., 2010). The diversity of integrin αβ heterodimers and the ability to
rapidly switch between high and low affinity states in response to intracellular stimuli
(inside-‐out) or extracellular ligand binding (outside-‐in) (Ginsberg et al., 2005; Hynes, 2002;
Takagi et al., 2002) is central to the versatility of integrin function. This integration of
dynamic cell adhesion with bi-‐directional signalling allows cellular behaviours such as
migration, growth, differentiation and deposition of basement membrane components
(Brown, 2000) to be guided by positional cues (Ramos et al., 1996; Streuli, 2009), providing
a basis for many of the key events in animal development.
The role of integrins in facilitating morphogenesis and tissue development is conserved
throughout the Bilateria (Brower, 2003; Knack et al., 2008; Reber-‐Müller, et al., 2001;
Whittaker & Desimone, 1993) and functional investigations have demonstrated that the
integrin signalling system is highly specific in terms of protein expression, ligand binding
and heterodimer combination (Humphries et al., 2006; Hynes, 2002). Whereas protein
expression is governed by transcriptional regulation, integrin heterodimer combination and
ligand binding are inextricably linked. The α and β integrin subunits make an unequal
contribution to ligand interactions, with the α-‐subunit holding the greatest influence over
ligand specificity and the β-‐subunit being primarily responsible for signal transduction
through cytoplasmic interactions and formation of cytoskeletal connections (Berman et al.,
2003; Legate, et al., 2006; Martin-‐Bermudo et al., 1997; Nishiuchi et al., 2003). As a single β-‐
subunit may associate with many α-‐subunits (Kinashi, 2005), different αβ integrin
combinations are capable of eliciting similar cellular behaviours on a range of substrates
through specific ligand binding. This is particularly significant to developmental processes
as diversity in the ligand specificity can facilitate behaviours such as cohesion, polarisation
Chapter Six Integrins of Acropora millepora
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and migration on basement membranes with various physical properties, thereby
contributing to the co-‐ordination of many distinct tissues during development.
The most diverse integrin complement belongs to mammals, containing 18α and 8β
subunits and although only 24 combinations are expressed as functional integrin receptors
(Hynes, 2002), the complexity of this complement is far greater than other bilaterians such
as sea urchin (3α & 4β), fly (5α & 2β), and worm (2α & 1β) (Brower, 2003; Putnam et al.,
2007; Schmitt & Brower, 2001). Although there are differences in the number of integrin
subunits between bilaterians, ligand specificities from different model systems are often
similar, allowing all described integrin receptors to be broadly categorised as a Laminin,
RGD tripeptide, Collagen or Leukocyte-‐specific type according to their ligand binding
properties (Huhtala et al., 2005; Hynes, 2002). Mammals, flies, worms and sea urchin all
contain at least 1 integrin receptor that binds Laminin and 1 that binds RGD tripeptide
containing proteins (eg. Fibronectin, Tiggrin), suggesting the last common ancestor of
bilaterians exhibited similar ligand specificities. Collagen binding and leukocyte-‐specific
integrin receptors are found only in chordates and appear to be independent diversifications
linked to the evolution of circulating adaptive immune cells and complex collagen structures.
In contrast to the wealth of information regarding integrin function in higher animals, the
expression, heterodimer formation and ligand binding properties of lower animal integrins
have received little attention. Recent genomic surveys of basal metazoan phyla have
discovered that the cnidarian and sponge integrin complement is similar to that of other
invertebrates, possessing up to 2α & 4β, and 5α & 7β respectively (Sebé-‐pedrós et al., 2010);
Chapter 4). Despite similarity in gene numbers between lower animals and bilaterian
invertebrates, phylogenetic analyses including sequences from Nematostella have failed to
determine a clear relationship between the integrins of cnidarians and bilaterians (Knack et
al., 2008; Magie & Martindale, 2008) offering little insight as to the function of cnidarian
integrins.
Although there are differences in integrin receptors between phyla, a number of
publications suggest integrin cytoplasmic signalling is remarkably similar to that of higher
animals, in even the simplest metazoans. Investigations into integrin systems of
representative cnidarians, sponges and lower metazoans (eg. Trichoplax adhaerens) have
identified the presence of complete integrin subunits and all of the major cytoplasmic
components required for canonical integrin signalling in every animal model considered.
Investigations extending beyond the animal kingdom also identified a complete signalling
complement in Capsaspora owczarzaki, a filose amoeboid nucleariid serving as an out group
Chapter Six Integrins of Acropora millepora
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to the Metazoa (Nichols et al., 2006; Sebé-‐pedrós et al., 2010). Despite the integrin system
traditionally being considered an animal novelty (Rokas, 2008), the presence of canonical
integrin signalling in such a distant Metazoan relative suggests that the cellular signalling
events following integrin ligand binding are highly constrained and have been conserved
since before the first metazoans evolved.
In addition to maintenance of integrin signalling from a basal Opsithokont ancestor, the
distribution of integrin gene expression is also conserved throughout the animal kingdom.
The jelly-‐fish Podocoryne carnea (Reber-‐Müller et al., 2001), the coral Acropora millepora
(Knack et al., 2008), and all bilaterian models investigated to date each express α and β
integrins in the presumptive mesendoderm prior to gastrulation (Bökel & Brown, 2002;
Davidson, Hoffstrom, Keller, & DeSimone, 2002; Marsden & Burke, 1998; Ramos, Whittaker,
& Desimone, 1996). Despite diverse modes of gastrulation (See Chapter 7.1), expression of
integrins in mesendodermal cells crosses the diploblast – triploblast divide, demonstrating
an unusually high degree of conservation in the pattern of expression consistent with
integrins playing an indispensible role during animal development.
Whereas evidence is available for the conservation of cytoplasmic signalling and expression
of integrins in at least one aspect of metazoan development, gastrulation, there is no
evidence to suggest ligand specificity is equally conserved between cnidarian and bilaterian
integrins. Investigation of cnidarian integrin ligand specificity aimed to address the lack of
functional data from non-‐bilaterian models and clarify the roles of integrins in the
ureumetazoan ancestor. As phylogenetics is demonstrated to be of limited predictive value
(Knack et al., 2008), the ligand binding specificities of cnidarian integrins must be
determined empirically using an ancestrally informative model. Anthozoan cnidarians such
as the coral Acropora millepora offer a unique perspective on the ancestral role of integrins
during development, having retained much of the genetic complexity from the common
eumetazoan ancestor (Technau et al., 2005). They are also the most basal phylum to possess
true tissues and are therefore likely to be highly informative regarding the genetic origins of
tissue morphogenesis, a process which is strongly influenced by integrin mediated adhesion.
Chapter Six Integrins of Acropora millepora
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Investigation of integrin-‐extracellular matrix interaction in higher animals, such as flies, is
routinely performed by assessing structural changes of cultured cells in response to contact
with potential ECM ligands (Jannuzi et al., 2002; Nieves et al., 2010). The maintenance of
coral cells in culture has not yet been established and developing such a system is beyond
the scope of the present investigation. To overcome this obstacle, it was necessary to turn to
an established experimental system already proven to be useful in assessing the structure
and function of integrins. Cultured Drosophila S2 cells have often been employed to conduct
such investigations and hold several advantages over other model systems including access
to a wide variety of genetic markers, allowing exploration of cellular affects, availability of
inducible expression vectors and convenient & efficient knockdown of endogenous
integrins. Investigation of coral integrin ligand specificity was therefore conducted using
transgenic expression of coral αβ subunit combinations in Drosophila S2 cells and exposing
transfected cells to a variety of known Drosophila integrin ligands. These studies provide a
basis for direct comparison between higher animal ligand specificity and that of
developmentally significant coral integrins. The phylogenetic distribution of integrin α and β
subunits from a wider range of cnidarians was also considered in order to determine the
broader relevance of coral ligand specificity in an evolutionary context.
Chapter Six Integrins of Acropora millepora
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6.2 Methods 6.2.1 Phylogenetic analyses integrins from Acropora millepora and Nematostella vectensis were identified using the
method described in Chapter 3. Maximum likelihood analysis of β-‐Integrin subunits was
performed using MolPhy with 1000 bootstrap replicates (Knack et al., 2008). Maximum
likelihood analysis of α-‐Integrin subunits was performed using PhyML (1000 bootstrap
replicates) and includes cnidarian sequences (AmItgα2, AmItgα3, NvItgα2) not present in
published phylogenetic analyses. integrin phylogenies are based on extensively edited
alignments presented in Supplementary Figure 6.1 (α-‐Integrin alignment) & 6.2 (β-‐Integrin
alignment).
6.2.2 Preliminary ligand binding assay Clones of AmItgα1, ItgβCN1 and AmItgβ2 were isolated from the Acropora millepora EST
project and RNA isolates as described in Chapter 2.2 & 2.3. Cloning of the 9 expression
constructs used throughout the following experiments was performed using a series of
overlap PCRs as detailed in Chapter 2.5. Procedural aspects of the following experiments,
including the maintenance of Drosophila S2 cell cultures, are presented in Chapter 2.6-‐2.9.
Preliminary assessment of the ligand binding properties of coral integrins was performed
using cell spreading on Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow, PacI,
Trimeric Laminin (Drosophila), and sucrose fractionated Laminin. Wells pre-‐incubated with
PBS were used as negative controls for each cell types. Cells expressing ItgαPS1 ItgβPS or
ItgαPS2 ItgβPS were used as positive controls for interaction with Laminin and RGD type
ligands respectively. Combinations of coral integrins (excluding tagged β constructs) were
co-‐transfected (Cellfectin – Invitrogen) with a constitutively expressing eGFP construct,
pH8CO vector (Rebay et al., 1991) and 500ng Mysopheroid (ItgβPS) RNAi. Cell spreading on
Laminin type substrates was performed using 5x 1:5 serial dilutions of stock laminin. Cell
spreading was assessed by phase contrast microscopy under an inverted light microscope
(Zeiss).
Chapter Six Integrins of Acropora millepora
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6.2.3 Optimisation of cell spreading conditions Serum free-‐ M3 medium (no added cations), BES-‐Tyrodes (1mM Mg2+, 10µM Ca2+, 1% BSA),
Robb’s Saline (1mM Mg2+, 10µM Ca2+, 1% BSA) and PBS (1mM Mg2+, 10µM Ca2+, 1% BSA)
were compared for their ability to support specific integrin mediated cell spreading on Rbb-‐
Tiggrin. Cells expressing AmItgα1 AmItgβ2 or ItgαPS2 ItgβPS (positive control) were
included in the cell spreading assay, conducted in replicate as described in Chapter 2.8.
The impact of Mg2+ and Ca2+ concentration on the percentage of cells exhibiting integrin
mediated cell spreading and the morphology of spreading cells was assessed using the
following concentrations of cations in Robb’s saline with 1% BSA (Table 6.1). Cells
expressing AmItgα1 AmItgβ2 or ItgαPS2 ItgβPS (positive control) were included in the cell
spreading assay, which was conducted in replicate as described in Chapter 2.8.
Combination Mg2+ (MgCl2)
Concentration
(mM)
Ca2+ (CaCl2)
Concentration
(mM)
1 50 10
2 5 0.2
3 0.1 0.2
4 0.01 0.2
5 0 (EDTA) 0 (EDTA)
6 1 0.002
7 1 0.02
8 1 0.2
Table 6.1 Concentrations of Mg2+ (MgCl2) and Ca2+ (CaCl2) added to Robb’s Saline for optimisation of cell spreading conditions. Integrin activation and ligand interaction is dependant on the availability of divalent cations in the surrounding medium. The divalent cation concentration in the extracellular fluid surrounding coral integrins (sea water) is expected to differ from that for Drosophila integrins (hemolymph), therefore the concentration of divalent cations required for optimum integrin activation may also differ. Comparison of S2 cell supporting media identified Robb’s Saline to give the highest degree of integrin mediated cell spreading. The divalent cation concentration included in preparation of Robb’s Saline was altered according to the 8 combinations shown in the table before use as a supporting medium for cell spreading assays.
Chapter Six Integrins of Acropora millepora
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6.2.4 Analysis of integrin surface expression integrin expressing cells were prepared for flow cytometry as described in Chapter 2.9.
Secondary detection at 546nm was used for cells transiently transfected with integrin (α
and β) and eGFP expression constructs. Cells exhibiting detectable levels in the GFP range
were considered successfully transfected, whilst cells exhibiting detectable emission in both
the GFP and 546nm ranges were considered integrin+. Integrins tagged with the HA epitope
were detected by direct immuno-‐fluorescence at 488nm, therefore cells were not
transfected with the eGFP expression vector.
Chapter Six Integrins of Acropora millepora
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6.3 Results 6.3.1 Integrin identification and phylogenetic analysis Investigation of integrin diversity in four representative cnidarians conducted using
JCUSMART (See Chapter 3) identified similar numbers of α and β subunits in Hydra, Clytia,
Nematostella and Acropora. Whilst the complete complement of Nematostella has
previously been reported (Putnam et al., 2007; Srivastava et al., 2010), the present analysis
identified 2 novel α-‐subunits from Acropora millepora and previously undescribed integrins
from Hydra (1α & 2β) and Clytia (1α & 1β). Due to limitations of the available data, the
number of integrins identified in Hydra and Clytia are likely to represent only a portion of
the total complement, however the Acropora (3α and 2β) and Nematostella (2α & 4β)
integrin complement is expected to encompass all distinct genes present in each genome.
Maxmium Likelihood analysis performed on a selection of integrin sequences was broadly
consistent with previous investigations (Ewan et al., 2005; Hughes, 2001; Huhtala et al.,
2005; Knack et al., 2008). Sequences from Clytia and Hydra, which are potentially
informative, were excluded from the analysis as gene models were not of sufficient length to
allow accurate phylogenetics. Integrin phylogenetics is complicated by homoplasy and poor
conservation of primary sequence, making unambiguous alignment difficult, therefore the
trees presented here are based upon extensively edited alignments (Supplementary Figure
6.1).
Analysis of integrin α-‐subunits shows grouping of sequences with RGD and Laminin binding
properties into 2 distinct clades, with the Cnidarian sequences forming a separate clade.
Deuterostome and protostome sequences were present in both the RGD and Laminin clades
suggesting the capacity to bind both ligand types was already established in the common
bilaterian ancestor. The position of the cnidarian α sequences in an indpendent clade is not
informative as to the ligand binding specificities of basal metazoans. This analysis also
revealed that NvItgα2 and AmItgα3 are closely related, forming the Cnidarian Minor Clade
however, association with the other cnidarian sequences (Cnidarian Major Clade) is poorly
supported.
Phylogenetics of β-‐Integrins is also consistent with published data, showing clustering of
chordate β1, β4 and β3 families and independent grouping of non-‐chordate sequences
(urchin, Fly, Cnidarian, Sponge) according to taxonomy. This pattern demonstrates the
independent expansion of β-‐Integrins in several lineages, including the Cnidaria where
Nematostella encodes 2 sequences orthologous to each of the Acropora β-‐Integrins.
Chapter Six Integrins of Acropora millepora
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Figure 6.1 Maximum likelihood phylogenetic analysis of representative α-‐Integrins. Numbers at branch points indicate the percentage of 1000 bootstrap replicates supporting the topology resolved using PhyML. Integrin sequences α7, α6, α3, α5, αV, α8 and αIIb taken from human. Bilaterian sequences are resolved into Laminin, RGD and Alpha 4/9 clades consistent with previous analyses. The ambiguous position of ItgαPS3 is likely due to the highly derived nature of this protein when compared with other Drosophila integrins. The position of GcItgα away from the distinct clades is suggested to result from early divergence. This position would be further resolved by inclusion of other sponge sequences. In contrast to the bilaterian sequences, which group in a ligand specific manner, the cnidarian sequences form an independent clade. This grouping suggests functional divergence had already occurred in the Urbilateria. Two novel cnidarian sequences (NvItga2 and AmItga3) are only weakly associated with the other cnidarian sequences, reflective of their sequence divergence. The presence of two distinct cnidarian clades suggests that cnidarian integrins may interact with multiple ligands.
Chapter Six Integrins of Acropora millepora
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Figure 6.2 (Excerpt from Knack et al., 2008) Maximum likelihood phylogenetic analysis of representative β-‐Integrins. Numbers at branch points indicate the percentage of 1000 bootstrap replicates supporting the topology resolved using MolPhy. In contrast to the α-‐integrin phylogeny, the grouping of the β-‐subunits in lineage specific clades suggests beta integrins have evolved through a number of lineage specific expansions, as is consistent with previous publications. Cnidarian sequences clade together with strong bootstrap support. Within the Cnidarian clade, Acropora and Nematostella sequences show 2 sub-‐clades consisting of ItgβCn1, NvItgβ3, NvItgβ4, and AmItgβ2, NvItgβ1,NvItgβ2, PcItgb. The position of Acropora and Nematostella sequences into two sub-‐clades suggests the sea anemone possess 2 β-‐Integrins for each coral β-‐Integrin, which may be the result of a recent duplication in Nematostella.
Chapter Six Integrins of Acropora millepora
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6.3.2 Ligand Binding of coral integrins
Preliminary ligand binding Assay The preliminary cell spreading assay to assess the ligand binding properties of coral
integrins was performed under non-‐optimised conditions using the following ligands:
• Rbb-‐Tiggrin
• Vitronectin
• Fibronectin
• Tennectin
• Twow
• PacI
• Drosophila Trimeric Laminin
• Sucrose fractionated Drosophila Laminin
Assessment of transfection efficiency by visualisation of eGFP expression prior to cell
spreading showed that ~30% of the total cell population expressed GFP 2 days after
transformation. Accordingly, ~30% of control cells for RGD tripeptide ligands (transformed
with αPS2 + βPS) spread on Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow, and
PacI. Positive control cells for Laminin interaction (transfected with αPS1 + βPS), spread
weakly on the trimeric Laminin and sucrose fractionated Laminins, indicating the quality of
the available Drosophila Laminin substrates was less than ideal. Cells transfected with coral
integrins showed both a low percentage of spread cells (~ 2-‐5% above empty vector
transfected controls) and a small degree of spreading on all substrates, suggesting
interactions with the tested substrates may only be weak. The highest degree of spreading
was observed in AmItgα1β2 and AmItgα1β1 transformed cells on Rbb-‐Tiggrin, a 53 amino
acid recombinant portion of the RGD containing Drosophila Tiggrin protein (Jannuzi et al.,
2002), consisting of 25 bases up and down stream of the RGD sequence. Rbb-‐Tiggrin also
facilitates a particularly strong interaction with the primary Drosophila RGD-‐binding
integrin (αPS2βPS), resulting in a highly spread morphology. Rbb-‐Tiggrin was therefore
used to assess RGD-‐ligand binding in all subsequent experiments.
Chapter Six Integrins of Acropora millepora
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0ptimisation of Cell spreading conditions In order to determine the optimal buffer for cell spreading assays, 4 buffers, Serum free-‐ M3
medium (no added cations), BES-‐Tyrodes (1mM Mg2+, 10µM Ca2+, 1% BSA), Robb’s Saline
(1mM Mg2+, 10µM Ca2+, 1% BSA) and PBS (1mM Mg2+, 10µM Ca2+, 1% BSA), were compared
for their impact on cell morphology and the capacity to allow coral integrin mediated
spreading on Rbb-‐Tiggrin. More than 95% cells stably transfected with αPS2βPS spread to a
diameter of no less than 3x that of the nucleus in all media. By comparison, very few of the
coral expressing cells demonstrated notable integrin mediated cell spreading. In all
transfections, cells appeared to have a more regular morphology (consistent with healthy
cells) in M3 media than saline based media, with PBS causing the highest degree of cell
death. Although the most protective media for S2 cells was M3, cells in this media showed
the smallest degree of spreading on Rbb-‐Tiggrin. The highest degree of cell spreading and
the most acceptable level of negative impact on cell health was observed in cells spread in
Robb’s saline, therefore subsequent experiments were conducted using only this medium.
Investigations into the optimal divalent cation concentration for integrin activation were
also assessed morphologically. High concentrations of divalent cations (5mM Mg2+ + 1mM
Ca2+ and above) resulted in formation of numerous cell processes, which were inconsistent
with integrin mediated adhesion. Concentrations of less than 1mM Mg2+ or 0.2mM Ca2+
facilitated cell spreading in only a small proportion of the cell population, whereas 1mM
Mg2+ and 0.2mM Ca2+ allowed optimal activation of coral integrins with a maximum of 7% of
cells in the AmItgα1 + AmItgβ2 transfected sample spreading beyond background levels on
Rbb-‐Tiggrin. Spreading in all other coral samples was observed to be between 2% and 5% of
the population under the same conditions compared to over 30% in αPS2βPS control cells
(transient transfection).
Chapter Six Integrins of Acropora millepora
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Surface expression of integrins following transient transfection Expression of integrins on the cell surface assessed by flow cytometry of duplicate cell
transfections showed that for each of the 8 transfections, 30-‐35% of the total cell population
expressed GFP (Table 6.2), indicating transfection was successful in all samples. The number
of transfected (GFP+) cells staining with anti-‐PS1 and anti-‐PS2 antibodies was higher in
ItgαPS1 (9.8%) and ItgαPS2 (10.0%) expressing cells than pHSMCS transfected negative
controls (1.5% and 0.4% respectively), indicating that approximately 10% of the total
population expresses detectible levels of Drosophila integrins 2 days after stress induction
of the heat shock promoter (ie. during transfection). Immunostaining of coral integrin
transfected cells with antibodies against AmItgα1, AmItgβ1, and AmItgβ2 showed no
detectable levels of surface expression (secondary detection).
Flow cytometry 1 day after transfection and in the presence of Mn2+ performed on a replica
set of transfections demonstrated 5-‐10% of the population was GFP+, however also failed to
detect coral integrins using the anti-‐ AmItgα1, AmItgβ1, and AmItgβ2 antibodies (data not
shown).
Expression of Acropora β-‐Integrins containing epitope (HA or c-‐MYC) tagged Drosophila βPS
serine loops inserted into the extracellular hybrid domain was assessed by flow cytometry 2
days after transfection (Table 6.3). Cells transfected with αPS2 + βPS-‐HA showed expression
in ~24% of the total population above empty vector (pHSMCS) transfected controls.
Similarly, cells transfected with αPS2 + βPS-‐MYC exceeded expression in controls by ~16%.
In contrast to the clear expression of Drosophila integrins, the percentage of the total cell
population expressing AmItgα1AmItgβ1-‐HA was only 1.8% above empty vector transfected
controls and AmItgα1AmItgβ2-‐MYC was detected in only ~1% of the total population above
the control level. Together, this suggests that whilst coral integrins are expressed on the
surface of transfected cells, they are only present in a minimal proportion of the transfected
cell population
Chapter Six Integrins of Acropora millepora
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Cell Line % of Total GFP+
Antibody % of Total GFP+/integrin+
pHSMCS 34.5 α-‐ItgαPS1 α-‐Mouse 546 1.2
ItgαPS1 ItgβPS 31.3 α-‐ItgαPS1 α-‐Mouse 546 9.8
pHSMCS 35.2 α-‐ItgαPS2 α-‐Mouse 546 0.4
ItgαPS2 ItgβPS 23.3 α-‐ItgαPS2 α-‐Mouse 546 10.0
pHSMCS 35.4 α-‐AmItgα1 α-‐Rabbit 546 0.0
AmItgα1 AmItgβ1 34.9 α-‐AmItgα1 α-‐Rabbit 546 0.1
AmItgα1 AmItgβ2 30.8 α-‐AmItgα1 α-‐Rabbit 546 0.1
AmItgα1-‐PS2 AmItgβ1-‐PS 34.6 α-‐AmItgα1 α-‐Rabbit 546 0.0
AmItgα1-‐PS2 AmItgβ2-‐PS 31.7 α-‐AmItgα1 α-‐Rabbit 546 0.0
AmItgα1-‐PS2 AmItgβ2L>D-‐PS 35.7 α-‐AmItgα1 α-‐Rabbit 546 0.1
pHSMCS 34.4 α-‐AmItgβ1 α-‐Rabbit 546 0.1
AmItgα1 AmItgβ1 34.6 α-‐AmItgβ1 α-‐Rabbit 546 0.0
AmItgα1-‐PS2 AmItgβ1-‐PS 35.1 α-‐AmItgβ1 α-‐Rabbit 546 0.0
pHSMCS 34.7 α-‐AmItgβ2 α-‐Rabbit 546 0.1
AmItgα1 AmItgβ2 31.0 α-‐AmItgβ2 α-‐Rabbit 546 0.1
AmItgα1-‐PS2 AmItgβ2-‐PS 31.9 α-‐AmItgβ2 α-‐Rabbit 546 0.1
AmItgα1-‐PS2 AmItgβ2L>D-‐PS 36.9 α-‐AmItgβ2 α-‐Rabbit 546 0.1
Table 6.2 Percentage of cells expressing detectable α and β integrins on the cell surface following transient transfection (as detected by subunit specific antibodies and anti-‐rabbit Alexa546 secondary antibodies). The Antibody column shows the specificity of the primary (top) and secondary (bottom) antibodies used for detection of integrins. Secondary antibodies were conjugated with the Alexa546 fluorophore. The percentage of GFP+ cells in each transfection indicates 30-‐35% of the total population were transfected effectively. Cells transfected with Drosophila integrins show integrin expression in 8-‐10% of the total cell population above empty vector controls. Integrin expression was not detected in cells transfected with coral integrins, suggesting primary anti-‐bodies against coral integrins did not bind to target epitopes.
Chapter Six Integrins of Acropora millepora
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.
Cell Line % of Total GFP+
Antibody % of Total integrin+
% of Total Expressing integrin
pHSMCS -‐ α-‐HA 488 3.17 ItgαPS2 ItgβPS-‐HA -‐ α-‐HA 488 29.46 26.29 AmItgα1 AmItgβ1-‐HA -‐ α-‐HA 488 4.97 1.8
Cell Line % of Total GFP+
Antibody % of Total GFP+/integrin+
% of Total Expressing integrin
pHSMCS 19.13 α-‐MYC α-‐Rabbit 546
0.21
ItgαPS2 ItgβPS-‐MYC 35.01 α-‐MYC α-‐Rabbit 546 18.14 17.84
ItgαPS2 ItgβPS-‐MYC 34.15 α-‐Rabbit 546 0.27
AmItgα1 AmItgβ2-‐MYC 27.17 α-‐MYC α-‐Rabbit 546
1.23 1.02
AmItgα1 AmItgβ2-‐MYC 27.8 α-‐Rabbit 546 0.18
Table 6.3 Percentage of cells expressing epitope tagged β integrins on the cell surface following transient transfection (as detected by anti-‐HA or anti-‐MYC antibodies). The percentage of GFP+ cells in each transfection indicates 20-‐35% of each population were transfected effectively. Anti-‐HA antibodies were tagged with Alexa488, which possesses the same peak emission wavelength as GFP, as such cells transfected with HA tagged integrins were not co-‐transfected with GFP. The percentage of the total cell population expressing Drosophila integrins (26% for HA-‐tagged integrins and 18% for MYC tagged integrins) was much higher than that of coral (1.8% for HA tagged integrins and 1% for MYC tagged integrins), indicating that whilst expressed in a low percentage of the population, coral integrins were present on the cell surface.
Cell spreading of GFP+/AmItgβ+ cells The ~1% of cells expressing both GFP and surface β-‐Integrins spread poorly on all
substrates follow cell sorting. All samples exhibited between 20% and 25% dead or
damaged cells which is likely due to extended periods in divalent cation free Robb’s saline
during cell sorting and the physical stress of the sorting process. Only 70% of healthy
αPS2βPS expressing cells spread on Rbb-‐Tiggrin, 25% less than expected. Spreading of coral
integrin expressing cells was similarly poor with ~2% of healthy cells spreading in each
sample, indicating that the cell sorting processes had a substantial negative impact on cell
viability and spreading.
Chapter Six Integrins of Acropora millepora
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6.4 Discussion The range of substrates that stimulate integrin mediated cellular behaviours such as
migration, differentiation and matrix deposition, is strongly influenced by the diversity of
integrin subunits expressed by the cell. These cellular behaviours govern a wide variety of
developmental processes. As such, understanding the diversity of integrins throughout the
Metazoa provides insight into the developmental roles, ancestral function and evolution of
the integrin family.
6.4.1 Novel Coral integrins may interact with 2 distinct ligand
types Examination of large protein datasets for Acropora, Nematostella, Hydra, and Clytia has
demonstrated that cnidarians possess similar numbers of α and β subunits to higher
invertebrates, which may appear somewhat surprising given the relative simplicity of the
cnidarian body plan. However, the relationship between morphological complexity and
genetic complexity is not linear (Ball et al., 2004) and phylogenetic analysis of integrins
provides another example of this disparity. Maximum-‐Likelihood analyses (Figures 6.1 and
6.2) show that cnidarian sequences form a phylum specific clade in both α and β integrin
phylogenies, indicating that integrin diversity among the Cnidarian integrin complement has
resulted from a number of independent duplications occurring after the divergence of
cnidarians from bilaterian evolution. Several lineage specific expansions have also occurred
in the Bilateria as evidenced by the existence of 3 clades of vertebrate β-‐Integrins and
grouping of α-‐subunits from each bilaterian phyla according to their ligand binding
specificity. Despite the clear evolutionary path of bilaterian integrins, a high degree of
primary sequence divergence and homoplasy effects have obscured the evolutionary
relationship between cnidarian and bilaterian sequences, leaving little clue as to the function
of basal cognates.
Inclusion of 2 novel coral sequences in the α-‐subunit phylogenetic analysis has
demonstrated that NvItga2 and AmItga3 form a clade only weakly associated with the other
cnidarian α-‐Integrins, suggesting they share a common origin independent of other
described cnidarian α-‐subunits. By contrast, the 2 coral β-‐subunits are closely associated
with each other and have a clear relationship to the integrins of Nematostella (2 β-‐Integrins
orthologous to each Acropora sequence), which could suggest 2 duplication events within
the cnidarian lineage or maintenance of 2 ancestral genes. The number of sequences in
Nematostella (2α & 4β) and Acropora (3α & 2β) combined with the phylogenetic
distributions suggest the ureumetazoan ancestor contained 2α integrins and 1 or 2β
Chapter Six Integrins of Acropora millepora
103
sequences. As the ligand binding specificity of integrin receptors is largely dependant on the
α-‐subunit, the independent origin of the 2 cnidarian α-‐subunits clades implies the cnidarian
integrin complement facilitates interaction with 2 distinct ligand types. Given cnidarians are
considered informative as to the genetic complement of the Eumetazoan ancestor, this
animal is also likely to have possesed the capacity to bind 2 distinct ligand types. Whether
these ligands bare any relation to bilaterian ligands was the focus of the functional studies
presented here.
6.4.2 Integrins containing AmItgα1 interact with RGD tripeptide
ligands Preliminary investigation of ligand binding showed that none of the 5 αβ combinations of
coral integrins spread significantly on RGD or Laminin based substrates compared to cells
expressing Drosophila integrins. This result could have been observed for a number of
reasons including failure of transfection and failure of integrins to bind efficiently to
substrates resulting from sub-‐optimal activation or vastly different ligand specificity. Co-‐
transfection with constitutively expressed GFP demonstrated the transfection efficiency to
be consistently 30-‐35% of the total population, a figure which was confirmed by flow
cytometry and supported by the proportion of Drosophila ItgαPS2 expressing cells that
spread on RGD peptides. Poor cell spreading mediated by coral integrins is therefore not
likely due to failure of transfection.
The capacity for integrins to switch between low and high affinity conformations is highly
influenced by the availability of divalent cations, which interact directly with amino acid side
chains to facilitate activation (Takagi et al., 2002). The cell media (M3 media) used to carry
out previous investigations of integrin structure-‐function using the Drosophila S2 cell
system is specifically designed to mimic the extracellular fluid contacting Drosophila cells in
vivo. This media contains 18mM Mg2+ and 6.8mM Ca2+ and allows efficient activation of
Drosophila integrin receptors, however, the extracellular fluid encountered by coral
integrins is seawater, which is vastly different in composition and contains 50mM Mg2+ and
10mM Ca2+ in a common artificial preparation. Performing cell spreading experiments on
Rbb-‐Tiggrin using range cation concentrations and a simplified saline media allowed up to
8% of cells transfected with AmItgα1 + AmItgβ2 to spread on Rbb-‐Tiggrin. This figure was
7% above spreading of empty vector control cells and provided an indication that coral
integrin receptors containing the AmItgα1 subunit bind to RGD tripeptide ligands.
Chapter Six Integrins of Acropora millepora
104
In order for the observed spreading on RGD ligands to be attributed to interaction with coral
integrins, the presence of coral integrins on the cell surface had to be confirmed. The
complete lack of GFP+/integrin+ cells in all samples detected with coral specific antibodies
(Table 6.2) strongly suggests the antibodies do not bind to target epitopes. These polyclonal,
peptide derived antibodies have confirmed specificity for their target peptides (data not
shown), however appear unable to recognise the folded integrin structure despite the
location of the target epitope on an exposed surface of the protein structure in both high and
low affinity conformations. Detection of epitope tagged β-‐subunits using anti-‐HA or anti-‐
MYC antibodies was able to detect expression in 1% of the cells transfected with AmItgα1-‐
AmItgβ1-‐HA and AmItgα1-‐AmItgβ2-‐MYC constructs compared to 26% or 18% of cells
transfected with ItgαPS2-‐ItgβPS-‐HA/MYC respectively (Table 6.3). Although expression only
occurs in a small percentage of the total population, the detection of epitope tagged β-‐
subunits confirms that coral integrins were successfully expressed on cell surface.
The percentage of coral integrin transfected cells that spread on Rbb-‐Tiggrin (2-‐7%) was
higher than that of cells expressing detectable levels of epitope tagged integrins on the cell
surface (1%), which is consistent with control transfections using the same epitope (18%
surface expression, 30% spreading on Rbb-‐Tiggrin). The presence of a higher percentage of
cells spreading than exhibiting detectable surface expression in both coral integrin and
control transfected cells suggests that spreading in coral integrin transfected populations
was in fact mediated by integrin-‐ligand interaction.
Although only a small percentage of cells expressed coral integrins successfully, exploration
of the ligand binding specificity of AmItgα1 containing heterodimers has provided the first
indications that like bilaterians, cnidarians also possess integrins which demonstrate affinity
for RGD tripeptide containing ligands. RGD sequences are common in bilaterian extracellular
matrix proteins, occurring in a range of developmentally expressed proteins including
Fibronectin, Vitronectin, Osteopontin, Easter, Tiggrin Fibrinogen, Fibrillin, and
Thrombospondin (Barczyk et al., 2010; Humphries et al., 2006). Recent data has revealed
that many of these proteins known to act as integrin ligands are not present in cnidarians
(see chapter 4), which limits the range of conserved potential ligands. Cnidarians possess
multiple representatives of Fibrinogen, Fibrillin and Thrombospondin containing xGD
sequences that may facilitate integrin binding in place of RGD sequences. Such tolerance to
changes in the first position of the recognition motif has previously been demonstrated in
the case of Drosophila thrombospondin where a KGD sequence facilitates binding of αPS2
integrins (Bentley & Adams, 2010). Identifying which of the matrix proteins conserved in
cnidarians and higher animals are capable of mediating integrin adhesion requires
Chapter Six Integrins of Acropora millepora
105
identification of xGD motifs in a wider range of proteins and comprehensive functional
assays encompassing the recently identified AmItgα2 and AmItgα3 subunits.
6.5 Conclusions Whilst the number of α and β integrins present in some representatives of lower animals
has recently been acknowledged, the true complexity of the basal metazoan integrin
repertoire is only now coming to light. The relationship of integrin genes from 2 anthozoans
within the context of the broader metazoan complement has for the first time suggested that
integrins from outside the Bilateria also demonstrate affinity for 2 types of ligand. The
distinct ligand specificities found in anthozoans are likely to reflect the binding capacity of
integrins from the Ureumetazoan ancestor, although the depth of their origin in metazoan
evolution is unclear, requiring further data from the Porifera. Furthermore, the indication
that AmItgα1 specifically binds RGD tripeptide sequences, combined with the ubiquity of
xGD sequences in conserved matrix proteins, such as thrombospondin, implies that RGD
tripeptide proteins represent one of the two predicted ancestral integrin ligand types. The
developmental roles of the full range of cnidarian integrins is yet to be explored, however,
understanding the ligand specificity of one cnidarian α-‐subunit expressed during
gastrulation has provided a solid basis for future investigations into potentially conserved
roles for integrins in diploblastic and triploblastic development.
Chapter Seven General Discussion
106
Chapter 7: General Discussion
7.1 Modes of gastrulation in cnidarians Cnidarians are considered the earliest branching (“lowest”) members of the animal kingdom
to display true gastrulation owing to their formation of a single gastric cavity (archenteron)
via ordered re-‐arrangement of cellular structure. Despite these re-‐arrangements resulting in
only two embryonic germ layers and a relatively simple body plan, morphological evidence
has revealed that all methods of gastrulation known from studies of triploblastic
development are also displayed within the phylum Cnidaria (Byrum and Martindale, 2004;
(Kraus & Technau, 2006). These methods include invagination, immigration/ingression,
delamination and epiboly as well as mixed modes of gastrulation (Figure 7.1 ).
Acropora millepora and Nematostella vectensis are the best studied examples of cnidarian
gastrulation in terms of morphology and genetic control. Both species are anthozoans, the
basal class of cnidarian (Figure 7.2) and have shared phylogeny to the level of sub-‐class
(Hexcorallia). As such, Acropora and Nematostella are considered closely related on an
evolutionary time-‐scale relative to classical models of comparative embryology (eg.
Drosophila, Xenopus, Gallus), and are ideally positioned for investigations of evolution and
development (Ball et al., 2002; Ball et al., 2004). However, in spite of their close evolutionary
relationship A.millepora and N.vectensis exhibit significant differences in gastrulation
strategy.
Chapter Seven General Discussion
107
Figure 7.1 Cnidarians exhibit a wide variety of modes of gastrulation including delamination, invagination, epiboly and ingression (a). The diversity of cnidarian modes of gastrulation encompass all observed bilaterian modes of gastrulation. Images of gastrulation in selected bilaterian models are shown (b-‐f). Mixed modes of gastrulation are common in bilaterians as demonstrated in X.Laevis, which combines epiboly of presumptive ectodermal cells with invagination of the presumptive mesoderm (e). Invagination and ingression are combined in amniotes such as chick (f) and human, where the primitive streak is formed by invagination of the presumptive ectoderm. As cell division drives ectodermal cells towards the primitive streak, they begin to lose epithelial character, with completion of the epithelial to mesenchymal transition marked by ingression / immigration into the blastocoel. Adapted from Byrum & Martindale, 2004 (a); Fujimoto et al., 2004 (b); Ball et al., 2004 (c); Wu & McClay, 2007 (d); Gilbert, 2003 (e); Wolpet et al., 2010 (f)
Chapter Seven General Discussion
108
Figure 7.2 Anthozoans (eg. Acropora millepora and Nematostella vectensis) are accepted to be basal among the Cnidaria (Ball et al., 2004; Technau et al., 2005). Unlike other classes of cnidarians, anthozoans possess a circular mitochondrial genome, which is consistent with the mitochondrial genome of bilaterians (Bridge et al., 1992). The basal position of anthozoans is also supported by phylogenetic studies of rRNA sequences (Odorico & Miller, 1997). As the basal class, anthozoans are most likely to reflect developmental aspects of the Eumatezoan ancestor, making them useful comparators for investigating evolution and development. The diversity of anthozoan developmental strategies complicates this comparison, however, consideration of data from developmentally distinct anthozoans that are closely related on an evolutionary time scale, such as Nematostella vectensis and Acropora millepora, assists in resolving many potential ambiguities of anthozoan development.
Gastrulation in Nematostella vectensis primarily occurs by invagination, with limited
evidence for a coinciding immigration of a minor cell population (See Kraus and Technau,
2006; Magie and Martindale, 2007 for discussion). Prior to gastrulation, the 128 cell stage
embryo somewhat resembles a prawn-‐chip stage embryo of A.millepora (Figure 7.3; Figure
1.2), which develops further into a spherical pre-‐gastrulation blastula. At the
commencement of gastrulation, the blastula invaginates, creating a blastopore. As the
invagination deepens, cells of the presumptive endoderm migrate towards the aboral pole,
whilst the cells adjacent to the presumptive ectoderm maintain contact with the inner
surface of the presumptive ectoderm and exhibit a polarised morphology typical of
migrating cell types. At the conclusion of gastrulation, the presumptive endoderm is in
proximity to the interior of the presumptive ectoderm, separated by an acellular connective
tissue layer known as the mesoglea. By this stage, the blastopore has developed pharyngeal
structure derived of ectodermal tissue and is now considered an oral pore.
Chapter Seven General Discussion
109
Whereas invagination is common in the Bilateria (eg. fly, frog and sea urchin) and the
dominant mode of gastrulation in anthozoans (Kraus & Technau, 2006), the mode of
gastrulation exhibited by A.millepora is remarkable and peculiar even among cnidarians. In
contrast to Nematostella and other animals that form a spherical blastula prior to
gastrulation, cells of the A.millepora embryo are organised into a flat bilayer (prawn-‐chip
stage) prior to gastrulation that persists beyond the 128 cell stage. At the onset of
gastrulation, the cells thicken leaving a concavity on the side of the presumptive endoderm.
As the concavity deepens, the blastopore becomes apparent and eventually closes to make a
spherical bi-‐layer embryo (Hayward et al., 2004). Opening of the oral pore and elongation of
the body column then marks the commencement of the planula larva stage, which is
morphologically similar to that of other cnidarians in which the mesoglea is apparent
(Figure 7.3)
Figure 7.3 Gastrulation strategies of Acropora millepora and Nematostella vectensis are distinct despite a close evolutionary relationship. Gastrulation in Acropora (A) is preceded by formation of a flat cellular bilayer, which reduces in circumference and thickens at the onset of gastrulation. The edges then begin to fold upward producing a concavity on the side of the presumptive endoderm. As the concavity deepens (gastrula) the blastopore becomes apparent and eventually closes to make a sphere (Hayward et al., 2004). By contrast, Nematostella gastrulation (B) is preceded by blastula formation and occurs by involution of presumptive endoderm from one side of the blastula (Lee et al., 2007). Unlike Acropora, the blastopore does not close and becomes the larval oral pore. Involvement of a conserved set of developmental genes in both modes of gastrulation implies the existence of differences in gene expression and the involvement of downstream effectors of morphological change, such as cell adhesion molecules.
Chapter Seven General Discussion
110
7.2 Genetic determinants of cnidarian gastrulation Most studies of cnidarian early development have focussed on assessing the expression of
transcription factors such as Snail, Twist, Brachyury, Otx, Pax, Sox genes and the Wnt system
(Hayward et al., 2004; Martindale et al., 2004; Momose & Schmid, 2006; Shinzato et al.,
2008; Smith et al., 1999). Transcription factors represent the classical targets of
developmental genetic investigation largely owing to the broad scale
developmental/morphological affects following expression of some transcription factors.
For example, Pax-6 (Eyeless) knockdown in Drosophila produces an eyeless phenotype and
over-‐expression results in ectopic eyes (Halder et al., 1995). Although the effects of other
transcription factors may be more subtle, the ability to influence the activity of whole gene
networks make transcription factors convenient targets for investigating the development of
atypical model systems such as cnidarians.
Comparative analyses between cnidarians and bilaterians into the diversity and expression
of transcription factors have identified two important points: (1) many transcription factors
involved in bilaterian gastrulation were already established at the time of cnidarian
divergence (although examples of specification and diversification are evident within
specific gene families) (Nichols, W. Dirks, Pearse, & King, 2006; Putnam et al., 2007;
Srivastava et al., 2010; Technau et al., 2005) and (2) many of the transcription factors that
function in bilaterian gastrulation also contribute to cnidarian gastrulation (de Jong et al.,
2006; Martindale, 2005; Martindale et al., 2004; Matus et al., 2006). Although these
principles are well evidenced, the diversity of gastrulation strategies observed among
cnidarians implies that genes responsible for cellular re-‐organisation during gastrulation are
dissimilar in expression and/or function between species -‐ even in closely related and
genetically similar species such as Acropora millepora and Nematostella vectenesis. This
complex relationship between genetic complement and morphological events is highlighted
by the substantial differences observed in the mode of gastrulation utilised by Acropora and
Nematostella (Figure 7.3). To further understand how networks of similar genes function to
produce vastly different phenotypes, investigations must consider not only the expression &
function of transcription factors, but also the function of downstream genes that facilitate
cellular behaviours such as division, apoptosis and migration.
Chapter Seven General Discussion
111
Cell adhesion molecules are the primary effectors of a number of cellular behaviours
including cell migration and cell & tissue polarisation, which are likely to contribute to re-‐
organisation in all modes of gastrulation. The most comprehensive survey into the diversity
of cnidarian adhesion molecules, presented in Chapter 4, revealed that cnidarians contain at
least 8 of the major adhesion genes demonstrated to function during bilaterian gastrulation
(Table 7.1). Members of the Cadherin and integrin families are of particular importance
among the genes identified as they have well defined roles in facilitating cell movements
during bilaterian gastrulation (Bökel & Brown, 2002; Halbleib & Nelson, 2006; Shimizu et al.,
2005; Yagi & Takeichi, 2000). The identification of cnidarian orthologues to so many critical
gastrulation genes supports the early evolution of not only the transcriptional genes
implicated in gastrulation, but also a wide variety of the terminal effectors, more than might
be expected from morphological comparison.
Although identifying the conserved adhesion proteins with potential roles in gastrulation is
an important first step, the simple presence of these genes in cnidarians provides little
insight into their function and significance to cnidarian gastrulation. In addition to protein
identification, the JCUSMART analysis also allowed the domain structure of proteins with
potential roles in cnidarian gastrulation to be assessed. Where complete models were
available, it was found that the domain structure of cnidarian adhesion proteins are
consistent with those of higher animal homologues, suggesting a similar capacity for protein
level interactions, particularly of regulatory mechanisms. This implies some degree of
functional similarity between cnidarian and bilaterian orthologous adhesion proteins (Table
7.1), which is further evidenced by motif conservation including the CCD & PCCD complex in
type-‐III cadherins (Chapter 5.4.1) and the MIDAS, ADMIDAS & NPxY/F motifs in integrins
(Knack et al., 2008).
In determining the influence of conserved cnidarian adhesion proteins on gastrulation, the
temporal and spatial patterns of gene expression must also be considered. In situ RNA
hybridisation of Acropora millepora embryos has demonstrated that integrins, planar cell
polarity implicated cadherins, and components of the Wnt system (eg. β-‐catenin) are all
expressed during gastrulation (Knack et al., 2008; Chapter 5). Whilst these genes are also
expressed during bilaterian gastrulation, several aspects of their expression in Acropora are
not consistent with bilaterian modes of gastrulation and allow a new model to be proposed
for adhesion protein function during gastrulation of Acropora millepora.
Chapter Seven General Discussion
112
Gastrulation related adhesion Gene
Maximum Number of Models Identified
in cnidarians
Pathway Functions Molecular Function Reference
Flamingo (Starry Night / CELSR)
1 • Homophilic cell adhesion • Selective Recruitment of Fz and Vangl
Chen et al., 2008;
Usui et al., 1999; Wu & Mlodzik, 2009
Frizzled 4
• Receptor for Wnt Ligands • Recruits DSH to plasma membrane during PCP
• Binds cytoplasmic DSH • ligand binding of Vangl prior to cell-‐autonomous PCP
Wu & Mlodzik, 2008; Guo et al., 2004; Wang et al., 2006;
Van Gogh (Strabismus
) 1
• Planar cell polarity (Fmi-Fz group)
• Invagination • Convergent extension • Directional cell division
• Interaction with FAT-Ds group
• ligand binding of Fz prior to cell-‐autonomous PCP
Bastock et al., 2003; Wu & Mlodzik, 2008; Jessen et al.,
2002
FAT 3 • Heterophilic ligand binding of Ds
Seifert & Mlodzik, 2007
Dachsous 1
• Planar cell polarity (FAT-Ds group)
• Invagination • Convergent extension • Directional cell division Interaction with Fmi-Fz group
• Heterophilic ligand binding of Ft
Seifert & Mlodzik, 2007
Type –III cadherins 2-‐3
• Adherins junction component
• Invagination • Mobility of presumptive mesoderm
• Mesoderm specification
• Neural development
• Homophilic ligand interactions Stabilise tissue (DE/G Cadherin)
• Increase tissue mobility (DN-Cadherin)
• Binds β-catenin at plasma membrane
Wu & McClay, 2007; Byrum et al 2009; Iwai et al., 1997
α-‐Integrins 3
• Heterodimer formation with β-‐Integrins
• Primary influence over ligand binding
• Major Ligand types: RGD, Laminin, Collagen, Leukocyte specific
Oloumi et al., 2004; Schmidt & Friedl, 2010; Hynes et al.,
2002; Marsden and Burke, 1998
β-‐Integrins 4
• Cell motility • Mesoderm migration • Stable adhesion to basement membranes
• Extracellular matrix deposition
• Influence proliferation, differentiation & apoptosis
• Heterodimer formation with α-‐Integrins
• Cytoskeletal connections • Binds ILK
Oloumi et al., 2004; Schmidt & Friedl, 2010; Hynes et al.,
2002; Marsden and Burke, 1998
Table 7.1 Adhesion gene families demonstrated to function during bilaterian gastrulation are conserved in cnidarians. Examination of the cnidarian adhesome has revealed at least 8 of the gene families that have conserved function throughout the Bilateria are also present in cnidarians. These families include the cell adhesion components of the 2 planar cell polarity pathways (Fmi-Fz, Ft-Ds), integrins and Type-‐III cadherins. The high degree of conservation of protein architecture and functional motifs between cnidarian models and bilaterian proteins suggest that cnidarians are capable of a similar range of cellular re-‐arrangements such as convergent extension, directional cell division, planar cell polarity and integrin mediated motility. Involvement of these proteins during cnidarian gastrulation is likely, however the variety of cnidarian modes of gastrulation suggest these processes are demonstrated selectively across a range of cnidarian taxa.
Chapter Seven General Discussion
113
7.3 Acropora millepora exhibits an inside-out mode of
gastrulation 7.3.1 The mobile presumptive ectoderm and un-coupling of β-
catenin from mesendoderm development
In most bilaterians, the regions of an embryo fated to become endoderm, mesoderm and
ectoderm are determined well before morphological changes of gastrulation have
commenced. Asymmetrical distribution of maternal mRNA, proteins, organelles and
cytoskeletal elements define the anterior-‐posterior (AP) axis prior to fertilisation and
subsequent signalling cascades then guide cell fate (Lee et al., 2007). Although the site of
gastrulation is variable among bilaterians with respect to the AP and DV axes it is consistently
influenced by a conserved network of signalling genes including β-catenin, bmp2/4/dpp,
Brachyury, Forkhead, Otx, Snail, and Twist (Martindale, 2005). Among these influential genes,
the role of β-catenin appears to be particularly well conserved with the accumulation of
nuclear β-catenin protein marking the site of cell migration and the future blastopore (Lee et
al., 2007; Logan et al., 1999; Range et al., 2005).
The influence of nuclear β-catenin accumulation appears to be conserved from prior to the
cnidarian-‐bilaterian divergence, as both cnidarians and bilaterians that gastrulate by
invagination exhibit a similar domain of nuclear β-catenin localisation prior to gastrulation.
Blastula stage embryos of Nematostella vectensis (Sea anemone) (Matus et al., 2006) and
Clytia hemispherica (Hydrozoan -‐ jelly fish) (Momose & Houliston, 2007) exhibit high levels of
nuclear β-catenin in the presumptive mesendoderm, at the site of gastrulation. This finding is
consistent with protein localisation analyses in bilaterians including Lytechinus variegates
which shows nuclear β-catenin in mesendodermal cells (Weitzel et al., 2004; Wu & McClay,
2007). Like β-‐catenin, the domain of Dishevelled (Dsh) expression is also indicative of the site
of mesendoderm formation in Nematostella and Lytechinus, as well as the scleractinian corals
Pocillopora meandrina, and Fungia scutaria (Marlow & Martindale, 2007). Inhibition of Dsh
expression is sufficient to inhibit the onset of gastrulation in both Nematostella and
Lytechinus embryos (Lee et al., 2007), as is β-catenin inhibition. The domains of expression
for Dsh and β-catenin overlap and the similarity in phenotypes produced upon knockdown are
explained by their cellular activity and protein level interactions.
Chapter Seven General Discussion
114
In Acropora millepora however, the domain of β-catenin expression (as indicated by in situ
RNA hybridisation) and nuclear localisation of β-catenin (and Dishevelled) is restricted to the
presumptive ectoderm. This domain of expression is opposite to what might be expected from
investigation of blastula forming animals, where expression occurs in the presumptive
meso/endoderm. Furthermore, the expression centres around the pole furthest the site of
blastopore development. To understand the implications of these expression patterns, the
cellular function of β-catenin must be considered.
Translocation of β-catenin to the nucleus is a key component of the canonical Wnt signalling
pathway in which binding of soluble Wnt protein to Frizzled receptors results in decreased
ubiqutination of β-catenin (mediated by Dsh). Subsequently, free (unbound) β-catenin
accumulates in the cytoplasm and nuclear translocation occurs. In the nucleus β-catenin is an
obligatory co-‐factor to the TCF/LEF-‐1 transcription complex, which activates a network of
genes including the Snail family. The net result of this activation is down-‐regulation of
epithelial characteristics such as cell adhesion and proliferation, accompanied by a marked
increase in cell mobility, survival and matrix deposition consistent with a mesenchymal
phenotype. Restriction of β-catenin and Dsh expression to the presumptive ectoderm of
Acropora embryos is therefore suggestive of an increased mobility of the ectoderm rather
than meso/endoderm prior to gastrulation.
Up-‐regulation of the Snail family of transcription factors downstream of nuclear β-‐catenin-‐
TCF/LEF activation is conserved among bilaterians and is widely considered a necessary step
in the formation of the archenteron and mesenchymal development. In Acropora however, the
domain of β-catenin expression in the presumptive ectoderm (Shinzato unpublished; Chapter
5.3.3) opposes the expression Am_Snail (Hayward et al., 2004), demonstrating a clear
uncoupling of this conserved mechanism, which would otherwise be expected to play a major
role in facilitating the tissue mobility required for gastrulation. A similar uncoupling of
mesendoderm specification from archenteron formation has also been suggested in
Nematostella. In Nematostella, the domains of β-catenin and snail expression overlap,
however, Kumburegama et al (2011) have demonstrated embryos inhibited in β-‐catenin
signalling still undergo invagination but fail to express AmSnail and specify endoderm.
Whether such an uncoupling is common among the Cnidaria remains to be determined, as do
the factors that drive endoderm specification both up and down-‐stream of Snail expression.
Chapter Seven General Discussion
115
7.3.2 Maintaining stability in the presumptive endoderm In addition to its transcriptional role down-‐stream of canonical Wnt signalling, β-catenin is
also implicated in the regulation of stable cell adhesion and planar cell polarity. In chordates,
E-Cadherin is the predominant epithelial marker responsible for cell-‐cell adhesion and
maintaining the stability of tissues/ cell sheets through lateral homophilic interaction. An
analogous role is played by DE-Cadherin in Drosophila and G-‐Cadherin in sea urchin, despite
significantly varied structures. At the plasma membrane, β-catenin binds directly to the
cytoplasmic region of active catenin binding Cadherin (E/DE/G), linking it to cytoskeletal
Actin. The tissue stabilising activity of catenin binding cadherins is therefore linked to the β-
catenin balance. Additionally, nuclear translocation of β-catenin in response to canonical Wnt
signalling results in down-‐regulation of E-Cadherin expression via TCF and Snail mediated
repression, as well as protein level Cadherin degradation. Conversely, stabilisation of
adherens junctions (AJ ; the junction type where E-Cadherin is most prevalent) by over
expression of E-Cadherin is sufficient to inhibit β-catenin translocation and gastrulation.
The change in epithelial phenotype from stable epithelium to migratory (Epithelial to
mesenchymal transition) is also influenced by the expression of another catenin-‐binding
Cadherin, N-Cadherin. During gastrulation, down-‐regulation of E-Cadherin occurs
concurrently with up-‐regulation of N-Cadherin (DN-Cadherin in Drosophila), which is both
necessary & sufficient for producing a migratory cell phenotype. Expression of catenin
binding cadherins can therefore produce either stable or invasive phenotypes in chordates
and protostomes although an analogous Cadherin has not been identified in sea urchins.
Given the domain of nuclear β-catenin in Acropora prawnchip stage embryos, it would be
expected that a catenin-‐binding Cadherin functionally analogous to E/DE/G-‐Cadherin (ie.
Stabilises the epithelium) is expressed in the presumptive mesendoderm. This pattern would
be inverse to what is observed in bilaterians where E/DE/G-‐Cadherin expression is restricted
to the presumptive ectoderm, outside the nuclear β-catenin domain. Alternatively, a migratory
type catenin binding Cadherin functionally analogous to N-Cadherin would have an expected
expression restricted to the presumptive ectoderm. However, only a single catenin-‐binding
Cadherin, Am_ACadherin, was identified in Acropora (Chapter 4.3.1) and no mRNA expression
could be detected during gastrulation (Chapter 5.3.3). This result suggests the unique absence
of a tissue stabilising catenin binding Cadherin during Acropora gastrulation and raises the
question of how presumptive endoderm stability is maintained throughout morphogenesis.
Chapter Seven General Discussion
116
The Acropora integrins, AmItgα1, ItgβCN1, and AmItgβ2, are each expressed specifically in the
presumptive endoderm throughout gastrulation (Knack et al., 2008). In bilaterians, integrins
have been shown to interact with a range of cell surface and matrix proteins to produce both
stable interactions and cell motility. Integrin signalling through integrin Linked Kinase (ILK) is
also critical to matrix deposition. Acropora contains all of the genes required for integrin
signalling through ILK including ILK, PINCH, Parvin, and FAK (Chapter 4.3.2) and although the
precise mechanism is unknown, the production of the mesoglea, a thin connective tissue layer
between the ectoderm and endoderm, is apparent towards the end of gastrulation, suggesting
matrix accumulation occurs during gastrulation.
The prospect that Acropora integrins stabilise the endodermal tissue through interaction with
and deposition of extracellular matrix proteins in a positive feedback loop (as observed
higher animals) is therefore not unreasonable. Identifying which matrix proteins present in
the mesoglea are likely candidates for integrin interaction is an area for further study,
although this task is greatly aided by results in Chapter 6, which indicate AmItgα1 containing
integrin heterodimers are likely to interact with RGD type ligands.
7.3.3 Co-ordinating expansion of the presumptive ectoderm The folding of presumptive ectoderm around the presumptive endoderm, the hallmark
feature of Acropora millepora’s unique mode of gastrulation, is accompanied by 2 important
observations: 1) The cells of each germ layer continue to divide throughout gastrulation (as
detectable by examination with a light microscope), and 2) the ectodermal tissue maintains a
relatively constant depth of 1-‐2cells (Ball et al., unpublished). Together these observations
imply expansion of the ectodermal tissue occurs by a co-‐ordinated process that maintains a
single cohesive epithelial sheet.
Maintenance and expansion of an epithelium in a single plane commonly results from planar
cell polarity (PCP), in which the orientation of individual cells are directionally co-‐ordinated
(polarised) throughout a tissue. Cellular polarisation is characterised by asymmetrical
localisation of PCP adhesion proteins and associated regulatory proteins. These proteins
function in 2 parallel groups; the core group consisting of Frizzled (Fz), Dishevelled (Dsh), Van
Gogh (Vang; also known as Strabismus -‐Stbm), Prickle (Pk), Flamingo (Fmi), Diego (Dgo); and
the Dachsous group consisting of FAT (Ft), Dachsous (Ds) and Four-‐jointed (Fj). Both groups
are conserved throughout the Bilateria (with lineage specific expansion of some members),
however their distribution among the Cnidaria has not previously been investigated. In
Chapter 5.3.1, JCUSMART analysis allowed orthologues of all major planar cell polarity
Chapter Seven General Discussion
117
components to be identified in cnidarians, with the exception of the cytoplasmic regulation
proteins Diego and Four-jointed.
In addition to local polarisation of individual cells, broad gradients of expression and protein
activity result in global polarity across the tissue. The Drosophila wing is among the classic
examples of both global and cellular polarity. Cellular polarity is detected by localisation of Fz-
Dsh-Dgo-Fmi to the distal plasma membrane and Vang-Pk-Fmi at the proximal plasma
membrane, whilst global polarity is demonstrated by clear gradients of Dachsous expression
(high proximal, low distal) and Four-jointed expression (low proximal and high distal) across
the wing. (Wu and Mlodzik, 2009).
Expression of Am_Daschous and Am_Van_Gogh in gradients across the presumptive ectoderm
of gastrulating Acropora embryos (Chapter 5.3.3) is consistent with the global expression
gradients observed in polarised epithelia such as the Drosophila wing (Dachsous) and mouse
phalangeal limb buds (Van Gogh; (Gao et al., 2011). The expression patterns of these Acropora
genes suggest PCP is involved in co-‐ordinating the expansion of the presumptive ectoderm
during gastrulation. PCP is commonly implicated during bilaterian gastrulation in facilitating
convergent extension (Shindo et al, 2008; Wang et al., 2006) and has recently been implicated
in Nematostella gastrulation, where expression of Vang at the animal pole is required for
archenteron invagination as demonstrated by morpholino knockdown (Kumburegama et al.,
2011). In both Acropora and Nematostella, Vang expression is restricted to the germ layer
exhibiting nuclear β-catenin localisation (the proposed mobile germ layer), which supports a
role for PCP in directing cell mobility across different modes of cnidarian gastrulation.
However, in contrast to Acropora where AmVangl is expressed in a gradient along a potential
second axis, Nv_Vang expression centres around the animal pole and forms a gradient along
the oral-‐aboral axis. This inconsistency in expression implies that whilst PCP is involved in the
morphogenic movements of both Acropora and Nematostella, mobility in the Acropora
presumptive ectoderm is not a simple translocation of the gene expression and cell
movements leading to archenteron invagination in blastula forming animals.
The precise role of planar cell polarity during Acropora gastrulation is not yet clear, although
one common function of PCP stands out as a favourable possibility. The prospect of
convergent extension (CE; the PCP mediated extension of a tissue along a single axis by
cellular intercalation) occurring in the presumptive ectoderm seems unlikely, as intercalation
of cells has not been reported. Furthermore, the region of PCP gene expression is quite broad,
encompassing the entire tissue, which is in contrast to expression patterns during bilaterian
CE events. The existence of a PCP in order to orientate auxiliary structures (eg. hairs, cilia) as
Chapter Seven General Discussion
118
observed in both the Drosophila wing and Mouse cochlea, is another possibility, although cilia
are not evident on the ectodermal surface until after gastrulation in Acropora. Whilst
expression of PCP genes prior to gastrulation may aid in co-‐ordinating ciliary beat in later
development, the delay in assembly of cilia implies a more fundamental function for PCP in
early development.
PCP has been demonstrated to influence the axis of cell division in a range of model systems,
including Drosophila and Sea Urchin. In Drosophila wing development and during sea urchin
gastrulation, PCP gene activity restricts cell division to a single direction, allowing the cell
sheet to expand in a single plane. Restriction of division to a single plane is consistent with
the morphological observations of Acropora development, hence the expression of AmVangl
and AmDachsous throughout the presumptive ectoderm and across a potential second axis,
may function to maintain the direction of cell division. This rationale also offers a mechanism
for maintaining flexible intercellular connections in the proposed mobile germ layer, which
shows no sign of classical stabilising proteins such as N-Cadherin and integrin. Whilst an
active PCP directing the axis of cell division and maintaining the stability of the presumptive
ectoderm is at present the most attractive prospect, understanding of PCP in cnidarians is in
its infancy. Further investigations into the Acropora PCP system, its influence on the direction
of cell division and potential roles in cilia mediated motility are required. Focusing on protein
localisation and functional inhibition analyses would provide the most direct and informative
evidence into the presence and function of planar cell polarity during Acropora development.
Investigating these aspects in Nematostella and Clytia as well as Acropora would provide a
solid basis for drawing more general conclusions regarding the role of PCP in cnidarians.
The inside-‐out model of coral gastrulation presented here represents a consolidation of much
of the available molecular and morphological evidence obtained from Acropora millepora,
however, each aspect of the model holds potential for further investigation. Questions remain
as to the transcriptional targets of nuclear β-catenin in conjunction with TCF/LEF such as the
manner in which nuclear β-catenin influences the profile of adhesion molecule expression,
and the consequences of this relationship for ectodermal mobility. The uncoupling of nuclear
β-‐catenin signalling from Snail expression and mesendoderm specification also requires
exploration in Acropora and may offer a new perspective on mechanisms of tissue re-‐
organisation involving differential adhesion in cnidarians. Other questions surround the
purpose of Dachsous and Van Gogh expression in the presumptive ectoderm. Are other PCP
genes expressed? Are the patterns of protein localisation as expected for PCP or do they have
additional roles in the development basal cnidarians not seen in higher animals? The
influence of canonical and non-‐canonical Wnt signalling on β-catenin nuclearisation and
Chapter Seven General Discussion
119
establishment of PCP also remains to be explored in Acropora. Investigating these aspects of
Acropora development require substantial methodological development to allow creation of
gene knockdowns, detailed exploration of sub-‐cellular protein localisation and profiling of
changes in gene expression during development and in response to genetic manipulation.
Chapter Eight General Conclusion
120
Chapter 8: General Conclusions
The importance of cell adhesion and dynamic changes in adhesive state, which are accepted to
be essential aspects of normal bilaterian development and function, have received little
attention in the context of cnidarian development. This project has addressed some of the
major deficits surrounding understanding of cnidarian adhesion systems, including the nature
of the cnidarian adhesion complement or “adhesome”, the involvement of cadherins in early
coral development, and the identification of the major integrin ligand type. Consideration of
these aspects of cnidarian adhesion along with previously published data has also facilitated a
new model of Acropora gastrulation to be proposed.
Investigating the nature of the cnidarian adhesome in a manner that would incorporate data
from multiple sources required development of a single platform for identification and
analysis of adhesion molecules. Chapter 3 details the development of such a platform, called
JCUMSART, which allows multiple large protein datasets to be assessed for protein models
with specific features. Although the analysis and data storage pipeline was originally designed
for identification of adhesion molecules, it has a demonstrated capacity for broader
application, being used for identification of selenium processing proteins as well as NOD and
Caspase related proteins.
The survey of cnidarian adhesomes (Chapter 4) encompassed data from 4 model cnidarians
(Acropora millepora, Hydra magnipapillata, Clytia hemispherica and Nematostella vectensis),
exhibiting different developmental strategies and life cycles. The differences between these
species allowed both an overview of the capacity of adhesion in cnidarians and comparison of
specific aspects of their adhesomes. A high degree of similarity between cnidarian and
bilatarian adhesomes suggested many of the adhesion components affecting developmental,
innate immune and defensive processes were already established in the ureumetazoan
ancestor. All 4 cnidarian species possessed an abundance of secreted pattern recognition
proteins with possible immune function, which is a clear advantage in an aquatic environment
where microbial transfer is easily facilitated.
Chapter Eight General Conclusion
121
Novelty among the cnidarians also centred around secreted pattern recognition proteins with
2 families (Collectin and Coll-‐IG) of PRR’s being identified for the first time in cnidarians. It is
unclear, whether the generalised expansion of pattern recognition receptors in the Cnidaria is
primarily associated with humoral immunity or symbiont uptake, which would hold
significant implications for studies assessing the genetic component of coral bleaching.
Type-‐III cadherins were among the proteins identified for the first time in cnidarians using
the JCUSMART. Cadherins with type-‐III structure include DE and G Cadherin which stabilise
embryonic germ layers during embryogenesis of invertebrates. An analogous role is played by
Type-‐I cadherins in vertebrates (eg. E-Cadherin). These proteins are of particular interest
during development due to conserved roles in β-catenin regulation, central to germ layer
specification. Sequence analysis shows that a single type-‐III Cadherin, Am_ACadherin, is
present in Acropora millepora, providing an opportunity to assess conservation of the
adhesion-‐germ layer specification mechanism in a model organism with a peculiar mode of
gastrulation (Chapter 5). Surprisingly, Am_ACadherin is not expressed during gastrulation of
Acropora millepora, demonstrating a significant departure from an otherwise conserved
specification system (Chapter 5.4.2). Another member of the Cadherin family, Dachsous, was
found to be expressed during coral gastrulation (Chapter 5.4.2), implying a role for planar cell
polarity in coral morphogenesis. This prospect is supported by the expression of another PCP
gene, Van Gogh, which is expressed in a similar pattern.
The cell adhesion survey also identified that the integrin pathway is strongly conserved in all
cnidarians considered. The complement of cnidarian α and β integrins is in alignment with
the complexity predicted by previous studies. In both Nematostella and Acropora, JCUSMART
allowed identification of novel α-‐Integrin subunits. Phylogenetic analysis demonstrates that
these sequences are divergent with respect to other coral integrins. The α-‐Integrin is accepted
to be the major contributor to integrin heterodimer ligand specificity and as such, the
divergence of the novel α subunits suggests cnidarians are capable of binding two distinct
ligands (Chapter 6.4.1). Ligand binding experiments conducted by transgenic expression of
coral integrins in a drosophila S2 cell system suggested that one of the ligands is an RGD
sequence (Chapter 6.4.2). The RGD sequence is common among extracellular matrix proteins
and the evidence provided here provides a solid basis for future investigation into the role of
integrins during coral development though manipulation of matrix proteins.
Chapter Eight General Conclusion
122
Consideration of results from each chapter of the project allowed a novel model of Acropora
gastrulation to be developed (Chapter 7.3). This model is inside-‐out with respect to blastula
forming animals, largely owing to the expression of β-catenin in the presumptive ectoderm,
rather than presumptive endoderm. The model suggests an increased mobility of the
presumptive ectoderm with respect to the presumptive endoderm. In the absence of a tissue
stabilising Type-‐III Cadherin, the presumptive endoderm is proposed to be stabilised through
integrin mediated interaction with and lay-‐down of extra-‐cellular matrix proteins. These
proteins, which are likely to possess accessible RGD sequences, ultimately form a component
of the mesoglea. Mobile interactions between cells in the presumptive ectoderm are proposed
to be facilitated by components of the planar cell polarity pathway, which may also act to co-‐
ordinate the direction of ectodermal cell division as the presumptive ectoderm folds around
the presumptive endoderm. Whilst each aspect of this model warrants future investigation,
this is the first such model to be proposed for what is a distinctive and unique mode of
gastrulation, which may hold broader implications for our understanding of endoderm
specification and the roles of cell adhesion in cnidarians.
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Appendix A: Supplementary Material
Chapter 4 Supplementary Figure 4.1
Protein Family HMMPFAM
Search Terms
BLAST
Search Terms
Cadherin 1. Cad 2. cad NOT dict NOT scad 3. CA NOT EGF NOT egf
NOT CAP NOT CAD_1 NOT CARD NOT ICAR NOT Dicty NOT ICAT NOT CAS NOT Pro_CA NOT SCAMP NOT SCA7
4. Protocad 5. cadg
6. Cadherin 7. Cadherin 8. Protocad 9. protocad 10. ProtoCad 11. Proto-cad 12. Proto-Cad 13. Proto-CAD 14. Dachsous 15. FAT NOT Fatty 16. Desmoglein 17. Desmocollin 18. CELR 19. CELSR 20. Selectin 21. selectin 22. falmingo 23. Flamingo 24. Calsyntenin 25. Calsyntenin 26. Starry 27. starry 28. E_value < 1E-25
integrin 29. ILK 30. ntegrin 31. INB 32. PSI 33. psinew 34. Talin 35. Talin 36. ICAP-1 37. FG-GAP 38. fg-gap 39. DISIN 40. LWEQ (Talin
diagnostic) 41. Int_alpha 42. int_alpha 43. ADAM
44. ntegrin 45. isintegrin 46. Talin 47. Talin 48. ntergrin AND inked 49. E_value < 1E-15
lectin 50. lectin 51. lectin 52. PA-I 53. Intimin 54. intimin 55. APT
68. CELIII 69. ndosialin (Endosialin) 70. lectin 71. lectin 72. c-type 73. Attractin
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56. Agglutinin 57. LECT 58. Knot1 59. PLEC 60. TECPR 61. ftp 62. ricin 63. Ricin 64. CRD 65. F5_F8 66. blect 67. B_lectin
74. attractin 75. Polycystin 76. polycystin 77. PKD 78. Pkd
Adhesion LRR 79. LRR 80. LRR, 7tm_1
Class B Adhesion GPCR 81. 7tm_2 82. Frizzled
83. Frizzled 84. Frizzled 85. FRIZZLED 86. Smoothened 87. smoothened 88. SMOOTHENED 89. E_value < 1E-10
Immunoglobulin
Superfamily
90. titin 91. -set 92. ig NOT Lig NOT signal
NOT align NOT lig 93. Ig 94. IG NOT PIG NOT AIG 95. Adhes 96. adhes 97. CAM NOT SCAMP
NOT calmodulin NOT Calmodulin
98. cell AND adhesion 99. CAM NOT |CAM (ie.
"pipe"CAM - this occurs in some identifiers) NOT almodul
100. cam NOT camp NOT came
101. mmunoglobulin 102. robo 103. Robo 104. oundabout 105. Neogenin 106. neogenin 107. DCC 108. Netrin AND eceptor 109. olorectal AND
cancer 110. ontactin 111. BOC 112. rother AND of 113. CDO 114. ysteine AND
ioxygen 115. pinin 116. Pinin 117. 1E-20 118. Toll NOT olloid AND
recept 119. toll NOT olloid AND
recept 120. 1E-10
Extracellular Matrix 121. LY NOT zf-LYAR 162. ollagen
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NOT LYR NOT CAP_GLY - LDL RECEPTOR
122. NtA - AGRIN 123. Reeler 124. reeler 125. Reelin 126. Reelin 127. NIDO 128. REJ 129. PLAT / LH2 130. MANEC 131. manec 132. FAP 133. fn3 134. FN 135. LamN 136. aminin_N 137. aminin_I 138. collagen 139. Collagen 140. C4 NOT HC4 NOT
C48 NOT PC4 NOT -C4 141. fn1 142. TB NOT PTB NOT
BTB NOT TBP NOT TBC NOT TB2 NOT PNTB NOT TBPIP
143. Fib_ 144. fib_ 145. fibrinogen 146. FBG 147. G2F 148. COLFI 149. C8 NOT PLAC8 150. steopontin 151. OSTEO 152. LINK 153. COLFI 154. ECM1 155. Col_cut 156. FReD 157. FRED 158. fred 159. Fred 160. TSP 161. tsp
163. aminin 164. ibulin 165. ibrillin 166. ibrin 167. nectin 168. steopontin 169. enascin 170. yaluron 171. xtracellular AND
atrix 172. thrombospondin
Other 173. Dicty (Dicty_CAD) 174. dicty 175. C4 NOT HC4 NOT
DC4 NOT C4d 176. CD36 177. Spondin 178. AMOP 179. VWA
194. ZO1 195. mucin binding 196. collagen binding 197. osteonectin 198. fibrillin binding 199. notch 200. Titan 201. Nidogen
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180. Sushi 181. notch 182. zu 183. MucBP 184. Collagen_bind 185. fn_bind 186. FBP 187. FAP 188. Fb_signal 189. amelogenin 190. ZP 191. FOLN 192. Kazal 193. LamNT
202. Usherin 203. Tensin 204. Slit 205. CSMD 206. Sidekick 207. sema 208. NL 209. cell AND adhesion 210. Cell AND adhesion 211. cell AND Adhesion 212. Cell AND Adheison 213. CAM NOT |CAM
NOT almodulin 214. cam NOT camp
NOT came 215. mmunoglobin 216. Robo 217. robo 218. oundabout 219. eogenin 220. DCC 221. Netrin AND receptor 222. olorectal AND
cancer ontactin 223. BOC 224. rother AND of 225. CDO 226. ysteine AND
ioxygen 227. pinin 228. Pinin
Supplementary Figure 4.1 Terms used to identify adhesion molecules during searches of JCUSMART annotation of Nematostella, Acropora, Clytia and Hydra datasets. Independent searches and search terms are used for HMMPFAM and BLAST. Boolean search terms were used in the following hierarchy: “OR” > “AND” > “NOT”. Each bullet point in the Figure constitutes an “OR” statement. E-‐value limits (where present) apply to all terms in the search statement.
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Supplementary Figure 4.2
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Supplementary Figure 4.2 Multiple sequence alignment of Talin proteins from representative metazoans used for maximum likelihood analysis (Figure 4.1). Proteins alignment only includes the region of overlap between Clytia talin1 and Clytia talin2. Alignment is coloured by percentage identity between all sequences included in the alignment.
Genbank Accessions: Human_Talin1 NP_006280.3; Human_Talin2 NP_055874.2; Ciona_Talin (Blom, Gammeltoft, & S Brunak, 1999); Urchin_Talin XP_001193631.1; Danio_Talin1 NP_001009560.1;Danio_Talin2 NP_957487.2; Drosophila_Rhea NP_648238.1; Apis_Rhea XP_391944.4
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Supplementary Figure 4.3
Supplementary Figure 4.3 Multiple sequence alignment of metazoan haemolytic lectins used for maximum likelihood analysis (Figure 4.2). Alignment is coloured by percentage identity between all sequences included in the alignment. Acropora sequences are labelled according to their EST of origin (Grasso et al., 2008) and Clytia sequences correspond to Contigs IL0ABA2YE22RM1 (1) and IL0ABA8YL09RM1 (2).
Genbank Accessions: CEL-III BAC75827.1
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Supplementary Figure 4.4
Supplementary Figure 4.4 In situ hybridization of haemolytic lectin A036-‐E7 from Acropora millepora. Expression commences in planula stage larvae and continues into adult life as demonstrated by cell specific staining (A & B). In larvae (A) and polyp stages (B) haemolytic lectin is expressed in the oral half of the animal, which is exposed to the environment after settlement. Cell specific staining suggests expression is restricted to nematoblasts, the precursor cells to nemtaocysts. In situ hybridization of sectioned adult tissue (C) demonstrates expression in cells basal to nematocysts, which hold a similar shape to nematocysts and are also expected to be nematoblasts. Expression of haemolytic lectin in nematoblasts, suggests a role in cellular defenses in coral. Image C courtesy of T..Ainsworth (unpublished).
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Supplementary Figure 4.5 Am_LRIG3 1 ------MPRSNMASVNIVTAWIVMCLVLFG-------------AALR-KDPNAKACAFAK Nv_LRIG3 1 -----------MASWFSTFSWLVLYIWILC-------------VFVSSQQTEGTACLEIK A_mel_LRIG3 1 MSNTEQIFFDHRFVWKYKWAIVILPILFLT--------SSNFIKANKISYGNIKDNQCPV Dr_LRIG3 1 ------------MPQPARAVSSVLLLLLLS----------GFTAVTGDSRRT-EHEPCPS Xl_LRIG3 1 -----------MCTMPLSWRRIIYLLLLFT------PLVLGFKSLERRVRRD--RGPCPS Mm_LRIG3 1 -----------MGAPGLRAATAALGLLLCAGLGRAGPAGSGGHGAPGQLLDDDAQRPCPA Am_LRIG3 41 PCICFGTKVDCKNRSLRMIPEGIPRNTTHLDLSVNQLTTLDMTKLSRLTKLRVLL----- Nv_LRIG3 37 PCYCLGNRVNCEGKHLTAIPVGIPSDITALNLGYNNIVDLDPKQLLKFTALKELFNPIPG A_mel_LRIG3 53 ECDCLGNVVDCINLQLIGAPSGLPPWTEILGLKGNNIASLEPDVLLHLTKLKELDLSGNK Dr_LRIG3 38 VCSCTGNLADCSRLKAGRTVERLPARITRLDLSHNKLRVFPEALFSSLPQLSEIKLSNN- Xl_LRIG3 42 PCRCLGDLLDCSRRKLTVVPSNLPEWLVQLDLSHNKLSSIKASSMNHLHNLRELRLNNN- Mm_LRIG3 50 ACHCLGDLLDCSRRRLVRLPDPLPAWVTRLDLSHNRLSFIQTSSLSHLQSLQEVKLNNN- Am_LRIG3 96 ------------------------------------------LHKNQLREVPPWQALPSS Nv_LRIG3 97 SFNPIPGSFNPIPGGFNPIPGGFNPILVASIPFLVASILSCHLHHNNIRGIPPWDSFPST A_mel_LRIG3 113 FGDDFKIILSEGTHLQMLKVNKNQLTQVPDMFFVKNITHLALAHNSITDINGTALLNLQR Dr_LRIG3 97 ---EFESIPDLGP-------------------NAGNLSSLILASNRIGRVSSERLSPLLT Xl_LRIG3 101 ---ELQIIPDLGP-------------------LSANITLFSLTNNKIEVILPEHLTPYQS Mm_LRIG3 109 ---ELETIPNLGS-------------------ISANIRQLSLAGNAIDKILPEQLEAFQS Am_LRIG3 114 LQRLWLHQNLISVVPSQRLTKKMSLAMLSLNNNNISVIEPYAFANLTSIWSL-------- Nv_LRIG3 157 LMTLTLHHNKIVAIPSLNTTKQHALRNLYLNSNKISSIGHHAFTNLSNLQNLYSIAYLRY A_mel_LRIG3 173 LQNLDLSXNKISVIRNGSFLAPNCLHNRNLNKNQIKVIENGSLDNLTS-LEELRLN---- Dr_LRIG3 135 LETLDLSNNNIVDVYAGAFPPIP-LKNLFMNNNRISTLEHGCFSNLSSSLLVLKLN---- Xl_LRIG3 139 LETLDLSNNLLAELKAGSFPTLQ-LKYLYINNNRISTMQSGAFDNLSATLQVLTLN---- Mm_LRIG3 147 LETLDLSNNNISELRT-AFPPLQ-LKYLYINNNRVSSMEPGYFDNLASTLLVLKLN---- Am_LRIG3 166 -------------KLGRNKLEAIPVAALSQVTGLEILDLTKNSIREIRVRLSHSIKSVIK Nv_LRIG3 217 TSLTEYKKEDCIKKLGKNRLTSVPSDALSQLQSLKRLDLSRNFFTSILASAFNRLSSLEV A_mel_LRIG3 228 ----------------KNYLTQLK-DLFTNLKKLRILEINRNELQTIQGLSLRGLKNLKE Dr_LRIG3 190 ----------------KNRLNSIP-AKIFSLPHLQHLELSRNRLRRVEGLTFQGLHGLRS Xl_LRIG3 194 ----------------KNRISHIP-SKMFKLSNLQHLELNRNRIKEILGLTFQGLDSLKS Mm_LRIG3 201 ----------------RNRISAIP-PKMFKLPQLQHLELNRNKIKNVDGLTFQGLGALKS Am_LRIG3 213 LKLSKNKIYNISAFAFWEMEKMQELHLDNNNLTSISKTWFFGVPMLRILNFDNNRIRVIE Nv_LRIG3 277 LKLSKNRISTIRG-AFWGQNKLQQLYLDRNNFTAIQTSSFFGLRDLQNLYLQSNQISTII A_mel_LRIG3 271 LHLKKNKIETLDDGAFWPLENLTILELDFNLLTMVRKGGLFGLEHLQKLTLSHNRIRTIE Dr_LRIG3 233 LKMQRNGISRLMDGAFWGLNNMEVLQLEFNNLTEVSKGWLYGLLTLQQLHLSHNSISRIK Xl_LRIG3 237 LRIQRNSIARLMDGAFWGLSTMEVLQLDHNRLTEITKGWLYGLLMLQKLHLSQNAISSIS Mm_LRIG3 244 LKMQRNGVTKLMDGAFWGLSNMEVLQLDHNNLTEITKGWLYGLLMLRELHLSQNAINRIS Am_LRIG3 273 ESKMPGVNWKLAFRLQILILANNEFTHIYSTTFAHLKYLRRLILNTNRIHYLADEAFSDL Nv_LRIG3 336 TTKFA--DWRSFPVLRKLNLERNRLSHIQDTTFKHLLALKVLNLANNRIYHISQGSFSDL A_mel_LRIG3 331 -----IQAWDRCKEIIELDLSYNEISTIERDTFEFLEKLKKLKLDHNQITYIADGAFSST Dr_LRIG3 293 -----PDAWEFCQKLAELDLSWNQLSRLEEGSFVGLSVLEQLHIGNNRISFIADGAFRGL Xl_LRIG3 297 -----PDAWEFCQKLSELDVSFNQLTRLEESSFGGLGLLSGLHIGNNKINFIADGAFRGL Mm_LRIG3 304 -----PDAWEFCQKLSELDLTFNHLSRLDDSSFLGLSLLNALHIGNNKVSYIADCAFRGL Am_LRIG3 333 LSLQTLDLRYNALPSTALEG-GVFANLNSVWLVRLDGNRITRISASSFHGLTSAISMNLS Nv_LRIG3 394 RSLEGLDLSNNDISWTVEEMNGPFRGLTNLASLRLDGNRITAIAATAFLGLENIKYLNLS A_mel_LRIG3 386 PNLQILELKFNKISYMVEDINGAFDPLGQLWKLGLAHNRIKSINKNAFTGLSNVTELDLS Dr_LRIG3 348 TNLQTLDLKFNEISWTIEDMNGPFSALDNLRKLFLQGNRIRSVTRKSFTGLEMLEQLDLS Xl_LRIG3 352 SSLNSLDLKSNDISWTIEDMNGTFSGLERLQRLTLQDNRITSITKKAFSWLDALEYLDLS Mm_LRIG3 359 TSLKTLDLRNNEISWTIEDMSGAFSGLDRLRQLILQGNRIRSITKKAFAGLDTLEHLDLS Am_LRIG3 392 ANAISSIDRFAFYEMTSLQYLYFDTEKLICNCEIKWLPTWLREK-NIQDDVRGLCSYPEN Nv_LRIG3 454 ANIITSIQENSFQGMDKLQKLWLNTSQLMCDCKIKWFGSWLRSRPSTRHTVRARCLHPQT A_mel_LRIG3 446 GNNITSIQENAFVSMTRLTKLRMNSSVLVCDCGLQWLSMWLREH-SYTD-AEVYCGFPHW Dr_LRIG3 408 NNAIMSLQANAFSQMKKLSELHLNTSSLLCDCQLKWFSLWVAEQ-AFLALLNASCAHPHL Xl_LRIG3 412 DNAITSMQTNAFSQMKSLQQLYLNTTSLLCDCQLKWLPKWLAEN-NFQTFVNASCGHPQI Mm_LRIG3 419 GNAIMSLQSNAFSQMKKLQQLHLNTSSLLCDCQLRWLPQWVAEN-NFQSFVNASCAHPQL
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Am_LRIG3 451 LAGRSILNISLSNLECDNSGSSPEVIKHPQGKKVLLGQNVTLRC---VVSWKNYSITVNW Nv_LRIG3 514 LYMKSVFNITPDAFVCDTSNPIPQISVHPPSQKAMRGDNVTLRCRVQVVQRNSSSVRLQW A_mel_LRIG3 504 LQGMSLTQLHHKNFTCDEYPK-PRIIEEPKSQMGIKGDNVTLVCR--ATSTAIARLHFTW Dr_LRIG3 467 LKGRSVFSIAQDEFVCDDFPK-PQITVQPETQSAIKGNNVTFVCS--AASSSDSPMTFAW Xl_LRIG3 471 LKGKIIFAVSPDDFVCDDFPK-PQITVQPETQSAIKGSNVTFICS--AASSSESPMTFAW Mm_LRIG3 478 LKGRSIFTVSPDGFVCDDFPK-PQITVQPETQSAIKGSDVSFTCS--AASSSDSPMTFAW Am_LRIG3 508 TKNGRPLKGAHLKIHTQSKDDY-----RGAYTSELHLRSVSRRDSGKYQCVAYSTEFVPV Nv_LRIG3 574 RLGYEVISERDITDFVRTDDDD-----IVTYNSDLFLISVDFSDAGLYQCIASN-KYGSQ A_mel_LRIG3 561 KHDNIEINDDNLQINTDSTENG-----VTEATSILHLTNVTHANAGKYQCMVTN-TYGTT Dr_LRIG3 524 KKDNELLSEPEIQNQAHVRAAAGETGELTEYTTTLQLRQVEFTSEGRYQCVISN-HFGSS Xl_LRIG3 528 KKDNELLHDSEIENFAHLRAQG---GDVMEYTTILRLRNVEFINEGKFQCVISN-HFGPT Mm_LRIG3 535 KKDNEALQDAEMENYAHLRAQG---GELMEYTTILRLRNVEFTSEGKYQCVISN-HFGSS Am_LRIG3 563 RSHWAQLSVLGFPKLTQTPTDVLVEPGKTFKLYCKAQGHPMPTLLWQKDGGRS-FPAADD Nv_LRIG3 628 FSKKARLEVLEFPVLTRKPHDVSVHVGGLIKLPCAATGYPVPVISWRMGDGKSKFPAAEE A_mel_LRIG3 615 YSAKAKLSILIYPSFSKIPHDIRVIAGSTARLECSAEGQPSPQIAWQKDGGND-FPAARE Dr_LRIG3 583 YSNKAKLTVNMLPFFTKTPMDLTIRAGATARLECAASGHPSPQIAWQKDGGTD-FPAARE Xl_LRIG3 584 YSVKAKLTVNMLPLFTKKPMDLTIRAGSTARLECAAVGHPTPQIAWQKNGGTD-FPAARE Mm_LRIG3 591 YSVKAKLTINMLPSFTKTPMDLTIRAGAMARLECAAVGHPAPQIAWQKDGGTD-FPAARE Am_LRIG3 622 RRIKYAEGLEVCEIRNAQYKDSGKYTCFAKNVAGSANASATVTVLEPPGFTGPWRKKVTV Nv_LRIG3 688 KRIEHLPSEHLFIIRNARGVDTGAYTCTATNGAGSINATAYVTVLEVPRFMQSMVSKR-V A_mel_LRIG3 674 RRMHMMPTDDVLFIVDVKTADSGVYSCTAQNLAGLIVANATLTILETPSFVKPMENKE-V Dr_LRIG3 642 RRMHVMPRDDVFFIVDVKTEDIGVYSCTAQNTAGAISANATLTVLETPSFLRPLLDRA-V Xl_LRIG3 643 RRMHVMPEDDVFFIVNVKTEDIGVYSCTAQNSAGSISANATLTVLETPSFLRPLMDRT-A Mm_LRIG3 650 RRMHVMPEDDVFFIVDVKIEDIGVYSCTAQNSAGSVSANATLTVLETPSFLRPLLDRT-V Am_LRIG3 682 SAGDSLVLECYVRGAPQPLVIWFKDGIKIQMNDRVVLTESRQLLVITKATDDDGGRYDCE Nv_LRIG3 747 RTGESAVLECKASGSPMPRFTWYKDDKKVTLSARVVAHG--QLLVFVTVLRDDEGTYTCQ A_mel_LRIG3 733 TVGGSIVLECMASGMPRPKLSWRKNGNPLLATERHFFTAEDQLLIIVDTRISDAGSYECE Dr_LRIG3 701 AKGETAVLQCIAGGSPPPRLNWTKDDSPLQATERHFFAAGNQLLIIVDAAEGDAGTYTCE Xl_LRIG3 702 SKGETTVLQCIVGGSPTPRVNWTKDDSPLVVTERHFFAAGNQHLIIVDTDLEDAGIYTCE Mm_LRIG3 709 TKGETAVLQCIAGGSPPPRLNWTKDDSPLVVTERHFFAAGNQLLIIVDSDVSDAGKYTCE Am_LRIG3 742 VANSQGNDTR-TMMVAIEPEKCTGVV----KKSNSNNSNYVKYDKKTFLGIIVVVVVACI Nv_LRIG3 805 VSNSLGTARQNTRLTVVEGSELQTCQ----DKES-------RYDKKTFLGIIVISVVTCV A_mel_LRIG3 793 MSNSLGSVVGASHLTVKPAPISTP-----------SPGSVNEDD---ILGLIIITVVCCA Dr_LRIG3 761 MSNPLGTERGNLRLSVLPNPNCDQGPAGGGAVGAAGSGRGPEDDGWTTVGIVIIAVVCCV Xl_LRIG3 762 VSNILGTERGNIHLTVLPNPTCDS---------PVNAIQTAEDDGWATAGIVIIAVVCCV Mm_LRIG3 769 MSNTLGTERGNVRLSVIPTPTCDS---------PHMTAPSLDGDGWATVGVVIIAVVCCV Am_LRIG3 797 VVTSMVWLFIQYNTLSCGKRRAPRRSLHTGPFGVDYSDESNSKALN-QEISYIPLKLSSS Nv_LRIG3 854 VGTSLVWLLVIYCARRGSHRR--RHKLRARPFQADGTDTTNSKLTHRDELSYIPLKSSSS A_mel_LRIG3 839 VGTSIVWVVIIYQTRRR-----LNNVAQGRPHAQPTPTLT--GTVADTQ----------- Dr_LRIG3 821 VGTSLVWVVIIYHTRRRNEDCSVTNTDETNLPADIPSYLSSQGTLAERQDGYMLPSESGS Xl_LRIG3 813 VGTSLVWVVIIYHTRRKNEDCSVTNTDETNLPVDTPSYLSSQGTLAERQDGYGS-SETGS Mm_LRIG3 820 VGTSLVWVVIIYHTRRRNEDCSITNTDETNLPADIPSYLSSQGTLADRQDGYIS-SESGS Am_LRIG3 856 SSTQTSRESPRSTATFLTASEAAQGRCAVATLAGCSSENASGSEEKSSGNDNISSENSLK Nv_LRIG3 912 GSTQTSRDSPRSTATFLASSDSHG----AAFPVSASIDTHSGSE-KTTG--NISSENSLK A_mel_LRIG3 881 THIYLETS------------SQHSKDSGTGDSTNPSSDQLQLCLP--------------- Dr_LRIG3 881 SHQFISSSIGGFYMPPKDMNSLCQLDTGSEADLEAAIDPLLCHYQGPVGSLLTPGAHYSA Xl_LRIG3 872 -HQFIASSMSGYFLQQRD-NGACNLDNGSEADLEVATDPLLFNYTGVPGPLYLRGNPYDP Mm_LRIG3 879 HHQFVTSSGGGFFLPQHDGAGTCHFDDSSEADVEAASDPFLCPFVGSTGPVYLQGNLYSP Am_LRIG3 916 ESRSSLASFCRSESSLPCCQEVVTSAQVHGSDSDSEKSRPTLAMFARNTS---------- Nv_LRIG3 965 GSHCSLTSSCPSVESEPVRQVVH--VQIHSSDSDSEKCKETSPTRRKETANL-------- A_mel_LRIG3 914 -----------------------------EEIVTCSVNNE-------------------- Dr_LRIG3 941 ELPDTYTVCVSEPR------LLSDSYSRKRDFYTCSSSLDPCDHMMLPHDIP-------- Xl_LRIG3 930 DAYEIFHAGYSMDRRTPNANFYESEYLKQKELGLFGHQHDDCYKIACSHGVQSLAGRIVG Mm_LRIG3 939 DPFEVYLPGCSSDPRTALMDHCESSYVKQDRFSCARPSEEPCERSLKSIPWP-HSRKLTD Am_LRIG3 966 --------------------------CSD---HDPGEHKLTFPDCIPYNKKTCTDHEKFS Nv_LRIG3 1015 -------------------------ACSQSDCEDASEHCGSAAQHCGTNAGYCGIPSNHC A_mel_LRIG3 925 --------------------------EEPSAVVNVGAPLLRYTNHERIVHENKDCAV--- Dr_LRIG3 987 -------------SCVDD------CETDQCVLPRSGSYMGTFGKAAWRPTQDHSAVILHE Xl_LRIG3 990 PTCSHKEDIEIK-MSLDTDILGLKHTVDQGILTSCSTYLGTFGKPVWRPQLDSPCGYVQP Mm_LRIG3 998 STYPPNEGHTVQTLCLNKSSVDFSTGPEPGSATSSNSFMGTFGKPLRRPHLDAFSSSAQP
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Am_LRIG3 997 SK--DVTPDVCVTFYPPSEKETSCDTDVVESTLILT------------------------ Nv_LRIG3 1050 GTPLETSGTSSGHYRTFRGLGPEYDSNDVGSVLIKSKEMIVRTPSSEHFSTTHV------ A_mel_LRIG3 ------------------------------------------------------------ Dr_LRIG3 1028 N-----------------PYAALHAEEELTHTPSHTHSSVYEQPFDSRTIDSNPTTPLGS Xl_LRIG3 1049 SFSQLT-SHTIPQTKLNMQLENGKDESQRTNIPDENATFFPKITSDYQRTSGFQCYELDT Mm_LRIG3 1058 PDCQPRPCHGKSLSSPELDSESEENDKERTDFREENHRCTYQQIFHTYRTPDCQPCDSDT Am_LRIG3 1031 SK--DVTPDVCVTFYPPSEKETSCDTDVVESTLILT------------------------ Nv_LRIG3 1104 GTPLETSGTSSGHYRTFRGLGPEYDSNDVGSVLIKSKEMIVRTPSSEHFSTTHV------ A_mel_LRIG3 ------------------------------------------------------------ Dr_LRIG3 1071 N-----------------PYAALHAEEELTHTPSHTHSSVYEQPFDSRTIDSNPTTPLGS Xl_LRIG3 1108 SFSQLT-SHTIPQTKLNMQLENGKDESQRTNIPDENATFFPKITSDYQRTSGFQCYELDT Mm_LRIG3 1118 PDCQPRPCHGKSLSSPELDSESEENDKERTDFREENHRCTYQQIFHTYRTPDCQPCDSDT
Supplementary Figure 4.5 Boxshade alignment of full length Am_LRIG3 protein with representative metazoan LRIG3 orthologues demonstrates a reasonable degree sequence conservation throughout the protein. Shading demonstrates >50% consensus to Am_LRIG3 (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig16713 as the Acropora orthologue of LRIG3, which was initially established by JCUSMART analysis.
Genbank Accesions: Mm_LRIG3 NP_796126.4; Dr_LRIG3 NP_001103817.1; Xl_LRIG3 NP_001103840.1 A_mel_LRIG3 XP_001121890.2
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Supplementary Figure 4.6 Am_PTPRD 1 MLFFKVAWLLALATIGVAVRAFRLTAIKNVITVNVRENVTLRWTYEVSRGERYTITWGTR Nv_PTPRD 1 ------------------------------------PTVTLTPKDFAGVTRGFVYMICNV Sp_PTPRD 1 --------------------------------ITQEQGEFMVTFPFGYHSGFNHGYNCAE Dr_PTPRD 1 ------------------------------------------------------------ Xl_XPTP-D 1 ------MQVPSCQTMNIARPVVVLLCFLLHAGAETPPKLTRTPVDQIGVSGGVASFICQA Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 61 ADLLFTQESWQKTAQPSPKMPTKYANRVKIVGKASLFIQRVDLSDDGFYNCHIQGDFVST Nv_PTPRD 25 TGSPMPTLTWYKNGSVIRNSR--IQIFTMPYGGLIRIGPLRAKIHKATYECEAD-NGVGP Sp_PTPRD 29 STNFASLRWIN---------------------------------YGKRASCLER------ Dr_PTPRD 1 ------------------------------------------------------------ Xl_XPTP-D 55 TGDPRPKIVRNKKGKKVSNQRFEVIEFDDGSGSVLRIQPLRTPRDEAIYECVASNSVG-- Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 121 SK----------DINLTVIDPPRILTQLPLELTWIEGNRQEINCDADGKPKPRVTWHKDG Nv_PTPRD 82 P-LRDSSTLIVHTASTKPAGFPRIAKHP-SFKQRRSGDDVTFTCAAVGTPTPNITWFKDR Sp_PTPRD 50 -VVDDGRKMVWVEADSLPAGYPEITSNPRRYMVVENNRPATIYCGATGNPEPEILWFKEF Dr_PTPRD 1 --------------DQLPAGFPTIDMGP-QLKVVERTRTATMLCAASGNPDPDISWFKDF Xl_XPTP-D 113 -EVATTTRLTVLREDQIPRGFPTIDMGP-QLKVVERTRTATMLCAASGNPDPEITWFKDY Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 171 SVVKSGHRR-----------------ATLEFKSVSYKDEGSYKCVAKN-IGGKKEKQ--- Nv_PTPRD 140 LPIVLNDP--RVSVLSD--------GRKLRITDLRESDNGKYSCVARNALGMVFSSQAMP Sp_PTPRD 109 VPIEANDR---ISMTDS----------GLQFSRAQLSDQGRYECAAKNSLGT-RYS--VE Dr_PTPRD 46 LPVNTSNN-GRIKQLRSESFGGTPIRGALQIEQSEESDQGKYECVATNNDGT-RYS--AP Xl_XPTP-D 171 LPVDTSNNNGRIKQLRS---------GALQIEQSEESDQGKYECVATNSAGT-RYS--AP Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 210 ----VKVNVLYAPKETNITTNLPQDTVDDGSIITITCEALGNPPPSYKFFINGKPIQDKK Nv_PTPRD 190 ARLFIYLTLSVVRSKPEFTLRPQDKTVDPDTDVELKCSARGSPTPSIVWEVDGSRISGQN Sp_PTPRD 153 AQVYVKD----RQVEPRFTILPENQEVVPGGSVNLTCAAYGSPMPRVRWMKAGMDLDDMD Dr_PTPRD 102 ANLYVRELREVRRVPPRFSIPPTDNEIMPGGSVNITCVAVGSPMPYVKWMLGSEDLTPED Xl_XPTP-D 219 ANLYV----RVRRIAPRFSIPPTNHEIMPGGSVNITCVAVGSPMPYVKWMLGSEDLTPED Mm_PTPRD 1 ---------------------------MPGGSVNITCVAVGSPMPYVKWMLGAEDLTPED Am_PTPRD 266 FERSGILSITAIGFKQRGIYSC-------------------------------------- Nv_PTPRD 250 ----G--ELIIRGIQRSGNYSCIADSNMGRVQAYAYVTVRLLPFAPSRPNAASITSDAIK Sp_PTPRD 209 DLPIGRNVLQLRDIYESANYTCVATSLLGTIDTSARVTVRTRPSVPNSPVVTGYTSSSLT Dr_PTPRD 162 DMPIGRNVLELTDVRQSANYTCVAMSTLGVIEAVAQITVKALPKPPGVPQVTERTATSIT Xl_XPTP-D 275 DMPIGRNVLELTDVRQSANYTCVAMSTLGVIEAIAQINVKALPKPPGTPMVTESTATSIT Mm_PTPRD 34 DMPIGRNVLELNDVRQSANYTCVAMSTLGVIEAIAQITVKALPKPPGTPVVTESTATSIT Am_PTPRD 288 ----------------------------------------------RPENDRGTGPISEV Nv_PTPRD 304 ITWHPRGAHVVTSYEVQYRLKG-KTEWQEITN-VRGMQSLVVSGLR----PFSSYEFRVF Sp_PTPRD 269 VEWSTLSSDTVTSHVLEYKRSRSGGSFTEMEIPSPQDTSFTIEGLL----PYTEYAVRLL Dr_PTPRD 222 LTWDSGNPEPVSYYIIQHKPKNSEDSFKEIDG--VATTRYSVGGLS----PYSDYQFRVV Xl_XPTP-D 335 LTWDSGNPEPVSYYIIQHKPKSSEEQYKEIDG--VATTRYSVAGLS----PYSEYEFRVV Mm_PTPRD 94 LTWDSGNPEPVSYYIIQHKPKNSEEPYKEIDG--IATTRYSVAGLS----PYSDYEFRVV Am_PTPRD 302 AV-YVRYA--PRITHPPGNKIVDENNPVGVRCEAEGYPPPLIKWVKLLGNKNVANGTNWV Nv_PTPRD 358 AVNSLGRSRPSLMAEYTTSETKPGSAPRNIEAMGVSDTSFKAGWLPPISANGRIRGYRLY Sp_PTPRD 325 AVNSIGRSNPSDEVTAMTGEDVPSQ-PEQFEGVAISASQIRLTWMMR-DTEPQIIRYELY Dr_PTPRD 276 AVNNIGRGPPSEDIEAKTAEQAPSTAPRQVRGRMLSATTAIIHWDEPEEANGQITGYRVY Xl_XPTP-D 389 AVNNIGRGPPSEPVMTRTSEQAPSSPPRNVQARMLSSTTILVQWEEPEEPNGQIQGYRVY Mm_PTPRD 148 AVNNIGRGPASEPVLTQTSEQAPSSAPRDVQARMLSSTTILVQWKEPEEPNGQIQGYRVY Am_PTPRD 359 LRSAQRTDQGRYRCIATNGFGRDAFAEFEINVYFAPVINRTASSQDVPA--WAGIPTNLS Nv_PTPRD 418 YSLDLLEDISQWRFMASS-TNSTTVTGLTRQTTHFFRILAYNIAGDGPLSEVVAVKTQKG Sp_PTPRD 383 YNVSTNDND---MHKTITPTTDYVLDNLRPNTLYHIRLAARSETGEGASSPVITVRTQQS Dr_PTPRD 336 YTTDPSQHVNQWEKQIVRTSNFLTIPGLTPNKTYYIKVLAFTSVGDGPLSSDLQIIAKTG Xl_XPTP-D 449 YTMDPTQHINSWTKHNVADSQITTIGNLEPQKTYSVKVLAFTSVGDGPLSNDIQVITQTG Mm_PTPRD 208 YTMDPTQHVNNWMKHNVADSQITTIGNLVPQKTYSVKVLAFTSIGDGPLSSDIQVITQTG Am_PTPRD 417 CV-ATANPAPRYE---------WTKASGVVATSEKSGWLQVTPQEG------------DG Nv_PTPRD 477 VP-GQPRSVQLSVLSSTAIRVTWSPP--RYPGDGIFGY-DVYYNKS-----KQDMDTVIG Sp_PTPRD 440 APGAPPQEVSGTVLSSTSIEVRWSPPPLEDQNGDITGY-KIIYRKMSLVSTNNPEMNVPV Dr_PTPRD 396 VP-SQPTDFKGEAKSETSILLSWNPPTQTGQDNQIIGY-ELLYKKG----DDKEEKRVSF Xl_XPTP-D 509 VP-SQPLNFKAEPESETSILLSWTPP----RSDTISSY-DLYYKDG----DHAEEV-ITI Mm_PTPRD 268 VP-GQPLNFKAEPESETSILLSWTPP----RSDTIASY-ELVYRDG----DQGEEQRITI
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Am_PTPRD 455 FE--PYTCTVENNKGSDSMVIRLVKVGIPVVQLKVVRRQSDSLFLEWLLLSDGATNSYI- Nv_PTPRD 528 AFQMNTKEIMGLTPYTVYKVAVAAKSDKGVGPMSFVLTARTDEDRPSDAPQNIQGRSRDS Sp_PTPRD 499 EADVRSYILEALRKYTLYDIRVVACTAIGDGPPSDSLSIRTAEDAPEGPPRKVRVRVHNS Dr_PTPRD 450 EPTT-TYLLKELKPFTTYMFQLAARSKHGIGAYTNEISAETPQTQPSAPPQEVKCTSHSS Xl_XPTP-D 558 DPAT-SYRLQGLKPNSLYYFRLAARASVGLGASTTEISARTMQSKPSAPPQDIRCNSQSS Mm_PTPRD 318 EPGT-SYRLQGLKPNSLYYFRLSARSPQGLGASTAEISARTMQS---------------- Am_PTPRD 512 -----SHYTLQYHTDHSNGGIERTIRISSEKTN--------------------------- Nv_PTPRD 588 TTLEISWDPPRPEHRNGLITHFTIKYRAKGQRG---AKYLTVDATKTSVVLHNLDKFTNY Sp_PTPRD 559 TTIRVQWQAPDPDLQNGEIRGYRIDYQTVTEE---------------------------- Dr_PTPRD 509 TSILVSWKPPPVELQNGIMTKYTIQYAATEGDDTSMRQVSDIPPEKYHYLLENLEKWTEY Xl_XPTP-D 617 TSILVSWLPPPVEKQNGIIIGYSLKYAGVDADDIKPHEILGISSETRQYLLEQLEKWTQY Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 540 ----------------YQLHDLQPYTKYTIELFATN------------------------ Nv_PTPRD 645 FIWVQASTKQGDGPFSDKHTVSTAEDVPNGAPRDVRIRVHNSTTMTVKWN-PPTTKQDGK Sp_PTPRD 591 ------------------------------------------------------------ Dr_PTPRD 569 RVTVNAHTEAGEGPESLPQLIRTEEDVPSGPPRKVEVEAVNSTSVKVLWRSPVPSRQHGQ Xl_XPTP-D 677 RIIVIAYTDVGPGPESSPILIRTDEDVPSGPPRKVEVEAVNSTSVKVSWRSPVANKQHGQ Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 560 ------------------------------------------------------------ Nv_PTPRD 704 ILGYMVFYTRVDDQGNQLRPPAQPESKDSMSEE-----NHKVHLTGLAPETTYQIEVAAY Sp_PTPRD 591 -------------GDPVGSPQVLFINQPDLR---------VAVLSDLLPKTFYSIEIAAF Dr_PTPRD 629 IRGYQVHYVRMVNGEPVGHPVIKDILMDDAQWEYDDSAEHELVLSDLHAETTYSVTVAAY Xl_XPTP-D 737 IRGYQVLYVRMENGEPKGQPMLKDVMLADA---------QEMIISGLQPETAYSITVTAY Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 560 -------KHFSSETSKVEAMTTEAAPSPPRNVKVKIINGTAVNVKWEAPLRPNGPLLFYT Nv_PTPRD 759 TIKGDGARSVTRLAKTMAQSPDPPFVYLERASGDPISDVTLAWRTSATGVIEYKVRYAKS Sp_PTPRD 629 TVRGDGIRSTTEIQQTPGEVPTTPVAFRVENNLDDTYTATWSPPVETHGDLV-------- Dr_PTPRD 689 TTKGDGARSKPKLVTTTGAVPEKPRLM-VSPTNMGTALLQWHPPPNTFGPLQ-------- Xl_XPTP-D 788 NTKGDGARSKPKIVSTSAAVPGKPRLV-ISHTQMNTALIQWYPPVETFGPVI-------- Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 613 VSYDKKEYIP-NGGTKILSVLPNITEVVIGALTPFSNYSIKVKVRNSLTVNESKPVTIST Nv_PTPRD 819 VRRLRGRDQGNLKMKEIKFKSHTTKQKFQSLANGIWYLFNVSLRTKAGWSPETSRWIEIP Sp_PTPRD 681 TYRLSYGPESGRPLYLDLRPSEFQSYTFDDLRLGTRYEFKLTASNEEGNSNEAIFYYTTH Dr_PTPRD 740 GYRLRFGRKDVEPLTVIEFSERENHYTTKEIHKGASYTFRLSARNKVGFGEETVKEISTP Xl_XPTP-D 839 GYRLKFGRKDLDMLTTFEFFEKEEHFTATDIHKGALYIFKLSARNKVGYGEEIVKELSIP Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 672 KMAAPSEPLAFEANMTGAHSATLWWRKPRQLNGKILLFKIRMRWRYKNVKGTY---RFIT Nv_PTPRD 879 PGPPSGPPLNVRAVAVSSTSVEVTWSAPDIWNRGGPILGYSVMYNPANKRGDVTVKNVTS Sp_PTPRD 741 EGTPSGSPMNVTASPLSSSSISVSWDPPLEEERNGLILKYTIRYYS----SAMPTELTNR Dr_PTPRD 800 EDTPAGYPQGIVAESSTTTTIQVSWQPVALAERNGAVVKYALQYKDINS-PRSPSELFIT Xl_XPTP-D 899 EEPPSGFPQSIHCDSTSSTSVLITWQHPNLEERNGLLTKYTLMYRDINL-PHYPIEVPIV Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 729 KKIPAKVTPRERRRLRRYTMDYRVLRLEPLREIQLTRIQPFAFISVHVSEGTGITENEVF Nv_PTPRD 939 PNIFKVVLKRLKKFREYEVRVRAIGMLGMGPASEPFIDRT-KEDGKCHCRDVRPRTVGSD Sp_PTPRD 797 TTETDIVLTSLVPNTAYSVEVRAHTSVGPGPYSTKDIATTPREAPASAPLDLRCAAPHGT Dr_PTPRD 859 APESTVTLDGLKADTTYDIKMCAFTSKGPGPYSPSVQFRT-QPVNQVFAKNFHVRAAMKT Xl_XPTP-D 958 PADTTVTLTGLKPDTTYDVKLRAHTSKGPGPFSPSVQFRT-LPVDQVFAKNFHVKAVMKT Mm_PTPRD 361 ----------------------------------------------MFAKNFHVKAVMKT Am_PTPRD 789 WSP--WSNNYTLQTLEG----APSAPRNVELKQNKG--ASVKVTWLPPEYPNGIIQK--- Nv_PTPRD 998 WVILEWLPPRIDAGIDT----PLTHADEVRVDALT--------ADVQTFKVQGLVPH-MK Sp_PTPRD 857 QLEVKWSHPSNIQRSGPGYNYLVYSTNSADNTDVSTWGRPVSAGEFTKLELRGL-QEETT Dr_PTPRD 918 SVLLTWEIPENYNPAQP---FTILYDNG-QSVEVD--------GKLTQKLIVNL-QPETQ Xl_XPTP-D 1017 SVLLSWEIPENYNSALP---FKILYDDGKMAVEVD--------GRATQKLITNL-KPETS Mm_PTPRD 375 SVLLSWEIPENYNSAMP---FKILYDDGKMVEEVD--------GRATQKLIVNL-KPEKS Am_PTPRD 838 ----------------YRIIYSNNSIKNYTAEVTK--GLKNTSLFYILSGLHKDSTYQ-- Nv_PTPRD 1045 YTVRLSASNAMGEG--PRAELSISTKKGKPPALAKPTMLRDEMNNGLIPVEL-HRAS--- Sp_PTPRD 916 YYVAVKLDTPEGNSPLSQIITCLTGTSAPSEPRSFDAHIASDNTINLVWSEP-SDIPGRL Dr_PTPRD 965 YSFLL---TNRGNS-AGGLQHRVSTMTAPDILRTKPFLISKTNADGMVTVEL-PGVQ--- Xl_XPTP-D 1065 YSFVL---TNRGNS-AGGLQHRVAAKTAPDVLKTKPVFIGKTNSDGMITVEL-PEVL--- Mm_PTPRD 423 YSFVL---TNRGNS-AGGLQHRVTAKTAPDVLRTKPAFIGKTNLDGMITVQL-PDVP---
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Am_PTPRD 878 -----------------------------INVQAFTSFPG-------EVSPTVRRDIKTP Nv_PTPRD 1099 --------EKNGPISYY----------LVVVVPLDKDG----------KYPQGSNPDEYF Sp_PTPRD 975 KDYVIKYHTSDDFRREYPDYYDGGGEIEILASEQRVSLSGQLFKPN-MEYEFNITARTEA Dr_PTPRD 1017 --------TAERVRAYY-----------IVVVPLKKQRPGKFIKLWENPDEMNLEELLRE Xl_XPTP-D 1117 --------VNDKIKGYY-----------IVIVPLKKSR-GKFIKPWESPDEMEFDELLKD Mm_PTPRD 475 --------ANENIKGYY-----------IIIVPLKKSR-GKFIKPWESPDEMELDELLKE Am_PTPRD 902 GDVLEQSKVD-------------------------------------------------- Nv_PTPRD 1131 KKVRRRS--AREAGQVKPYIAAKFLADQFPKKFLVGDEGFKNQYGYYNKRLVANSYYTVF Sp_PTPRD 1034 IRGPPASILVRTRIQALEHIPK----PTIQNSTMNGQS--IKIALPTITQNAALLDYLYV Dr_PTPRD 1058 INRTSVSLRFRRHLELKPYIAACF--HELPNEFTLGDA--KMYGEFQNKQLLNGQEYVFF Xl_XPTP-D 1157 ISRKRRSLRFRREAEQKPYIAAHF--DLLPTEFTLGDQ--KQYGGFENKQLQNGQEYVFF Mm_PTPRD 515 ISRKRRSIRYGREVELKPYIAAHF--DVLPTEFTLGDD--KHYGGFTNKQLQSGQEYVFF Am_PTPRD 912 --------------------------------------------TASLYGGVFAGIIAFV Nv_PTPRD 1189 SRAYVQTDDGDFVYTSSPFTDPVLITPPSAAG----LSGGGKGGLNLAYVIIPVVLLLVI Sp_PTPRD 1088 IVIGLRNKNG-EPVALTISPNDIKTEDLEVQSRGRRSAQHPRSRRAATGQDTEPYIAAKL Dr_PTPRD 1114 VLAVLEISDS-MLYAASPYSDPVVFADIDPQP---------IIDEEEGLIWVVGPVLAVI Xl_XPTP-D 1213 VLAVIEHSES-AMFATSPYSDPVVSMEIDPQP---------MTDEEEGLIWVVGPVLAVV Mm_PTPRD 571 VLAVMDHAES-KMYATSPYSDPVVSMDLDPQP---------ITDEEEGLIWVVGPVLAVV Am_PTPRD 928 FVIIIVALVIRNH-RKRRDGTDLSGKQYAVANSNDSGIGDKESLDRVPGVKCVQGV---- Nv_PTPRD 1245 VTVIVLAVLYVMRMRRRRR------------------SKKESKCKDEEKSEPSDPVEM-- Sp_PTPRD 1147 NGEEMPASFVI-------------------------GDGSEVNGYRNKPLKEGEYYTLFT Dr_PTPRD 1164 FIICIVIAILLY--KRKRAESEARK-------------GSLPSGKEMPSHHPTDPVEL-- Xl_XPTP-D 1263 FIICIVIAILLY--KRKRTESDSRK-------------SSLPNSKEIPSHNPTDPVEL-- Mm_PTPRD 621 FIICIVIAILLY--KRKRAESESRK-------------SSLPNSKEVPSHHPTDPVEL-- Am_PTPRD 983 RRGSEDVIGPGKFVTIKPIPIQRLPEYCAINHADRNKGFREEFLSIRVPGS--FTWENSQ Nv_PTPRD 1285 RRLNFQ--TPAMID--HP-PISVLDLPKHIAILKADNNSQFTQEYESIEPGQTFTADSSQ Sp_PTPRD 1182 RAVILSENGTAMMS--HP-PIPVSQFQEHIERLKGNDAMLFSQEYESIEPGQQFTWDHSN Dr_PTPRD 1207 RRLNFQ--TPGMAS--HP-PIPVMELADHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Xl_XPTP-D 1306 RRLNFQ--TPGMAN--HP-PIPILELEDHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Mm_PTPRD 664 RRLNFQ--TPGMAS--HP-PIPILELADHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Am_PTPRD 1041 KPENKAKNRFNSIIPYDHTRVTLTELEGSPGSDYINANFIDGYDHPFKFIATQGPVANTF Nv_PTPRD 1340 MECNKGKNRYPNIHAYDHSRVRLSYINGIEGSDYINANFCDGYRKERAYIATQGPMQHTA Sp_PTPRD 1239 LETNKPKNRYANVIAYDHSRVILSPMEGLPGSDYTNANYCDGYRKQNAYIATQGPLLETM Dr_PTPRD 1262 LEVNKPKNRYANVIAYDHSRVLLSAIDGIPGSDYINSNYIDGYRKQNAYIATQGALPETF Xl_XPTP-D 1361 LEVNKPKNRYANVIAYDHSRVLLSAIDGIPGSDYINSNYIDGYRKQNAYIATQGPLPETF Mm_PTPRD 719 LEVNKPKNRYANVIAYDHSRVLLSAIEGIPGSDYVNANYIDGYRKQNAYIATQGSLPETF Am_PTPRD 1101 GDFWRTVWEQGVCVVIMVTNIVEGGRIKCLKYWPSASPEMYGLVLVSPQGEEELADYVVR Nv_PTPRD 1400 ADFWRMVWEQRTFTIVMLTREEERGRVKCDQYWPTDGTDVYEGIEVSLVDWVELANYTIS Sp_PTPRD 1299 ADFWRMVWEQRTNTIVMMTKLEERNRVKCDQYWPTRDQEKYGFIQVTLLDTTELATYTVR Dr_PTPRD 1322 GDFWRMIWEQRSANIVMMTRLEERSRVKCDQYWPNRGTETYGLIQVTLLDTVELATYCVR Xl_XPTP-D 1421 GDFWRMMWEQRSATVVMMTKMEERSRIKCDQYWPSRGTETYGLIQVTLLDTVELATYTVR Mm_PTPRD 779 GDFWRMIWEQRSATVVMMTKLEERSRVKCDQYWPSRGTETHGLVQVTLLDTVELATYCVR Am_PTPRD 1161 KFSIQMISKSAEHSAREVTQYHFTAWPDQGVPTHATSLLAFLRKVRTSVPEDSGPILIHC Nv_PTPRD 1460 TL---QICKEGASQPREVKHFQFTGWPDHGVPAHPTPFLAFLRRVKFYNPPDAGPIVVHC Sp_PTPRD 1359 SF---ALVKNRSMEKREVKQFQFTAWPDHGVPEHATSVLAFISRVKSCNPPDAGPIVVHC Dr_PTPRD 1382 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKSCNPPDAGPMVVHC Xl_XPTP-D 1481 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKTCNPPDAGPMVVHC Mm_PTPRD 839 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKTCNPPDAGPMVVHC Am_PTPRD 1221 SAGVGRTGTYIVLDAMLDQIAAEGVVDIYGFISHIRQQRSFMVQTEGQYVFVHNALEEYV Nv_PTPRD 1517 SAGVGRTGCFIVIDSMLERLRHEETVDIYGHVTVLRTQRNYMVQTQEQYIFSHDAILEAV Sp_PTPRD 1416 SAGVGRTGAYIVIDSMLERIKHEKTVDIYGHVTCLRAQRNYMVQTEEQYIFIHEALYEAV Dr_PTPRD 1439 ------------------------------------------------------------ Xl_XPTP-D 1538 SAGVGRTGCFNVIDAMLERIRHEKTVDIYGHVTLMRAQRNYMVQTEDQYIFIHDALLEAV Mm_PTPRD 896 SAGVGRTGCFIVIDAMLERIKHEKTVDIYGHVTLMRAQRNYMVQTEDQYIFIHDALLEAV Am_PTPRD 1281 TCGSTEFPVSDLPERLRILNTVDPDSGDSLVVAEFKNLGLGASDGDSFIVASRQENKSKN Nv_PTPRD 1577 SCGNTEVHARNLLHHIKKLTELGKGE-VTGLEEEFKVVDPSQAKKHKYGAATLAINRPKN Sp_PTPRD 1476 ASGTTEVHARNLYGHIQKLTALEPGETITGMENEFKRLASQKAQPSRFVSANIPANKFKN Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1598 TCGNTEVPARNLYAYIQKLTQIEPGENVTGMELEFKRLASFKAHTSRFISANLPCNKFKN Mm_PTPRD 956 TCGNTEVPARNLYAYIQKLTQIETGENVTGMELEFKRLASSKAHTSRFISANLPCNKFKN
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Am_PTPRD 1341 RYA-ILPFDRTRVTLWPYTGMVGSDYINASFVDSFQQREAFIATQAPLENTVVDFWRMTW Nv_PTPRD 1636 RLANILPYETTRVHISPVRGVEGSDYINASFIDGYRQRGLFIATQGPLQDTVDDFWRMLM Sp_PTPRD 1536 RLLNIQPYEGNRVCLQPIRGVEGSDYINASHIDGYRQRNAYIATQGPLAETTEDFWRMLW Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1658 RLVNIMPYESTRVCLQPIRGVEGSDYINASFIDGYRQQKAYIATQGPLAETTEDFWRMLW Mm_PTPRD 1016 RLVNIMPYESTRVCLQPIRGVEGSDYINASFLDGYRQQKAYIATQGPLAETTEDFWRMLW Am_PTPRD 1400 EYEAYSIVMLTTMNELQEGQCA-YYLPRREEFIVYGLLMVEVETEDQRNGFCRRRLRVTN Nv_PTPRD 1696 EQNSNIIVMLTQLHEGEWEKCYKYWPTDRSARHQYYIVDPIAEHEYPQFVIRDFKVKDA- Sp_PTPRD 1596 EQNSTIIVMLSKLREMGREKCHQYWPAERSARYQYFVVDPMSEYNMPQYILREFKVTDA- Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1718 EHNSTIVVMLTKLREMGREKCHQYWPAERSARYQYFVVDPMAEYNMPQYILREFKVTDA- Mm_PTPRD 1076 EHNSTIVVMLTKLREMGREKCHQYWPAERSARYQYFVVDPMAEYNMPQYILREFKVTDA- Am_PTPRD 1459 TKSGEIRSIYHFHFKEWAERSIPLNGMGLLDMMKCITRVQQQTGN-GPIVIHCSDGSGRT Nv_PTPRD 1755 -RSDTVRNIKQFHFLGWPETGVPKSGEGIIDLIGQVQRAYEQQEEEGPITVHCSDGVGRT Sp_PTPRD 1655 -RDGQSRTIRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQEGPISVHCSAGVGRT Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1777 -RDGQSRTVRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQDGPISVHCSAGVGRT Mm_PTPRD 1135 -RDGQSRTVRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQDGPISVHCSAGVGRT Am_PTPRD 1518 GTFCAINVALERVKLDGTIDIFQTVRRLRTQRPLMVQTEEQYKLCYEITRLFVESFNDYS Nv_PTPRD 1814 GVFTALFIVLERMRSEGVVDLFQTVKLLRTQRPAMVQSQEQYLFCYKTALEYLGSFDHYA Sp_PTPRD 1714 GVFITLSVVLERMRYEGIVDMFQTVKMLRTQRPAMVQTEDQYQFCYHAALEYLGSFDHYT Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1836 GVFITLSIVLERMRYEGVVDIFQTVKMLRTQRPAVVQTEDQYQFCYRAGLEYLGSFDHYA Mm_PTPRD 1194 GVFITLSIVLERMRYEGVVDIFQTVKMLRTQRPAMVQTEDQYQFCYRAALEYLGSFDHYA Am_PTPRD 1578 DLK Nv_PTPRD 1874 I-- Sp_PTPRD 1774 --- Dr_PTPRD --- Xl_XPTP-D 1896 T-- Mm_PTPRD 1254 T--
Supplementary Figure 4.6 Boxshade alignment of full length Am_PTPRD protein with representative metazoan PTPRD orthologues demonstrates a high degree of sequence conservation C-‐terminal to the transmembrane region. Shading demonstrates >50% consensus to Am_PTPRD (Black –conservation, Grey – conservative substitution). The degree of conservation in the C-‐terminal region supports the assignment of Acropora protein model Contig12034 as the Acropora orthologue of PTPRD, which was initially established by JCUSMART analysis.
Genbank Accesions: Dr_PTPRD XP_002663075.2; Sp_PTPRD XP_001184195.1; Mm_PTPRD NP_001014310.1; Xl_XPTP-D NP_001083850.1
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Chapter 5 Supplementary Figure 5.1
Supplementary Figure 5.1 Aligned Nucleic Acid and Protein sequence of Am_ACadherin (Type-‐III Cadherin) supercontig (Contig6389 + Contig5439 in the 2010 Acropora millepora transcriptome assembly).
Forward Frame 3: 1 D C G T T S K C E A G L L K F S V E G T 3 GACTGTGGTACAACTAGCAAATGCGAGGCTGGTTTGTTAAAGTTCTCAGTTGAAGGAACA 21 S L F K I D P R T G V V S V G S T P L D 63 TCCCTGTTTAAAATTGATCCGCGCACTGGTGTTGTAAGTGTTGGCTCCACTCCTCTCGAT 41 Y E K Q R E H V F T V V V E D F G E K I 123 TATGAAAAGCAGAGAGAGCATGTGTTTACTGTCGTTGTGGAAGATTTTGGCGAGAAGATA 61 Y K S R G F V T I D V R N T D D E K P Q 183 TACAAATCCAGGGGATTTGTTACGATTGACGTGCGAAATACGGATGACGAGAAGCCACAG 81 F L E S D Y T I S I A E D A E T G R P L 243 TTCCTAGAGAGTGATTACACGATCAGTATTGCTGAAGACGCCGAGACGGGGAGGCCTCTA 101 A A I L A R D A D G D P V K Y S I T S G 303 GCAGCTATACTTGCCAGAGATGCAGATGGGGATCCTGTTAAGTACTCTATAACTAGCGGT 121 D S G G I F Q I N P T T G V L S L K S S 363 GATAGTGGTGGCATTTTCCAGATTAACCCAACAACTGGAGTTCTTTCTTTGAAATCAAGC 141 I K G N P R T Q Y T L Q V K A S N S A Q 423 ATCAAAGGAAATCCTCGGACACAGTACACGCTTCAAGTGAAGGCGTCGAATTCTGCGCAG 161 D S R F D E V R V V V N I E D S N D N R 483 GATTCTCGTTTCGATGAAGTGCGTGTTGTGGTCAACATCGAGGACAGCAATGATAATAGG 181 P V F T D C P P E V P V E E N K P R G H 543 CCAGTGTTCACTGATTGCCCGCCTGAGGTCCCAGTAGAGGAGAACAAGCCAAGAGGACAC 201 R V Y Q V V A Q D T K D R G R N K E I E 603 AGGGTCTATCAGGTCGTCGCTCAGGATACTAAGGACAGGGGAAGAAACAAAGAGATTGAA 221 Y F L V T G G E R L F E I D N T T G V I 663 TATTTTCTCGTTACCGGTGGAGAAAGGTTGTTTGAGATTGATAACACTACCGGAGTTATA 241 K T L T S L D R E T K D Q H T L I I M A 723 AAAACCTTAACCAGCTTGGACAGGGAAACCAAAGATCAGCATACATTAATTATAATGGCT 261 E D G G H G R N S A E R L L S Y C I L D 783 GAGGATGGAGGGCATGGAAGAAATTCGGCGGAGAGACTTCTTTCATATTGTATTCTTGAC 281 V K V V D Q N D N F P F F L T R T Y Y A 843 GTCAAAGTTGTAGACCAGAACGACAATTTCCCCTTCTTCTTGACCAGAACTTACTATGCC 301 S V W Q G A P V N T E V L T V R A A D M 903 AGCGTCTGGCAAGGAGCGCCTGTGAATACAGAGGTTTTAACCGTAAGGGCAGCGGACATG 321 D T R V N A N I D N S E V Q Y Q L V N A 963 GACACGAGAGTGAATGCCAATATTGACAATAGCGAAGTGCAGTATCAGTTGGTCAATGCT 341 D D K F Q V E L A T G V I K T K A T L V 1023 GATGACAAATTTCAAGTTGAACTGGCAACTGGAGTGATTAAAACCAAAGCCACGTTGGTC 361 S F V G K V Q L Q I R A I N K Q P M A I
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1083 AGCTTTGTGGGAAAAGTACAGTTGCAGATAAGAGCCATTAACAAACAACCCATGGCTATA 381 S E E R P R T S T T T V E I N V E K D K 1143 AGTGAGGAAAGACCACGCACGTCAACGACTACTGTGGAAATCAACGTCGAAAAAGATAAA 401 P P A F A P S A V Y K S A I N E D V K I 1203 CCACCCGCGTTTGCACCGTCGGCGGTTTATAAAAGTGCAATAAACGAAGACGTCAAAATA 421 G K E V Q Q I L A I S Q V D R K N K I V 1263 GGAAAGGAGGTTCAACAGATCTTAGCGATCAGCCAGGTAGACAGGAAAAACAAAATAGTG 441 Y S F V K S N P E G E Q K F Q I D P A T 1323 TACAGTTTTGTGAAGTCCAATCCAGAGGGAGAACAAAAATTTCAAATTGACCCGGCGACT 461 G N I T T A S T L D Y E Q V K E Y R L Q 1383 GGCAACATCACAACTGCTAGCACGCTGGATTATGAACAAGTGAAGGAGTACAGGCTTCAG 481 F R A T D I A T N L Y A T C V V I I S L 1443 TTCCGAGCTACTGATATTGCGACGAACCTTTACGCGACTTGCGTGGTCATCATTTCGCTG 501 I D V N D D T P T F K L E E Y T A R V P 1503 ATCGATGTCAACGACGACACGCCAACGTTCAAGCTAGAAGAATACACTGCTAGAGTTCCG 521 E N A A V D F N V I T I E A D D R D T A 1563 GAAAATGCAGCAGTAGATTTTAATGTGATCACTATAGAAGCCGACGACAGGGATACAGCT 541 L S G Q V G Y T L E V S D S S E G Q F F 1623 TTGTCTGGTCAAGTTGGTTACACCCTAGAAGTGTCCGACAGTTCCGAAGGACAATTCTTC 561 A I N G Q T G V M I T K N S F D R E D P 1683 GCCATCAATGGTCAAACCGGAGTGATGATTACGAAAAATTCGTTTGATCGAGAGGATCCG 581 K H I P K Y N V F A V A T D K G V P P L 1743 AAACACATTCCGAAGTACAATGTGTTTGCCGTGGCAACAGACAAGGGCGTTCCTCCTCTC 601 A G K L D F E T K K T Y N I S I T A T D 1803 GCTGGAAAACTTGACTTTGAAACGAAGAAGACGTACAACATCTCCATCACCGCAACAGAT 621 R K D S A T V P V V V N V L D T N D N I 1863 AGAAAAGACAGTGCTACAGTTCCAGTGGTGGTCAATGTTCTGGACACCAACGATAACATT 641 P Q F R N L I Y E A I I P E N S P P G Q 1923 CCTCAATTTCGCAATCTAATTTACGAGGCCATTATTCCAGAAAACAGCCCTCCTGGCCAA 661 R V A T V F A T D L D S P K I Q N D L R 1983 AGGGTGGCGACAGTCTTTGCCACTGACTTGGACAGTCCGAAAATCCAAAACGATCTCCGA 681 Y S L D A D G Q K N F A V D A V S G L I 2043 TATTCCCTCGACGCAGACGGACAGAAGAACTTTGCAGTTGATGCTGTGAGTGGTCTGATC 701 T T A N Q R L D R E V N P V V T F T A F 2103 ACCACTGCCAATCAAAGGCTGGATCGAGAGGTGAATCCAGTGGTTACCTTCACTGCTTTT 721 A F D G K H R G E A L I R V T L R D V N 2163 GCTTTTGATGGCAAACACAGAGGGGAAGCTTTGATTCGAGTCACGCTCCGAGATGTCAAC 741 D N S P Y F P N P P Y V G Y V E E N L D 2223 GACAACAGTCCATACTTTCCCAACCCTCCCTATGTCGGCTATGTAGAGGAAAACCTAGAT 761 P G A S V M V I Q A F D L D S G I D G E 2283 CCGGGGGCAAGTGTCATGGTTATTCAAGCGTTCGATCTGGATTCCGGCATTGACGGAGAA 781 I V Y S L D D S S N N K F K I D R N S G 2343 ATCGTTTACTCTTTAGATGACAGCTCCAACAACAAGTTTAAGATCGATCGTAATTCCGGC
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801 L V T T V E T M E K E T A V N S F T I V 2403 CTTGTAACAACTGTGGAAACTATGGAAAAAGAGACTGCTGTGAATTCGTTCACAATCGTT 821 V K A T D K G S P A L S G T V T A T I R 2463 GTGAAAGCAACCGACAAAGGAAGTCCAGCTCTGAGTGGAACTGTAACGGCTACCATCCGA 841 V S D G N D Q A P V F N P R E Y R Q K V 2523 GTATCTGATGGAAACGATCAAGCGCCAGTATTCAACCCCAGGGAATACAGGCAAAAAGTG 861 P E D S P P G F L V T Q V K A T D Q D E 2583 CCGGAGGATTCTCCCCCAGGATTCCTCGTCACGCAAGTGAAAGCTACCGACCAGGACGAG 881 G Y N A E L E F T I T A G N D P Y Q F Y 2643 GGATATAACGCAGAGCTCGAGTTCACCATTACGGCGGGCAATGACCCTTACCAGTTCTAC 901 I D P K T G E I L V S G M L D F D H G K 2703 ATCGACCCGAAAACAGGCGAAATACTTGTATCGGGTATGCTGGATTTTGACCATGGAAAG 921 K S Y N L T V M V S D R G V P P K Q A A 2763 AAGTCCTATAACCTGACAGTTATGGTTAGTGATCGCGGTGTACCACCTAAACAAGCTGCG 941 K P A F V Y I T I V D S N D N P P V F V 2823 AAACCAGCTTTTGTTTACATAACTATCGTGGATTCCAATGATAATCCGCCGGTCTTTGTG 961 P A E Y S I K V T E G T K P G D T V Q L 2883 CCTGCTGAATACAGCATTAAAGTCACCGAGGGTACAAAACCTGGAGACACTGTGCAACTG 981 V T A V D Q D T G T N A L F T F G I A D 2943 GTAACAGCGGTTGATCAGGACACGGGTACTAATGCTCTTTTTACGTTTGGCATCGCTGAC 1001 G D D A D M F G I R P D P K N S S I G F 3003 GGTGATGATGCTGATATGTTTGGTATAAGACCCGATCCCAAGAACAGCAGTATTGGGTTC 1021 I Y T V L Q L D R E T V P Q Y N L T V T 3063 ATTTACACGGTGCTGCAGTTGGATCGCGAAACCGTTCCTCAGTACAATCTCACCGTCACC 1041 A T D T G G L Q G V A V V R I T V L D T 3123 GCGACCGATACTGGCGGGCTCCAAGGCGTTGCTGTGGTGCGTATTACTGTCTTAGACACC 1061 N D N G P W F Q P R Y Y E G S I T V T S 3183 AACGATAACGGCCCGTGGTTCCAACCTCGCTACTATGAAGGTTCAATTACGGTGACTAGT 1081 D S N S Q Q E I T T V K V F D P D E P S 3243 GATTCGAACTCACAACAAGAAATTACCACAGTGAAAGTCTTTGACCCAGATGAGCCAAGC 1101 N G P P F S F S I E S T K P A T D A T R 3303 AATGGACCTCCGTTCAGCTTTTCGATTGAAAGTACAAAGCCTGCCACTGATGCAACTCGC 1121 F G L R K D P K E P Q T A N E V Y S I G 3363 TTTGGGTTGCGAAAAGATCCCAAAGAACCCCAAACCGCAAACGAGGTGTACTCAATCGGA 1141 A F T R Q V P E W E L T I K A I D S G K 3423 GCTTTCACGCGTCAAGTTCCTGAATGGGAACTGACAATCAAGGCTATTGATAGTGGAAAG 1161 P V A M F N S T L V F V W V V D D K N L 3483 CCGGTGGCCATGTTCAACTCGACGCTTGTGTTCGTTTGGGTTGTTGATGACAAGAACTTG 1181 N E P F D G A L T I I V N A Y D D K F A 3543 AACGAACCATTTGACGGAGCATTGACCATCATAGTAAACGCCTACGATGACAAATTTGCC 1201 G G I I G K A Y Y Q D V D Y M G D E N T 3603 GGCGGTATCATCGGAAAGGCCTACTACCAAGACGTGGACTACATGGGAGACGAGAACACA 1221 Y S M S E Q E Y F T L G E L T G D I S A 3663 TACTCTATGAGTGAGCAAGAGTATTTCACCTTGGGCGAACTTACTGGTGACATAAGTGCT
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1241 A A N I P V G L Y K F E I E V L E Q R L 3723 GCGGCAAACATTCCTGTGGGACTATACAAGTTCGAGATAGAGGTGCTTGAGCAACGGCTT 1261 R P N T N T F K T V T S S V S V I V Q S 3783 CGGCCAAACACCAACACTTTTAAAACCGTAACGTCTTCAGTATCGGTCATCGTGCAAAGT 1281 V P R A A I L Q S V A V Q I L A Y R R P 3843 GTGCCCAGGGCTGCGATTCTGCAAAGCGTGGCTGTGCAGATTCTCGCGTATCGCAGGCCG 1301 A L F V A D I Y T N F R Q K L A G I F G 3903 GCCCTCTTTGTGGCTGACATTTATACCAACTTCCGACAAAAGTTAGCTGGAATCTTCGGC 1321 V Q E A D I L I F S V Q R A P S K R V P 3963 GTTCAGGAAGCTGACATTTTGATCTTTAGCGTTCAAAGGGCTCCAAGCAAGCGTGTTCCT 1341 L A D V F G V E I Q L A V R S S G S S F 4023 CTCGCGGACGTGTTTGGAGTCGAGATTCAACTGGCTGTTCGTTCGTCGGGAAGTTCTTTC 1361 M D K M D V V R G I V E G K A E L E A L 4083 ATGGATAAGATGGATGTTGTCAGGGGAATAGTAGAAGGAAAGGCAGAACTAGAGGCATTG 1381 S L K I G D I G I D V C A A E R Q D V G 4143 AGCTTGAAGATCGGAGATATTGGTATTGATGTGTGTGCTGCGGAGCGACAAGATGTTGGC 1401 V C N N K V E A S S A F T V V S G D I G 4203 GTTTGCAACAATAAAGTGGAGGCTTCGTCAGCGTTCACTGTAGTCAGCGGTGACATTGGC 1421 Q I E P S K S S L T V V S I D I T L K A 4263 CAAATAGAACCGAGCAAGTCTAGTTTAACAGTAGTATCCATTGACATCACACTGAAAGCC 1441 V Y T S I L P P D I N C T T G T P C L H 4323 GTCTACACATCAATCCTTCCACCAGATATAAACTGCACAACAGGCACTCCCTGTCTACAT 1461 G G T C H N A V P K G I I C E C G R D Y 4383 GGGGGCACGTGTCACAACGCAGTTCCCAAGGGGATCATTTGCGAATGTGGACGAGATTAC 1481 L G P E C Q S T T R T F R G N S Y L W L 4443 CTTGGACCAGAGTGTCAGAGCACTACGAGAACGTTCAGAGGAAATTCATACTTGTGGCTT 1501 D K L A S Y E R S S I S L Q F M T G S A 4503 GATAAGCTCGCATCCTATGAACGGAGCTCGATTTCGCTACAGTTTATGACTGGCTCTGCC 1521 N G L L L Y Q G P L Y N G A N N G L P D 4563 AATGGCTTGTTGCTTTACCAAGGACCTCTGTACAATGGGGCCAACAATGGTCTTCCAGAT 1541 S I A L Y L V D G F A K L V I A L G P H 4623 TCGATTGCATTGTATTTGGTGGACGGATTTGCCAAACTGGTGATCGCCCTTGGGCCACAC 1561 P M T P L E L Y M N K G D R L D D R T W 4683 CCTATGACACCATTAGAGCTGTACATGAACAAGGGAGATCGTTTGGATGACAGGACGTGG 1581 H T V E V I R E R K K V V L R I D K C S 4743 CACACGGTTGAGGTCATTCGAGAACGCAAGAAAGTTGTGCTGAGGATCGACAAGTGTTCC 1601 Y S K I V E D Y G Q I V E D R S S C E I 4803 TATTCGAAAATTGTAGAGGATTATGGTCAAATTGTCGAGGACAGGTCCTCATGCGAAATC 1621 K G E I W G S A I Y L N G F G P L Q I G 4863 AAGGGAGAAATCTGGGGCTCCGCGATCTACTTGAATGGTTTTGGTCCTCTTCAAATTGGT 1641 G V E N S I S D M K I N F T G F S G C I 4923 GGCGTGGAAAACTCGATCAGCGATATGAAGATAAACTTCACTGGTTTCTCCGGCTGCATT 1661 R N I Y N N G R M Y D L F N P L K E V N
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4983 CGAAATATTTACAACAATGGCAGGATGTACGATTTGTTTAATCCATTGAAAGAAGTCAAC 1681 T E L G C R L N N Q C P N C N G R G Y C 5043 ACGGAGTTGGGATGCCGGCTCAATAACCAGTGTCCAAACTGCAATGGGCGTGGCTACTGC 1701 E P F W N Y A I C V C D L G F G G A N C 5103 GAACCCTTCTGGAATTATGCTATCTGTGTGTGTGATCTTGGTTTCGGTGGAGCTAATTGT 1721 D S R T Q A N W Y R A N S F T Q Y R V K 5163 GACTCAAGGACACAAGCCAACTGGTATCGTGCAAATAGCTTTACCCAGTACCGCGTAAAA 1741 Q V K R K R R E L V P A P V S M A N E F 5223 CAAGTTAAGAGAAAGCGTCGTGAACTTGTTCCAGCGCCGGTGTCGATGGCAAATGAATTC 1761 Y T N I A L Q V R V S P N S S N V V I F 5283 TACACAAACATTGCCTTGCAAGTCAGGGTGTCTCCGAATTCATCTAATGTTGTCATTTTC 1781 L A S N S L G T E F N R I D V K N H V L 5343 TTAGCGTCCAACAGTTTGGGAACTGAATTTAACAGGATTGATGTCAAGAATCATGTGTTG 1801 R Y A F R L G D R M K I L K I P Q L N V 5403 CGTTACGCGTTCCGATTGGGTGACCGTATGAAGATCCTCAAGATCCCTCAACTGAACGTG 1821 T D D K Y H T V I V K R E G N R A S L Q 5463 ACGGACGATAAGTATCACACTGTGATCGTGAAGAGGGAGGGTAACCGCGCCAGCCTTCAG 1841 I D Y R G K V E G T T G G L H K L L N M 5523 ATCGATTATAGGGGCAAAGTGGAAGGAACAACAGGAGGACTTCACAAGCTGCTAAACATG 1861 G G G S F F T G G L P N I T E V R V V E 5583 GGTGGAGGAAGTTTCTTTACTGGGGGCTTGCCTAATATTACTGAGGTTAGGGTAGTGGAA 1881 A F V N S G G N A V L R T A E G N I I S 5643 GCGTTTGTTAATAGCGGAGGCAACGCTGTCCTGCGCACAGCTGAGGGCAACATCATCTCA 1901 S G M G S A Y T G I G S Y M S N V I T V 5703 AGCGGGATGGGATCCGCATACACTGGCATCGGTAGTTACATGAGCAATGTAATTACTGTC 1921 N N F G G V D V S Y G V S G A P H V Q R 5763 AATAACTTTGGAGGCGTTGATGTATCGTACGGAGTTAGCGGTGCGCCACACGTTCAGCGG 1941 T I K T K S S I F T G S S G V I T R I S 5823 ACCATCAAAACAAAAAGCAGCATCTTTACAGGAAGCAGCGGTGTGATAACCAGAATCAGT 1961 V S R G H S V E F M N S R F F T R K T K 5883 GTCAGTAGGGGTCATAGTGTGGAATTCATGAACAGTCGTTTCTTTACACGCAAGACCAAG 1981 Q K Q K V I I S S S G G S V S G G S G G 5943 CAGAAGCAAAAGGTAATCATCAGTTCATCTGGAGGTTCAGTGTCCGGCGGAAGTGGAGGT 2001 A S G G S G G A S G S G G S V G V S G G 6003 GCCTCGGGAGGAAGTGGTGGTGCTTCAGGCTCGGGCGGAAGTGTTGGTGTATCTGGAGGA 2021 G G A S V G G S I L G S S A S M D T K G 6063 GGTGGAGCTTCCGTCGGAGGCAGTATATTGGGAAGTAGCGCATCCATGGATACAAAAGGC 2041 N L R S Y G S G F G T W T I A G A G P N 6123 AACCTTAGATCCTATGGCAGTGGTTTCGGGACTTGGACCATCGCGGGCGCAGGCCCTAAT 2061 E A G D V Q V I G D F G G C T A S N S Y 6183 GAAGCTGGAGATGTTCAAGTTATAGGTGATTTTGGAGGGTGCACTGCATCAAACAGTTAC 2081 N G L D L D S H P T I E A R R Q N V E F 6243 AATGGTCTGGATTTGGACAGCCATCCCACTATAGAAGCGCGCCGTCAGAATGTTGAGTTC
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2101 P C P C G S N F C R H G G T C V S A D P 6303 CCCTGTCCGTGTGGCTCAAACTTCTGTCGTCACGGAGGCACTTGTGTAAGCGCCGACCCG 2121 P Y C L C P V G W S G P V C E S I V K D 6363 CCTTATTGTTTGTGCCCAGTGGGCTGGAGCGGTCCAGTATGCGAGAGTATCGTCAAAGAT 2141 P R P G Q R P S S R W A N P A V I A C I 6423 CCAAGGCCAGGCCAACGGCCGTCAAGCAGATGGGCAAATCCCGCAGTCATCGCTTGCATT 2161 L V I L L A I L V I I G A V L L K R R P 6483 CTCGTCATTCTCCTGGCAATTCTCGTCATAATTGGCGCCGTACTTCTAAAGCGCCGTCCC 2181 Q P A V V A V V E D G H V H D N V R P Y 6543 CAGCCAGCCGTTGTAGCCGTCGTCGAAGACGGACATGTACACGACAACGTGCGTCCTTAC 2201 H D E G A G E E D N F G Y D I S T L M K 6603 CACGACGAAGGCGCAGGCGAGGAGGACAACTTTGGCTACGACATTTCAACGCTTATGAAG 2221 Y T Y V E N G V A G T G G V G H G K F K 6663 TACACTTACGTGGAAAACGGTGTGGCAGGCACGGGCGGTGTGGGCCATGGAAAATTCAAG 2241 N G G S S G E E E F T A T E T K P L L Q 6723 AACGGTGGTAGTAGCGGTGAAGAAGAGTTTACTGCGACCGAGACTAAGCCGTTGTTACAA 2261 G A M P D D D L H F K T T T I T K R K V 6783 GGTGCAATGCCCGACGATGACTTGCATTTCAAGACGACGACCATCACTAAGCGAAAGGTG 2281 V H P D S I D V K Q F I D T R V S E A D 6843 GTACATCCCGATAGCATCGACGTTAAGCAGTTTATTGATACACGTGTTTCTGAGGCGGAT 2301 G E Y I L S I D E L H I Y R Y E G D D S 6903 GGCGAGTACATCCTGTCCATCGACGAACTACATATTTATCGTTATGAGGGAGATGATTCG 2321 D V D D L S E L G D S D E E P D E E E E 6963 GATGTGGATGATTTGAGTGAATTGGGAGACAGCGACGAAGAACCCGACGAGGAAGAAGAG 2341 Q E F A F L Q D W G K K F D N L N R I F 7023 CAAGAATTTGCTTTCTTGCAAGATTGGGGCAAGAAATTCGATAACCTCAATCGCATTTTC 2361 N E D E - F M F S D E L C F V H N V C A 7083 AACGAGGACGAGTAGTTCATGTTCAGCGATGAACTATGTTTTGTACATAATGTTTGTGCT 2381 F - N H F - P Q I S T P G A N T A F A Y 7143 TTCTAGAATCATTTCTAGCCACAGATTAGTACACCGGGTGCAAATACGGCCTTCGCATAC 2401 K K N F F - V N S S R K W T - N N R C K 7203 AAGAAGAACTTTTTCTAGGTAAATTCTTCTCGTAAGTGGACTTAGAATAACCGTTGTAAA 2421 F F L K R K A T L V L C G L H H L V - M 7263 TTTTTTTTAAAACGGAAGGCTACACTTGTCTTGTGCGGCTTACACCACCTCGTGTAAATG 2441 P R I S Y L T S F G T V D V I G - T - A 7323 CCTAGAATTTCATATTTGACATCATTTGGAACGGTGGACGTGATTGGTTAAACTTAAGCC 2461 K H F V - H K A F A K R L S G T L W - H 7383 AAACATTTCGTTTAGCATAAAGCATTTGCAAAAAGATTAAGTGGAACACTCTGGTAACAT 2481 E I K N F Y F V C G A F D N T L K E N - 7443 GAAATTAAGAACTTTTATTTTGTGTGTGGAGCATTTGATAACACTCTCAAAGAGAACTAG 2501 L S S Y Y T G L V L F I A M Y M Q S L V 7503 CTGTCTAGTTATTACACAGGGTTGGTTCTTTTTATCGCAATGTACATGCAGTCTTTGGTT 2521 F R V D N V I S L E I F Q W Q F I L A - 7563 TTTCGTGTTGATAACGTAATTTCCCTAGAAATATTCCAATGGCAGTTTATTCTTGCATAA
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2541 W K C I L L - M R F S H C C R I Y F V W 7623 TGGAAATGCATTCTGCTATAAATGCGATTTTCTCACTGTTGTAGAATATATTTTGTATGG 2561 T L L I A F - Y R Y F Y S G E L Y F A S 7683 ACATTGTTAATAGCCTTTTAGTACCGTTATTTCTATTCTGGTGAGTTGTATTTCGCCAGC 2581 F K C - A V V N - M H I A C D P L P S W 7743 TTCAAATGTTAAGCCGTAGTTAACTGAATGCATATTGCATGTGATCCCTTGCCATCATGG 2601 R T - S T Q - A E H C A R S Q S F F G V 7803 CGGACCTAAAGTACTCAATAGGCAGAACACTGCGCTCGTTCACAGTCCTTTTTCGGCGTC 2621 I V S K K R - S R A - E S D V F E A Q T 7863 ATCGTCTCCAAGAAGAGATAATCGAGAGCTTAAGAAAGTGACGTTTTTGAGGCACAGACG 2641 E T R I E H F G H Q D S G P S Q I F K P 7923 GAGACCAGAATTGAACATTTCGGACACCAGGACAGTGGTCCCTCCCAGATTTTCAAACCA 2661 V V P R I F Y N I N V V V K R Q L K E E 7983 GTTGTCCCTAGGATATTTTACAATATAAATGTGGTCGTTAAAAGACAACTTAAAGAGGAA 2681 N S S L P L S V R G S K T S - A Q A R - 8043 AACAGCTCACTTCCGCTTTCCGTTCGTGGCTCAAAAACGTCGTGAGCTCAAGCTCGCTAA 2701 L Y Q C V A N I K P E I K C F H - F - S 8103 TTATACCAGTGCGTCGCAAACATCAAACCTGAAATTAAATGTTTTCATTGATTTTAAAGC 2721 Q E Y L L I L I K I S R - D L S R - P S 8163 CAAGAATATTTACTTATCCTAATTAAAATTTCGCGTTAAGATCTTTCTCGATAGCCGTCT 2741 N K - - T S - D S I N F S N F - R T R A 8223 AACAAGTGATGAACGAGTTAAGATTCAATTAATTTCAGTAATTTTTGACGAACGAGAGCT 2761 K I - F V V F S V S - Y P F L I V F H - 8283 AAGATTTGATTCGTCGTTTTCTCCGTATCTTGATATCCTTTTTTAATAGTTTTCCACTGA 2781 C R I Y T V F W K F K S A V - R E K P A 8343 TGTAGAATTTATACTGTTTTCTGGAAATTCAAATCTGCAGTTTGAAGGGAAAAACCGGCA 2801 L G R Q D A F Y T Q Y A F H C S T A C - 8403 CTCGGAAGGCAAGATGCATTTTATACCCAGTATGCATTCCATTGTTCTACAGCTTGTTGA 2821 A S E I V I A C L D G N V H - L G L I C 8463 GCAAGTGAAATAGTTATTGCATGTTTAGATGGAAATGTACACTAGTTGGGGCTAATATGC 2841 R H L H S L T Y C C S V V R Y F S M F L 8523 CGCCACCTTCATTCGTTGACGTATTGTTGCTCGGTCGTTAGATATTTTTCTATGTTCCTT 2861 L L C V I I - K Y M K P R L L V K I L C 8583 CTTTTATGTGTAATAATTTAAAAGTACATGAAACCTAGATTGCTAGTCAAGATACTTTGC 2881 - A F - N N Y E K G F - I 8643 TGAGCTTTTTAGAATAACTATGAAAAAGGCTTTTGAATA
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Supplementary Figure 5.2
Supplementary Figure 5.2 Boxshade Alignment of Representative Dachsous C-‐terminal domains. Conservation of the Catenin Binding Domain (CBD; Blue) but not the Juxta-‐Membrane Domain (JMD) distinguishes the Dachsous cytoplasmic region from that of TypeI-‐IV / “classical” cadherins. The Extracellular region in each of the proteins included in the above alignment contains only Cadherin repeat (EC) domains consistent with the canonical structure of Dachsous. Shading demonstrates >50% consensus to AmDachsous (Black –conservation, Grey – conservative substitution).
Genbank Accesions: Dm_Ds NP_523446.2; Mm_Ds1 NP_001156415.1; Dr_Ds XP_001921284.2
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Supplementary Figure 5.3
Supplementary Figure 5.3 Multiple sequence alignment of cadherin cytoplasmic domains used for maximum likelihood analysis presented in Figure 5.4. Analysis is based on the highly conserved Juxta-‐Membrane Domain (JMD) and Catenin Binding Domain (CBD). Shading corresponds to consensus of 30% or more. Analysis is based on 148 positions of 29 sequences.
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(Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum
Genbank accession numbers: Hs_CDH1 AAI46663.1; Mm_Cdh1 AAH98501.1; Hs_CDH9 AAI09385.1; Mm_Cdh9 NP_033999.1; Bm1-‐Cadherin BAD91049.1; Gb1-‐Cadherin BAD91050.1; Dm_shg NP_476722.1 ; Af1-‐Cadherin BAD91054.1; Fc1-‐Cadherin BAD91052.1; Bm2-‐Cadherin BAD91048.1; Gb2-‐Cadherin BAD91051.1 ; Dm_CadN AAZ66476.1; Af2-‐Cadherin BAD91055.1; LvG-Cadherin AAC06341.1; SpG-‐Cadherin XP_001175592.1; Ap-‐Cadherin BAC06834.1; SE-Cadherin BAC06836.1; Bs-‐Cadherin AAB88396.1; 1_Ci0100151854 NP_001121583.1; Cj-‐Cadherin BAD91056.1; LE-Cadherin BAD91057.1; At-‐Cadherin BAD91058.1; Dm_cadN2 ABI31322.2
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Supplementary Figure 5.4
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Supplementary Figure 5.4 Boxshade alignment of AmFlamingo protein with representative metazoan Flamingo (CELSR) orthologues demonstrates the degree of conservation. Highly conserved regions correlate with functional domains (coloured boxes). Although the current model of AmFlamingo is truncated at the N-‐terminus, all diagnostic C-‐terminal domains are present supporting the designation of this model as the Acropora orthologue of Flamingo, which was initially established by JCUSMART annotation. Shading demonstrates >50% consensus to AmFlamingo (Black –conservation, Grey – conservative substitution). Signal sequences have been removed from the N-‐terminus of each protein.
Genbank Accesions: Mm_CELSR2 NP_001004177.2; Dr_CELSR1a XP_002661517.2; Dm_Flamingo NP_724962; Nv_Flamingo JGI proteinID 84228
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Supplementary Figure 5.5 AmVangl 1 -----MPSHRSDRGHRDRDHDH------------------------NDKRERQRLADDTG Nv_Vangl 1 ------------------------------------------------------------ Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 1 ------------------------------------------------------------ Dm_Vang 1 MENESVKSEHSGRSRRSRNHNNNGGGGGGGGGGGGGSVNNGYHRERDRSRHSHRSTHSSK Mm_Vangl1 1 ---MDTESTYSGYSYYSSHSKKSHRQGER-TRERHKSPRNKDGRGSEKSVTIQAPAGEPL Dr_Vangl1 1 ---MDTDSIHSGYSHHSNRSRGSNKQSERSSRDRHKPHSRDSSSRSDKNVTISATSVQPQ Xl_Vangl1 1 ---MDTESNHSGYSHHS--------------RGRQQRERRHDGQKS-----VNVTVGQPL AmVangl 32 SQEILDEG---------------------IIQVQVIPQDDNWGETATSITETATSIVT-L Nv_Vangl 1 -------------------------------------QDDNWGETATTITETATSIVT-L Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 1 -MDDFDDG---------------------VIEVQVIPQDDNWGETATTITETATSVYSDL Dm_Vang 61 SAKGFQRGDMAPYQTSVNMTGDGSHDGQEVIEVQILPQDENWGENTTAVTGNTSEQSISM Mm_Vangl1 57 LANDSAR------------------------TGAEEVQDDNWGETTTAITGTSEHSIS-Q Dr_Vangl1 58 QPDQSS--------------------------APTDAQDDNWGETTTAVTGTSDHSLS-Q Xl_Vangl1 39 LGEDSGRG-----------------------EEDEEGQDDRWGETTTAVT--SERSAS-F AmVangl 70 SETSLSEVGKDKRSFSCSFLR-HIKLVPAAILSICAVVSPILFVVFPLV----------- Nv_Vangl 23 SEGDLSEIGDEKKGLAFGVCR-HFKLIPAAIISLIALASPILFLVIPVV----------- Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 39 DDTDPFGEEIEKHRFQCPNSKQALNILLSVILALMAVVSPIAFVIIPNV----------- Dm_Vang 121 EDINNMWHRESDKGFSFACRR-YVESSFYFLLGCGAFFSPVAMVVMPYVGFFPSAFDHPE Mm_Vangl1 92 EDIARISKD-MEDSVG-LDCKRYLGLTVASFLGLLVFLTPIAFILLPQILWR-------- Dr_Vangl1 91 EDLAGFGKD-TEGPVEKLNCIRFLPLALTLLLGLLVLVTPLSFLILPQLMWP-------- Xl_Vangl1 73 EDLAPVGTS-RIPDTVKPNTWIMIGFYLSCALGLFAILTPPAFIVLPQVFWG-------- AmVangl 118 -TWKP----EPCGYDCDGVFISFAVKELLLVIAIWALYFRRSRATMPRIFVLRVGMMLLG Nv_Vangl 71 -LWKP----PMCGIECDGLFLSLAVKELILVVGIWALYFRKSRVTMPRVFVLRVGVLVLA Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 88 -SSSLKVTGEECGSVCEGLYITIAIKEIVLIFGLWALYIRPNKVDLPRLNVFKVGMMVLV Dm_Vang 180 ITQTVRTQLLACSEQCKGQLVSLAARLLLLAIGLWAVFMRRTSATMPRIFLYRALVLLLV Mm_Vangl1 142 ------EELKPCGAICEGLLISVSFKLLILLIGTWALFFRKQRADVPRVFVFRALLLVLI Dr_Vangl1 142 ------ERLQTCGTACEGLFLSLAFKLLILLLAGWALFMRPTRASLPGIAVFRALLGLLT Xl_Vangl1 124 ------SQLEPCGVVCEGLYISLAFKLLLLFLASWAVFLRPPRCDLPRLVEFHALLIVLL AmVangl 173 FVVIVCFWLFYGLRIIA---------KQTSQFSDILRFAVNFTDTMLFLHYASLVVLWVR Nv_Vangl 126 FLVLVCYWLFYGLRIIA---------KRGQDFGEILKFAVTLVDSLLFLHYLSVIVLWLR Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 147 YVSIICFWLFYSIRMIG---------NGTNQY-LTVSFASSFLDAMLFLHYMALVLMWIR Dm_Vang 240 TICTFAYWLFYIVQVTNGAKIVVETGGDAVDYKSLVGYATNFVDTLLFIHYVAVVLLELR Mm_Vangl1 196 FLFVVSYWLFYGVRILD---------SRDQNYKDIVQYAVSLVDALLFIHYLAIVLLELR Dr_Vangl1 196 LLLLLSYWLFFGVRILD---------SQDDNYQGIVQFAVSLVDALLFIHYLAVVLLEIR Xl_Vangl1 178 FFFLSSYWLFYGVRVLG---------PQEKNLLGVVEYAVSLVDALIFIHYLALILLELR AmVangl 224 QTDPLFTLKVIRSTDGESKFYNIGPLSIQRTAAFVLEQYYKDFPEYNPFLMQT---PART Nv_Vangl 177 QSEPMFTLKVIRSTDGVNKFYNVGLLSIQRAAIYVLEQYYKDFPEYNPYMMSV---PARS Hm_Vangl 1 -HENIYNVSVIRNVDGSRKHYMIGQCSIQKAAVNVLEKYYIDFNEYNPYLPRP---TSRS Ch_Vangl 197 PMEKVYTVSIIRNVDGMRRYYNIGQSSIQKAAVFCLERYYIDFTEYNPYMPRP---QSRT Dm_Vang 300 HQQPCYYIKIIRSPDGVSRSYMLGQLSIQRAAVWVLQHYYVDFPIFNPYLERIPISVSKS Mm_Vangl1 247 QLQPMFTLQVVRSTDGESRFYSLGHLSIQRAALVVLENYYKDFTIYNP-NLLT---ASKF Dr_Vangl1 247 HLQPCFSLCVVRSTDGETHHYNMGQLSIQRAALVTLEHYYKDFTVHNP-ALLT---AAKS Xl_Vangl1 229 QLQPFFYLKVMRSSDGEMRFYSLGTLSIQRAAMFVLENYYKDFPVFRP-DPPV---VRKR AmVangl 281 SHKQFS-SLTFYDIDGK---QND--KVNPRARAILTAASTRRRDPARNDRFYEEAEFERK Nv_Vangl 234 SAKQFS-TLKFYDIDGK---LQDNSKVNPRTRAIITASSQGRRNPGRNDRFYEEAEFERK Hm_Vangl 57 KINKFS-NIKFYDLDNKMDMGNGKNFSQQASKAVIAAAALGRRKEGRNDRFYEELEIDRR Ch_Vangl 254 KINKLA-GLKIYDLDGK---GDG-TLTQQASKAFIAAAAAGRRKEGRNDRFYEEQELDRR Dm_Vang 360 QRNKISNSFKYYEVDGV-----SNSQQQSQSRAVLAANAR-RRDSSHNERFYEEHEYERR Mm_Vangl1 303 RAAKHMAGLKVYNVDGP-------SNNATGQSRAMIAAAARRRDSSHNELYYEEAEHERR Dr_Vangl1 303 RAAKHLAGLKVYNVDGAG------SDAATAQSRAKMAAAARQRDTSHNELYYEEAEHDRR
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Xl_Vangl1 285 --TRHNNHPQVYSVDGP-------NSSTVSQSQTLISTSTN-----YKERYYEEAEHARK AmVangl 335 IKRRKARLFNACEEAFGHIKRVQLE---RGPCVPMDPEETAQSLFPSFARPLQKYLRTVR Nv_Vangl 290 LKRRKARLVATCEEAFGHIKRVQLE---RGPSVPMDPEETAQSLFPSFARPLQKYLRTVK Hm_Vangl 116 VRKRKARLVAAAEEAFGHIARLNAFDTSKKANGSMNPDEAAQAIFPTLARPLQKYLRTTR Ch_Vangl 309 IRKRKARLVAAAEEAFGHVARLNAFDSSKKADGSMDPEEAAQAVFPTLARPLQKYLRTTR Dm_Vang 414 VKKRRARLITAAEEAFTHIKRIHNEP---APALPLDPQEAASAVFPSMARALQKYLRVTR Mm_Vangl1 356 VKKRRARLVVAVEEAFIHIQRLQAEEQQKSPGEVMDPREAAQAIFPSMARALQKYLRTTR Dr_Vangl1 357 VRKRKARLVVAVEEAFTHVRRLQDEEKKKPPGDEMDPREAAQAIFPSMARALQKYLRTTR Xl_Vangl1 331 VRRRKARLVMSVHDAFSQLKRLVQQDEEWKLPNTLHPREAAQSIFPLIAQSLQRYLRSTQ AmVangl 392 LHHRYTMDGIIKHLAHCLTFDMTPKAFLERYLKDQPCGEFT-TKRRCQSWSLVCEEQVTK Nv_Vangl 347 QHHRHNMDAILKHLAHCLTFDMSPKAFLERYLNDQPCIEYTGASVGPQSWSLVCEEQVTS Hm_Vangl 176 QQLHYPLESIMKHLAHCIMFDMSAR----------------------------------- Ch_Vangl 369 QQLYYPLESILKHLAHCISYELSSKAFIERYTCDQPCISYV-GYDGRQEWTLVTDSSPTR Dm_Vang 471 QQPRHTFESILKHLAHCLKHDLSPRAFLEPYLTESPVMQSEKERRWVQSWSLICDEIVSR Mm_Vangl1 416 QQHYHSMESILQHLAFCITNSMTPKAFLERYLSAGPTLQYDKDRWLSTQWRLISEEAVTN Dr_Vangl1 417 QQHCHSMDSIQAHLAFCITNNMTPKAFLESYLTAGPTLQYGRESSQTRHWTLVSEASVTS Xl_Vangl1 391 QAHLHSMEGIIQHLTLCLTHRMSPQAFLEQYLHPGPPVQYPSNPYG--VWTLVSEESVTS AmVangl 451 TLSSSTVFQLKGDDLSLVVTVRRLPYLDISEDEFDFESNKFVLRLNSETSV Nv_Vangl 407 GLSNSTVFQLKTEVLSLVVTVNNIPHFVVDEDEFDFENNKFVLKLNSETSV Hm_Vangl --------------------------------------------------- Ch_Vangl 428 QLNEGTVFQLKHEDISLVIAVSKLPVFAMSELPFNGDSNRFVFRLNSETSV Dm_Vang 531 PIGNECTFQLIQNDVSLMVTVHKLPHFNLAEEVVDPKSNKFVLKLNSETSV Mm_Vangl1 476 GLRDGIVFVLKCLDFSLVVNVKKIPFIVLSEEFIDPKSHKFVLRLQSETSV Dr_Vangl1 477 PLRNGSEFQLKSSDFSLVVTSKTIPHLKLSEEYVHPKSHKFVLQLQSETSV Xl_Vangl1 449 PLRSDLTFCLQCSDTQLLVTVCGIPFLKLSETFISPNSHRLIVSSKPETNL
Supplementary Figure 5.5 Boxshade alignment of full length Am_Van Gogh Like (AmVangl) protein with representative metazoan Van Gogh orthologues demonstrates a reasonable degree of sequence conservation. Shading demonstrates >50% consensus to AmVangl (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig17842 as the Acropora orthologue of Van Gogh, which was initially established by BLASTp analysis (E-‐value = 1E-‐100).
Genbank Accesions: Mm_Vangl1 NP_808213.2; Dr_Vangl1 NP_991313.1; Xl_Vangl1 NP_001089844.1; Dm_Vang NP_477177.1
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Supplementary Figure 5.6 Am_Dsh 1 -------MEETKVIYHIDDEDTPYLVKLPKSPTDVTLSDFKNVL-NRPS--YKFFFKSMD Nv_Dsh 1 -------MDETKVIYHIDDEDTPYLVKLGKTPDIVTLGDFKNVL-NRPN--YKFFFKSMD Xt_Dsh1 1 -------MAETRIIYHIDEEETPYLVKLPVPPEKVTLADFKNVLSNRPVHHYKFFFKSMD Mm_Dsh1 1 -------MAETKIIYHMDEEETPYLVKLPVAPERVTLADFKNVLSNRPVHAYKFFFKSMD Dr_Dsh1 1 -------MAETKIIYHIDEEETPYLVKLSVAPEKVTLADFKNVLSNRPVNSYKFFFKSMD Dm_Dsh 1 MDADRGGGQETKVIYHIDDETTPYLVKIPIPSAQVTLRDFKLVL-NKQNNNYKYFFKSMD Ch_Dsh 1 -----MAEKETKIIYHVDDEETPYLVKIPKPPDQVTLGDFKSVI-NRPN--FKFFFKSMD Hv_Dsh 1 ----MSVPDETKIIYHVDDEETPYLVKIPKSPSLVTLGDFKNVI-NRPN--YRFFFKSMD Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 51 DDFGVVKEEISDEEMNLPCFNGRVVAWLVTSDTSSVSDTG--RDDQQSVISG-TSDLHPP Nv_Dsh 51 DDFGVVKEEISDDETHLPCFNGRVVAWLVTSDTSSVSGGGDLASDQQSIISASPSDLHVP Xt_Dsh1 54 QDFGVVKEEISDDNAKLPCFNGRVVSWLVLAE-SSHSDGG-------SQSTESRTDLPLP Mm_Dsh1 54 QDFGVVKEEIFDDNAKLPCFNGRVVSWLVLAE-GAHSDAG-------SQGTDSHTDLPPP Dr_Dsh1 54 QDFGVVKEEVSDDNAKLPCFNGRVVSWLVLAE-SSHTDGM-------SVCTDSHTEHPPP Dm_Dsh 60 ADFGVVKEEIADDSTILPCFNGRVVSWLVSADGTNQSDNC-------SELPTSECELGMG Ch_Dsh 53 DDFGVVKEEIIDDDAPLPCFNGRVVSWVVPPE-DGSCDGQ-------SQHSGDGIFVPVQ Hv_Dsh 54 DDFGVVKEEIIDDDTILPCFNGRVVSWVVPPEEGSNDCNS-------------------T Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 108 MPRTVGIGDSRPPSFHHGGQSTAGDFDSEAESTVSSRRG--SRRREKHSIN-RNEVIHG- Nv_Dsh 111 VQRTGGIGDSRPPSFHHGGHSTAGDFDSEVDSTISSRRGGSSRRKERHSSRGIGHRSHR- Xt_Dsh1 106 IERTGGIGDSRPPSFHPNASSSRDGLDNETGTDSVVSHRRDRHRRKNRETHDDVPRINGH Mm_Dsh1 106 LERTGGIGDSRPPSFHPNVASSRDGMDNETGTESMVSHRRERARRRNR---DEAARTNGH Dr_Dsh1 106 LERTGGIGDSRPPSFHANAVNSRDGLDTETGSEPVLRHRRERERERTRRRRDDSERV--- Dm_Dsh 113 LTNRKLQQQQQQHQQQQQQQQQQHQQQQQQQQQQVQPVQLAQQQQQQVLHHQKMMGNP-- Ch_Dsh 105 SSNSNISRSSTMRSKERVQDSDAESIVSRRSSRSRSSRKYESDADRRSRRSHREHRN--- Hv_Dsh 95 NSGESALKQGRSNVSKKDRFPDNESVCSHKSVRSNRSRRLDTENENRSKVRNHRDHRSD- Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 164 ----RRRHESDRYESAS-VMSSDIETTSYQDSSDDQSSVSRFSSTTEGSQ--LLRGRHRR Nv_Dsh 170 ----SGVRQGERFDTTS-TMSSDIETTSYMDSSDDQ---SRFSSTTEGSH--LLSGRHRR Xt_Dsh1 166 PKLDRIRDPGG-YDSASTVMSSELESSSFVD-SDEDENTSRLSSSTEQSTSSRLIRKHKR Mm_Dsh1 163 PRGDRRRDLGLPPDSASTVLSSELESSSFID-SDEEDNTSRLSSSTEQSTSSRLVRKHKC Dr_Dsh1 163 -----VRDSAMGCDSGS-IMSSELESSSFID-SEDEEDASRLSSSTEQSSSFQLMKRHKR Dm_Dsh 171 ----LLQPPPLTYQSAS-VLSSDLDSTSLFG-TESELTLDRDMTDYSSVQ--RLQVRKKP Ch_Dsh 162 ---------YDQYDSAS-MMSSDLETTSFVD-SEEESQMS---SATESSR--YVGGNKRR Hv_Dsh 154 ------------FDSSS-IMSSDLETTSFVD-SDEESRISSTTENSKYGG-----ARQKR Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 217 RRRRPRMPRVQRCSSFSTITESTMSLNIITVTLNMEKVNFLGISIVGQSNKRGDGGIYVG Nv_Dsh 220 RRRRPRMPRVQRCSSFSTITESTMSLNIITITLNMDKVNFLGISIVGQSNKKGDGGIYVG Xt_Dsh1 224 RRRKQKMRQIDRSSSFSSITDSTMSLNIITVTLNMEKYNFLGISIVGQSNDRGDGGIYIG Mm_Dsh1 222 RRRKQRLRQTDRASSFSSITDSTMSLNIITVTLNMERHHFLGISIVGQSNDRGDGGIYIG Dr_Dsh1 216 RRRRHKVAKIDRSSSFSSITDSTMSLNIITVTLNMEKYNFLGISIVGQSNDRGDGGIYIG Dm_Dsh 223 QRRKKRAPSMSRTSSYSSITDSTMSLNIITVSINMEAVNFLGISIVGQSNRGGDGGIYVG Ch_Dsh 206 RRRRQRMPRVERCSS--------------------------------------------- Hv_Dsh 195 RRRKQRMPRVERCSSFSTITESTMSLNIITVVLNMDKINFLGISIVGQANKKGDGGIYVG Hm_Dsh 1 ------------------IYE----------------------------KKK-------- Am_Dsh 277 SIMKGGAVDLDGRIEPGDMLLQVNDVNFENMSNDDAVRVLREMVHKPGPITLTVAKCWDP Nv_Dsh 280 SVMKGGAVDLDGRVEPGDMLLQVNDVNFENMSNDDAVRVLREMVHKPGPITLTVAKCWDP Xt_Dsh1 284 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRVLREIVSKPGPISLTVAKCWDP Mm_Dsh1 282 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRVLREIVSQTGPISLTVAKCWDP Dr_Dsh1 276 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRILREIVSKNGPISLTVAKCWDP Dm_Dsh 283 SIMKGGAVALDGRIEPGDMILQVNDVNFENMTNDEAVRVLREVVQKPGPIKLVVAKCWDP Ch_Dsh ------------------------------------------------------------ Hv_Dsh 255 SVMKGGAVDADGRIEPGDMILAVGDVNFENMSNDDAVRVLRECVHKPGPIMLTVAKCWDP Hm_Dsh 7 SVLQS-------------TIPGVGDVNFENMSNDDAVRXPQRVRTQTWSHHADGAKCWDP Am_Dsh 337 TPKGYFTLPPSEPVRPIDTSAWVQHTTAMNQFGN---PVVQYGKQPNSQSITTMTSTSSS Nv_Dsh 340 TPKGYFTLPHSDPVRPIDTSAWVQHTTAMNQFAAQQGQFVPPAKPGNSQSLSTMTSTSSS Xt_Dsh1 344 TPRSYFTIPRAEPVRPIDPAAWITHTSALTGAYPRY------------------------ Mm_Dsh1 342 TPRSYFTIPRADPVRPIDPAAWLSHTAALTGALPRYGTSPCS-------------SAITR Dr_Dsh1 336 SPRSYFTIPRAEPVRPIDPAAWISHTTALTGSYPQN------------------------ Dm_Dsh 343 NPKGYFTIPRTEPVRPIDPGAWVAHTQALTSHDSIIAD---------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 315 NPKGYFTVPRNDVTRPIDPAAWMQHSEAVRASGGLLGGRTGS---------PSMSTMTST Hm_Dsh 54 NPKGYFTVPRNDVTRPIDPQLGCNIPEAVRASGGLLGGRTGS---------PSMSTMTST
Appendix A Supplementary Material
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Am_Dsh 394 MTSSLPESDIPGYTDIPDGEGLSLSTDMTTVVKAMAAPDSGLDVRDRMWLKITIPNAFIG Nv_Dsh 400 MTSSLPESDR--YTEIPEGEALTINTDMSTVVKAMAAPDSGLDIRDRMWLKITIPNAFIG Xt_Dsh1 380 --------------EQEDSPLSV-KSDMATIVKVMQLPDSGLEIRDRMWLKITISNAVIG Mm_Dsh1 389 TSSSSLTSSVPGAPQLEEAPLTV-KSDMSAIVRVMQLPDSGLEIRDRMWLKITIANAVIG Dr_Dsh1 372 --------------EFDELPLTMGKTDMATIVKVMQLPDSGLEIRDRMWLKITIANAVIG Dm_Dsh 381 --------------IAEPIKERLDQNNLEEIVKAMTKPDSGLEIRDRMWLKITIPNAFIG Ch_Dsh ------------------------------------------------------------ Hv_Dsh 366 SDSLTSSIPESDRYIDHSDLGLSIKTDMNTVIKVMASPDSGLVVRDRMWLKITIPNAFIG Hm_Dsh 105 SDSLTSSIPESDRYIDHSDLGLSIKTDMNTVIKVMASPDSGLVVRDRMWLKITIPNAFIG Am_Dsh 454 SDVVDWLYSRVDGFQDRREARKYACNLLKSGFIRHTVNKITFSEQCYYVFGDLCGNMAAL Nv_Dsh 458 SDVVDWLYTHVEGFMDRREARKYACNLLKAGYIRHTVNKITFSEQCYYVFGDLCGNLAAL Xt_Dsh1 425 ADVVDWLYTHVEGFKERREARKYASSMLKHGYLRHTVNKITFSEQCYYVFGDLCGNVAAL Mm_Dsh1 448 ADVVDWLYTHVEGFKERREARKYASSMLKHGFLRHTVNKITFSEQCYYVFGDLCSNLASL Dr_Dsh1 418 GDVVDWLYSRVEGFKDRRDARKYASSLLKHGYLRHTVNKITFSEQCYYTFGDLCQNMATL Dm_Dsh 427 ADAVNWVLENVEDVQDRREARRIVSAMLRSNYIKHTVNKLTFSEQCYYVV-NEERNPNLL Ch_Dsh ------------------------------------------------------------ Hv_Dsh 426 SDLVDWLFAHVDGFQDRRDARKYASKLLKAELIKHTVKKVTFSEQCYYVFGDLNSSMKGL Hm_Dsh 165 SDLVDWLFAHVDGFQDRRDARKYA------------------------------------ Am_Dsh 514 SIAEHEGDG--DTDTLGPLPVHPHGTPWMAG-PPSYGYVQMPPYPTAPPPVMVDGQTPGY Nv_Dsh 518 SLNENEGD----QDTLGPLPPQQQAIPWMTGGPPAYGYHQMPSYPPSMAP--SDAKTPGY Xt_Dsh1 485 NLNEGSSGT-SDQDTLAPLPHPAA-PWPLG-QGYSYQYPLAPPCFPPTYQEPGFSYGSGS Mm_Dsh1 508 NLNSGSSGA-SDQDTLAPLPHPSV-PWPLG-QGYPYQYPGPPPCFPPAYQDPGFSCGSGS Dr_Dsh1 478 NLNEGSSGAGSEQDTLAPLPPPSTNPWPMGGQPFPYPPFTAPPAFPPGYSDPCHSFHSGS Dm_Dsh 486 GRGHLHPHQLPHGHGGHALSHADTESITSDIGPLPNPPIYMPYSATYNPSHGYQPIQYGI Ch_Dsh ------------------------------------------------------------ Hv_Dsh 486 ILDDFESNELPLPHSSHCGSWVGNNQSSQYVMQMGGMPLIHPGMIVRGSPSITSSSEMGV Hm_Dsh ------------------------------------------------------------ Am_Dsh 571 GQSLY------------------------------------------------------- Nv_Dsh 572 AQSFYSGTSQHSGSQSPQSASG-------------------------------------- Xt_Dsh1 542 AGSQHSEGK----STYSGVFLP-------------------------------------- Mm_Dsh1 565 AGSQQSEGSKSSGSTRSSHRTP------------GREERRATG--------AGG-----S Dr_Dsh1 538 AGSHHSEGSRSSSSNPSIGRIQRAVQ--------REKERKSTGSESDSGKRAGGRRVERS Dm_Dsh 546 AERHISSGSSSSDVLTSKDISA-------------------------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 546 APSASNYSNVPPNYMDMYSQIYQPTFNPYFAHNSGSQGSQHTGSNSGSGGSFRGHQLNQQ Hm_Dsh ------------------------------------------------------------ Am_Dsh ------------------------------------------------------------ Nv_Dsh 594 SSRKGSERDRGAGDDHKSSGGSSSERSGS------------------------------- Xt_Dsh1 ------------------------------------------------------------ Mm_Dsh1 600 GSESDHTVPSGSGSTGWWERPVSQLSRGSSPRSQA-------------------SAVAPG Dr_Dsh1 590 ASQLSHRSHALSSRSHTHSRVPSQHSRTSFSYSHAPFTKYGHTSCALSERSHASSYGPPG Dm_Dsh 568 -SQSDITSVIHQANQLTIAAHGSNKSSGS------------------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 606 QQLVMQQQHYDYDQATIRSSSSSDKSRGSKSSLESKRVLLATTDHLASDKIISPDTVSLN Hm_Dsh ------------------------------------------------------------ Am_Dsh ----------------------------------------------------------- Nv_Dsh ----------------------------------------------------------- Xt_Dsh1 ----------------------------------------------------------- Mm_Dsh1 641 LPP---LHPLTKAYAVVGGPPGGPPVRELAAVPPELTGSRQSFQKAMGNPCEFFVDIM- Dr_Dsh1 650 LPPPYSLARLTPKGAVCSGPPGAPPVREMGAIPPELTASRQSFQHAMGNPCEFFVDIM- Dm_Dsh 596 ----------SNRGGGGGGGGGGNNTNDQDVSVFNYVL--------------------- Ch_Dsh ----------------------------------------------------------- Hv_Dsh 666 GIRESNISNNNYEKGENARNNTSREFGRLDTIPREISASKQSFRMAMGNSSNEFFVDVM Hm_Dsh -----------------------------------------------------------
Supplementary Figure 5.6 Boxshade alignment of full length Am_Dishevelled (Am_Dsh) protein with representative metazoan Dishevelled orthologues demonstrates a high degree of sequence conservation. Shading demonstrates >50% consensus to Am_Dishevelled (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig1475 as the Acropora orthologue of Dishevelled, which was initially established by BLASTp analysis (E-‐value = 1E-‐136). This Acropora sequence has since been verified by sequencing of cloned DNA (S.Ukolova, unpublished).
Genbank Accesions: Hm_Dsh XP_002162745; Hv_Dsh AAG13667.1; Dm_Dsh NP_511118.2; Mm_Dsh1 NP_034221.3; Dr_Dsh1 XP_698367.5; Xt_Dsh1 NP_001116886.1
Appendix A Supplementary Material
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Chapter 6 Supplementary Figure 6.1
Appendix A Supplementary Material
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Appendix A Supplementary Material
172
Supplementary Figure 6.1 Multiple sequence alignment of α-‐Integrin proteins used for maximum likelihood analysis presented in Figure 6.1. Analysis is based on extensively edited alignment of 843 positions. Shading corresponds to consensus of 30% or more between all sequences included in the alignment.
integrin sequences α7, α6, α3, α5, αV, α8 and αIIb -‐Homo sapiens; integrin αPS1-‐3 -‐Drosophila melanogaster; (Sp) Strongylocentrotus pupuratus; integrin-‐α Pat2 and integrin-‐α Ina1 -‐Caenorhabditis elegans; (Am) Acropora millepora; (Nv) Nematostella vectensis; (Pc) Podocoryne carnea; (Gc) Geodium cydonium; (Lv) Lytechinus verigatus
Genbank accession numbers: LvSU2 AAC23572; SpαP AAD55724; DmPS2 P12080; Itgα5 P08648; ItgαV P06756; Itgα8 P53708; Itgαllb P08514; Itgα6 P23229; Itgα7 Q13683; Itgα3 P26006; ItgαPS3 O44386; AmItgα1 EU239371; Itgα4 P13612; Itgα9 Q13797; NvItgα1 XP_001641435; ItgαPat2 P34446; ItgαPS1; Q24247; PcItgα AAG25993; ItgαIna1 Q03600; GcItgα CAA65943
Appendix A Supplementary Material
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Supplementary Figure 6.2
Appendix A Supplementary Material
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Appendix A Supplementary Material
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Appendix A Supplementary Material
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Supplementary Figure 6.2 Multiple sequence alignment of β-‐Integrin proteins used for maximum likelihood analysis presented in Figure 6.2. Analysis is based on extensively edited alignment of 991 positions. Shading corresponds to consensus of 30% or more between all sequences included in the alignment
(Hs) Homo sapiens; (Dm) Drosophila melanogaster; (Ce) Caenorhabditis elegans; (Sp) Strongylocentrotus pupuratus; (Am; ItgbCn1) Acropora millepora; (Nv) Nematostella vectensis; (Sd) Suberites domuncula; (Gc) Geodium cydonium; (ItgβPo1) Ophlitaspongia tenuis;
Genbank accession numbers: HsItgβ3 P05106; HsItgβ5 P18084; HsItgβ6 P18564 ; HsItgβ2 P05107 ; HsItgβ7 P26010 ; HsItgβ1 P05556 ; SpItgβG AAB39739 ; SpItgβL AAC28382 ; SpItgβC AAB39740 ; DmβPS P11584 ; CePat3 Q27874 ; AmItgβ2 EU239372 ; NvItgβ1 XP_001641468 ; NvItgβ2 XP_001627336 ; PcIntB AAG25994 ; ItgbCN1 AAB66910 ; NvItgβ3 XP_001637894 ; NvItgβ4 XP_001621822 ; OtItgβ1 AAB66911 ; SdItgβ CAB38100 ; GcItgβ CAA77071 ; HsItgβ4 P16144 ; Dmβ-‐nu Q27591 ; HsItgβ8 P26012
Appendix B JCUSMART survey of the cnidarian adhesome
177
Sub-Family Organism Sequence Id BLAST e_value HMMPFAM
Signal Peptide TM Conclusion Notes
Flamingo Nvec jgi|Nemve1|84228|e_gw.9.5.1 CELSR 0CA (8x), EGF (2-3), LamG, EGF, LamG, EGF (3x), HormR_1, GPS, 7tm_2 No 1 Flamingo start and stop codons present
Flamingo Acropora Contig3665cadherin EGF LAG seven-pass G-type receptor 1 [... 2.00E-98 HormR, GPS, 7tm_2 No 7 flamingo c-term best hit to Nvec flamingo; overlaps Contig8737
Flamingo Acropora Contig8737cadherin, EGF LAG seven-pass G-type receptor 2 (f... 1.00E-171 CA, EGF (2x), LamG, EGF,LamG, EGF(2x) No No flamingo middle best hit to Nvec flamingo; overlaps Contig3665
FAT Nvec jgi|Nemve1|197466|fgenesh1_pg.scaffold_5000272 FAT3 SP, CA (29x), EGF, LamG, EGF, LamG, EGF Yes No FAT 4 like start and stop codons presentFAT Nvec jgi|Nemve1|20161|gw.67.8.1 FAT1 CA (34x), LamG, EGF (3x), TM No 1 FAT 1 No start and no stop codonFAT Nvec jgi|Nemve1|20073|gw.193.26.1 FAT4 0 CA (11x), EGF (3x), LamG, EGF, LamG, EGF No No FAT 4, partial FAT4 has CA (25x) but the rest is identicial in structure. No start no stopCalsynetenin Nvec jgi|Nemve1|167342|estExt_gwp.C_860192 Calsyntenin 2 1.00E-84 CA (2x), CA (wobbly) LamG (wobbly) Yes 1 Calsyntenin 2 start and stop codons presentCalsynetenin Acropora Amil_rep_c147915 calsyntenin 1 [Danio rerio] 8.00E-79 CA (2x), LamG, TM No 1 CalsynteninCalsynetenin Acropora Contig31873 calsyntenin 1 [Danio rerio] 8.00E-76 CA, LamG Yes No Calsyntenin partialCatenin Binding Acropora Contig 6389 + Contig5394
Cj-cadherin [Caridina japonica] 1.00E-129
CA (6x) + CA (7x), EGF, LamG, EGF, LamG, EGF, TM, Cadherin_C No 1 Type III cadherin these two contigs overlap on the basis of DNA assembly
Catenin Binding Nvec jgi|Nemve1|239897|estExt_fgenesh1_pg.C_210083 FAT3 0 SP, CA (24x), EGF, LamG, LamG, EGF, TM, Cadherin_C Yes 1 Type III cadherin start and stop codons presentCatenin Binding Nvec jgi|Nemve1|244010|estExt_fgenesh1_pg.C_1040031 FAT3 0 SP, CA (30x), EGF, LamG, EGF, LamG,EGF, TM, Cadherin_C Yes 1
Type III cadherin Uli Technau's Endothelial Cadherin start and stop codons present
Catenin Binding Nvec jgi|Nemve1|239899|estExt_fgenesh1_pg.C_210088 No good hits EGF, TM, Cadherin_C (wobbly) No 1 Type III cadherin start and stop codons present combines with 239898 Extracellular region!Catenin Binding Nvec jgi|Nemve1|223294|fgenesh1_pg.scaffold_2546000001 Cadherin_C No No
Unknown - Classical Cadherin (partial) No start codon, stop codon present
Catenin Binding Hydra CL3352Contig1 No good hits Cadherin_C No No
Unknown - Classical Cadherin (partial) has a M but not likely to be the real start
Cad23/Dachsous Monosiga jgi|Monbr1|27065|fgenesh2_pg.scaffold_17000053 No good hits CA (2x) No No start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|206603|fgenesh1_pg.scaffold_71000072 No good hits CA (4x) Yes 1 Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|129879|e_gw.270.98.1 CELSR 3 1.00E-78 CA (4x) No No Cadherin start and stop codons presentCad23/Dachsous Acropora Contig12092
FAT tumor suppressor homolog 3 [Gallus gallus] 2.00E-49 CA (6x),TM Yes 1
Cad23/Dachsous Nvec jgi|Nemve1|243040|estExt_fgenesh1_pg.C_770089 FAT1 1.00E-59 CA (7x) Yes 1 Cadherin 17 start and stop codons presentCad23/Dachsous Acropora Contig8381 CA (7x), TM Yes 1Cad23/Dachsous Nvec jgi|Nemve1|129882|e_gw.270.7.1 CELSR 1.00E-30 CA (8x) No No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|27265|fgenesh2_pg.scaffold_18000084 cadherin23 1.00E-35 CA (8x) Yes no Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|214866|fgenesh1_pg.scaffold_212000008 FAT4 1.00E-80 CA (9x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|221587|fgenesh1_pg.scaffold_851000001 dachsous 1.00E-99 CA (9x) No No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|207189|fgenesh1_pg.scaffold_77000086 Hedgling 1.00E-178 CA (14x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|248422|estExt_fgenesh1_pg.C_5570001 Hedgling 0 CA (17x) No No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|29591|fgenesh2_pg.scaffold_36000032 fat 1.00E-51 Ca (20x) Yes No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|27264|fgenesh2_pg.scaffold_18000083 FAT4 1.00E-57 CA (24x) No 2 Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|31013|estExt_fgenesh2_pg.C_20691 FAT4/cadherin23 1.00E-173 CA (24x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|239898|estExt_fgenesh1_pg.C_210085 FAT1 0 CA (30x) Yes No Cadherin
start and stop codons present probably should be combined with 239899 to make one model duplicate of technau's
Cad23/Dachsous Monosiga jgi|Monbr1|37884|estExt_fgenesh1_pg.C_170117 FAT4 1.00E-160 CA (30x) Yes 2 Cadherin 16 like start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|207195|fgenesh1_pg.scaffold_77000092 dachsous 1.00E-153 CA (36x) Yes 1 dachsous start and stop codons present
Cad23/Dachsous Acropora Contig10872
dachsous 1 [Rattus norvegicus] >gi|149068442... 0 CA (19x), TM cadherin_C No 1 Dachsous Matches Unigene D041-C8. C-terminal region alignment places with Dachsous.
MBCDH1 Monosiga jgi|Monbr1|14707|e_gw1.3.170.1gb|AAP78679.1| MBCDH1 [Monosiga brevicollis] 0 EGF, CA (2x) No No
gb|AAP78679.1| MBCDH1 [Monosiga brevicollis] start, no stop
unidentified Hydra gb|DT613897.1 No good hits CA No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DT617585.1 No good hits CA No No Unknown - Cadherin (partial) No start no stopunidentified Clytia IL0ABA14YL05RM1 protocadherin gamma A2 1.00E-12 CA No No No start No stopunidentified Nvec jgi|Nemve1|110347|e_gw.100.165.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|127187|e_gw.236.58.1 Hydra Homolgue only CA No No No start, no stop ref|XP_002122245.1| PREDICTED: similar to Sushi, von Willebrand ... 4.00E-91unidentified Nvec jgi|Nemve1|127202|e_gw.236.61.1 No good hits CA No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|18675|gw.236.62.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|219496|fgenesh1_pg.scaffold_416000004 No good hits CA No No start and stopunidentified Nvec jgi|Nemve1|223469|fgenesh1_pg.scaffold_2837000001 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|225510|fgenesh1_pg.scaffold_9093000001 No good hits CA No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|225923|fgenesh1_pg.scaffold_14475000001 No good hits CA No No No start, stop presentunidentified Nvec jgi|Nemve1|225928|fgenesh1_pg.scaffold_14604000001 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|238641|estExt_fgenesh1_pg.C_70089 No good hits CA No No start and stopunidentified Nvec jgi|Nemve1|4597|gw.4224.3.1 starry night 1.00E-31 CA No No No start, no stopunidentified Nvec jgi|Nemve1|67148|gw.100.168.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|219501|fgenesh1_pg.scaffold_416000009 No good hits CA (wobbly), F5_F8_type_C No No start and stop
Appendix B: JCUSMART Survey of the Cnidarian Adhesome
Cadherins
Appendix B JCUSMART survey of the cnidarian adhesome
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Sub-Family Organism Sequence Id BLAST e_value HMMPFAM
Signal Peptide TM Conclusion Notes
unidentified Nvec jgi|Nemve1|244693|estExt_fgenesh1_pg.C_1310026 No good hits CA Yes No start and stopunidentified Hydra CL1102Contig1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|CO538479.1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN603165.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN811522.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN812369.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN815486.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DT619671.1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Nvec jgi|Nemve1|148827|e_gw.2646.1.1 No good hits CA (2x) No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|52838|gw.77.119.1 starry night 1.00E-30 CA (2x) No No No start, no stopunidentified Clytia SA0AAB45YD15CTG FAT tumor suppressor 1.00E-16 CA (2x) Yes No Start codon, no stop codonunidentified Hydra CL5439Contig1 No good hits CA (3x) No 1 Unknown - Cadherin (partial) No start no stopunidentified Hydra CL5681Contig1 No good hits CA (3x) No 2 Unknown - Cadherin (partial) No start no stopunidentified Nvec jgi|Nemve1|219493|fgenesh1_pg.scaffold_416000001 No good hits EGF, CA (3x) No No start and stopunidentified Nvec jgi|Nemve1|219500|fgenesh1_pg.scaffold_416000008 protocadherin 1.00E-24 CA (3x) No No start and stopunidentified Nvec jgi|Nemve1|220674|fgenesh1_pg.scaffold_557000003 CELSR 1.00E-39 CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|40605|gw.212.44.1 starry night 1.00E-59 CA (3x) No No No start, no stopunidentified Nvec jgi|Nemve1|98014|e_gw.48.27.1 No good hits CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|153449|e_gw.6181.1.1 CELSR 1.00E-47 CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|48709|gw.12488.2.1 fat (dros) 1.00E-60 CA (4x) No No No start, no stopunidentified Nvec jgi|Nemve1|70250|gw.100.177.1 CELSR 1.00E-47 CA (4x) No No No start, no stop
unidentified Nvec jgi|Nemve1|91648|e_gw.27.37.1gb|AAI01037.1| PCDHGC4 protein [Homo sapiens] 1.00E-70 CA (5x) Yes No
cadherin partial ( or PCDH-Gamma-C4) No stop codon
unidentified Nvec jgi|Nemve1|124570|e_gw.212.50.1gb|ABX84114.1| hedgling [Nematostella vectensis] 1.00E-67 CA (5x) No No No start no stop
unidentified Nvec jgi|Nemve1|41182|gw.270.64.1 CELSR 1.00E-59 CA (5x) No No No start no stop
unidentified Nvec jgi|Nemve1|118913|e_gw.157.87.1
ref|NP_059088.2| cadherin EGF LAG seven-pass G-type receptor 2 i… 4.00E-77 CA (5x) No No No start no stop
unidentified Clytia IL0ABA4YB04RM1cadherin EGF LAG seven-pass G-type receptor 3 [Ra... 1.00E-27 CA (6x) No 1 No start No stop
unidentified Nvec jgi|Nemve1|10520|gw.100.7.1 FAT 4 or Cadherin 23 1.00E-98 CA (7x) No No No start no stopunidentified Nvec jgi|Nemve1|40082|gw.212.41.1 CELSR 1.00E-120 CA (8x) No No No start no stopunidentified Monosiga jgi|Monbr1|13666|e_gw1.2.131.1 FAT4 1.00E-154 CA(12x) No No No start, no stopunidentified Nvec jgi|Nemve1|122785|e_gw.193.27.1 FAT 4 0.00E+00 CA (16x) No No start codon, No stop codonunidentified Nvec jgi|Nemve1|89517|e_gw.21.14.1 FAT1 0 CA (17x) No No No start no stopunidentified Nvec jgi|Nemve1|30053|gw.23.10.1 FAT 4 0 CA (18x) No No No start no stopunidentified Nvec jgi|Nemve1|107177|e_gw.85.10.1 dachsous/FAT4 0.00E+00 CA (24x) No No No M and no stopNovel Architecture Nvec jgi|Nemve1|216996|fgenesh1_pg.scaffold_280000006 No good hits CUB, EGF (3x), CA (2x) No No start and stop codons present. Scaffold 280. Ab inito model with no EST support.Novel Architecture Nvec jgi|Nemve1|247587|estExt_fgenesh1_pg.C_3180027 No good hits TSP1, CA (7x), EGF Yes No start and stop codons present. Scaffold 318. Ab inito with partial EST support.Novel Architecture Monosiga jgi|Monbr1|11672|fgenesh1_pg.scaffold_31000029 gb|ABX84114.1| hedgling 1.00E-40
EGF (3x), CCP, EGF, CCP (wobbly), EGF, CA (15x), EGF (2x), CCP (wobbly), EGF (2x) Yes 5 start and stop codons present
Novel Architecture Monosiga jgi|Monbr1|30335|fgenesh2_pg.scaffold_48000016 gb|ABX84114.1| hedgling 1.00E-180
Lam_NT (wobbly), LamEGF (4x), furin_3, LamG (wobbly), CA (46x), FN3 (wobbly), Y_Phosphatase Yes Yes start and stop codons present
Novel Architecture Monosiga jgi|Monbr1|11339|fgenesh1_pg.scaffold_28000057 No good hits
VWD, EGF, iptmega(wobbly), CA (6x wobbly), PKD (wobbly) SH) Yes 2 start and stop codons present
Novel Architecture Monosiga jgi|Monbr1|12200|fgenesh1_pg.scaffold_36000025 FAT4 0.00E+00 CA (58x) No 1 Cadherin novel or error start and stop codons present
Appendix B JCUSMART survey of the cnidarian adhesome
179
Integrins
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes Integrin Beta Hydra gb|DT609127.1 integrin beta chain [Podocoryne carnea] 1.00E-43 INB (wobbly) No Integrin beta - AmItgb2 homologue second best hit was AmItgb2 1E-43
Integrin Beta Nvec jgi|Nemve1|80868|e_gw.3.432.1 integrin beta 2 [Acropora millepora] 0INB, EGF (4x wobbly), Integrin_B_tail, Integrin_b_cyt 1 Integrin beta - AmItgb2 homologue
Integrin Beta Nvec jgi|Nemve1|123281|e_gw.198.1.1 integrin beta 2 [Acropora millepora] 0INB,EGF (4xwobbly), Integrin_B_tail,Integrin_b_cyt 1 Integrin beta - AmItgb2 homologue
Integrin Beta Clytia SA0AAB34YK24RM1 integrin subunit betaCn1 [Acropora millepora] 1.00E-33EGF (4x wobbly), Integrin_B_tail (wobbly) 1 Integrin beta - ItgbCN1 homologue
Integrin Beta Hydra CL4502Contig1 integrin subunit betaCn1 [Acropora millepora] 6.00E-86 INB No Integrin beta - ItgbCN1 homologue
Integrin Beta Nvec jgi|Nemve1|91193|e_gw.26.1.1 integrin subunit betaCn1 [Acropora millepora] 0INB, EGF (4x wobbly), Integrin_B_tail, Integrin_b_cyt 1 Integrin beta - ItgbCN1 homologue
Integrin Beta Nvec jgi|Nemve1|90281|e_gw.23.1.1 integrin subunit betaCn1 [Acropora millepora] 1.00E-168INB,EGF (4x wobbly), Integrin_B_tail Integrin beta - ItgbCN1 homologue This one is thought to be a splice varient
Integrin Beta Nvec jgi|Nemve1|143492|e_gw.826.1.1 integrin subunit betaCn1 [Acropora millepora] 1.00E-177INB,EGF (4xwobbly), Integrin_B_tail,Integrin_b_cyt 1 Integrin beta - ItgbCN1 homologue
Integrin Beta Acropora Contig12118 integrin subunit betaCn1 [Acropora millepora] 0 No AmItgB1Integrin Beta Acropora Contig17193 integrin beta chain [Podocoryne carnea] 1.00E-174 1 AmItgB2Integrin Alpha Clytia SA0AAB95YG15RM1 integrin alpha chain [Podocoryne carnea] 2.00E-74 int_alpha_rpt (4x) No Integrin alpha - partial
Integrin Alpha Nvecjgi|Nemve1|238305|estExt_fgenesh1_pg.C_50011
integrin, alpha 4 (antigen CD49D, alpha 4 subun... 2.00E-54
int_alpha_rpt (3x), Integrin_alpha2 1 Integrin alpha
Integrin Alpha Nvecjgi|Nemve1|196726|fgenesh1_pg.scaffold_3000047 integrin alpha 1 [Acropora millepora] 1.00E-149
int_alpha_rpt (4x), Integrin_alpha2,Integrin_alpha 2 Integrin alpha - AmItga1 homologue TMH 1 is in N-term therefore only 1 real TMH.
Integrin Alpha Hydra CL3123Contig1integrin, alpha 8 [Gallus gallus] >gi|124950|sp… 2.00E-35
int_alpha_rpt (2x), Integrin_alpha2 No Integrin alpha - partial
Integrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0 AmItga1Integrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0 AmItga2 -NEWIntegrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0.00E+00 AmItga3 -NEW
Integrin Alpha Repeat Containing Clytia SA0AAB7YH09RM1 AmItga1 1.00E-05Integrin_alpha2 (wobbly), Integrin_alpha (wobbly) integrin alpha related
Integrin Alpha Repeat Containing Hydra gb|DN813198.2 integrin alpha 1 [Acropora millepora] 6.00E-12 int_alpha_rpt integrin alpha relatedIntegrin Alpha Repeat Containing Hydra gb|CA302076.1 integrin alpha 2 [Pseudoplusia includens] 1.00E-10 No domains integrin alpha relatedIntegrin Alpha Repeat Containing Clytia IL0ABA2YN22RM1 Integrin alpha 4 [Mus musculus] 2.00E-14 int_alpha_rpt (2x wobbly) integrin alpha related
Integrin Alpha Repeat Containing Hydra CL6512Contig1integrin alpha 4 [Rattus norvegicus] >gi|149... 3.00E-14 int_alpha_rpt (wobbly) integrin alpha related
Integrin Alpha Repeat Containing Clytia SA0AAB95YG15NM1 integrin alpha chain [Podocoryne carnea] 2.00E-29 No Domains integrin alpha relatedIntegrin Alpha Repeat Containing Hydra CL6142Contig1 integrin alpha chain [Podocoryne carnea] 2.00E-20 int_alpha_rpt integrin alpha relatedIntegrin Alpha Repeat Containing Hydra gb|CV181210.1 integrin alpha chain [Podocoryne carnea] 3.00E-12 No domains integrin alpha related
Integrin Alpha Repeat Containing Hydra gb|DN244145.2integrin alpha-PS2 [Culex quinquefasciatus] ... 5.00E-15 int_alpha_rpt integrin alpha related
FG-GAP repeats occur in other proteins but int_alpha repeats are more restricted.
Integrin Alpha Repeat Containing Hydra gb|DT620726.1Integrin, alpha 2b (platelet glycoprotein IIb of ... 2.00E-08 No domains integrin alpha related
Integrin Alpha Repeat Containing Hydra CL10025Contig1 ITGA7 variant protein [Homo sapiens] 9.00E-13 int_alpha_rpt (wobbly) integrin alpha relatedIntegrin Alpha Repeat Containing Hydra CL10282Contig1 No good hits int_alpha_rpt (wobbly) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|13072|fgenesh1_pg.scaffold_55000008 No good hits int_alpha_rpt (2x wobbly) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|9496|fgenesh1_pg.scaffold_15000186 No good hits int_alpha_rpt (2x wobbly) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|10253|fgenesh1_pg.scaffold_20000037 quinoprotein (ISS) [Ostreococcus tauri] 1.00E-29 int_alpha_rpt (2-4x) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25795|fgenesh2_pg.scaffold_11000195 quinoprotein (ISS) [Ostreococcus tauri] 9.00E-15 int_alpha_rpt (2-4x) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25557|fgenesh2_pg.scaffold_10000207 quinoprotein (ISS) [Ostreococcus tauri] 8.00E-07 int_alpha_rpt (2x wobbly) integrin alpha related
Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25858|fgenesh2_pg.scaffold_12000007 quinoprotein (ISS) [Ostreococcus tauri] 3.00E-05 int_alpha_rpt (2x wobbly) integrin alpha related
Integrin Alpha Repeat Containing Nvec jgi|Nemve1|115107|e_gw.130.30.1glycosylphosphatidylinositol phospholipase D [Mus... 1.00E-160 int_alpha_rpt (6x) No Integrin alpha related BLAST hit has only 2 int_alpha_rpts. Start and stop codons present.
Integrin Alpha Repeat Containing Nvecjgi|Nemve1|224736|fgenesh1_pg.scaffold_5705000001
ref|NP_777241.1| glycosylphosphatidylinositol specific phospholi... 2.00E-45 1.00E-45 int_alpha_rpt (4x) No Integrin alpha - partial BLAST hit has only 2 int_alpha_rpts. No start and No stop codons.
Integrin Alpha Repeat Containing Acropora Contig19889 integrin alpha 1 [Acropora millepora] 1.00E-150 int_alpha_rpt (2) NoIntegrin Alpha Repeat Containing Acropora Contig33553 integrin alpha 1 [Acropora millepora] 1.00E-141 int_alpha_rpt (2) NoIntegrin Alpha Repeat Containing Acropora Contig7700 integrin alpha 1 [Acropora millepora] 4.00E-61 int_alpha_rpt (4) No
Talin Clytia SA0AAA26YF22RM1talin-1 [Culex quinquefasciatus] >gi|1678730... 2.00E-20
Talin_middle(wobbly), MA_2 (wobbly), ILWEQ Talin - middle
Talin Clytia IL0ABA5YH08RM1 talin [Podocoryne carnea] 1.00E-134 B41_5, PTBI_2 (wobbly) Talin - N-termTalin Hydra CL1Contig29 talin [Podocoryne carnea] 1.00E-118 B41/FERM_N Talin - N-term This may be the N-terminus of one of the other partial talins.
Talin Mbrevjgi|Monbr1|23461|fgenesh2_pg.scaffold_4000204 talin 2 [Mus musculus] 1.00E-127
B41, PTBI (wobbly), Talin_middle (wobbly), ILWEQ (wobbly) Talin 2 - N-term
Talin Clytia IL0ABA4YJ15RM1talin 1 [Danio rerio] >gi|55139380|gb|AAV413... 1.00E-155
MA (wobbly), VBS, ILWEQ (wobbly) Talin 1 - C-term
Talin Mbrevjgi|Monbr1|6317|fgenesh1_pg.scaffold_4000199 talin 1, isoform CRA_b [Homo sapiens] 1.00E-180 VBS, MA (wobbly), ILWEQ Talin 1 - C-term
Talin Hydra CL5745Contig1 talin 1, isoform CRA_b [Homo sapiens] 9.00E-72 ILWEQ Talin 1 - C-term
INB_2, EGF_2 (4), INB_2, EGF_2 (4),
Appendix B JCUSMART survey of the cnidarian adhesome
180
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
Talin Nvecjgi|Nemve1|159935|estExt_gwp.C_110123 Tln2 protein [Mus musculus] 0
B41, PTBI (wobbly), Talin_middle, 5bar (wobbly), MA (wobbly), VBS, ILWEQ Talin 2
Talin Hydra CL9211Contig1 talin 2 [Mus musculus] 3.00E-79 MA (wobbly), ILWEQ (wobbly) Talin 2 - C-term
Talin Clytia SA0AAB5YM11RM1 talin 2, isoform CRA_a [Homo sapiens] 1.00E-149VBS, MA (wobbly), ILWEQ (wobbly) Talin 2 - C-term
Talin Acropora Contig10699 talin 1, isoform CRA_b [Homo sapiens] 0 VBS, MA (wobbly), ILWEQ Talin1 - C-termTalin Acropora Amil_rep_c147660 talin 1, isoform CRA_b [Homo sapiens] 1.00E-65 ILWEQ Talin1 - C-termTalin-related Hydra gb|DT615285.1 Tln2 protein [Mus musculus] 2.00E-54 MA (wobbly) Talin 2 - partial
Talin-related Acropora Contig943talin 1 [Danio rerio] >gi|55139380|gb|AAV413 1.00E-111 MA (wobbly), ILWEQ (wobbly)
Talin-related Nvec jgi|Nemve1|123513|e_gw.200.72.1huntingtin interacting protein 1 [Homo sapiens] >... 6.00E-70 ILWEQ Talin related
Talin-related Mbrevjgi|Monbr1|36287|estExt_fgenesh1_pg.C_40453
huntingtin interacting protein 1 related, isoform... 9.00E-17 SH3, ILWEQ Talin related
Talin-related Mbrev jgi|Monbr1|14137|e_gw1.2.850.1 huntingtin-interacting protein 1 [Homo ... 2.00E-24 ILWEQ Talin related
Talin-related Hydra gb|CV659942.1talin 1 [Danio rerio] >gi|55139380|gb|AAV413... 9.00E-49 ILWEQ (wobbly) Talin related
Talin-related Clytia IL0ABA22YH03RM1talin 1 [Gallus gallus] >gi|81175199|sp|P54939.... 4.00E-58 ILWEQ (wobbly) Talin related
Talin-related Clytia SA0AAB72YJ22RM1 Talin 1 [Homo sapiens] 4.00E-30 ILWEQ Talin relatedTalin-related Hydra gb|DR435962.1 talin 2 [Homo sapiens] 1.00E-31 No domains Talin related
Talin-related Hydra CL1594Contig1 talin 2 [Xenopus (Silurana) tropicalis] >gi|... 1.00E-29 ILWEQ Talin related
I/LWEQ domains bind to actin. It has been shown that the I/LWEQ domains from mouse talin and yeast Sla2p interact with F-actin PUBMED:9159132. The domain has four conserved blocks, the name of the domain is derived from the initial conserved amino acid of each of the four blocks PUBMED:9159132. I/LWEQ domains can be placed into four major groups based on sequence similarity:1. Metazoan talin.2. Dictyostelium discoideum (Slime mould) TalA/TalB and SLA110.3. Metazoan Hip1p .4. Saccharomyces cerevisiae Sla2p .
Talin-related Clytia SA0AAB23YM10RM1 talin 2 [Xenopus (Silurana) tropicalis] >gi|... 4.00E-17 ILWEQ Talin relatedTalin-related Clytia IL0ABA6YH10RM1 Tln2 protein [Mus musculus] 4.00E-65 No Domains Talin relatedTalin-related Hydra CL3431Contig1 vinculin [Drosophila melanogaster] 1.00E-29 ILWEQ (wobbly) Talin related vinculin has its own domain called "vinculin". Integrin Linked Kinase Hydra CL2782Contig1 Integrin-linked protein kinase [Salmo salar]... 1.00E-124 Ank (3x), tyrkin Integrin-linked kinase
Integrin Linked Kinase Clytia SA0AAA16YD20RM1integrin-linked kinase [Gallus gallus] >gi|1050... 1.00E-145 Ank (3x), tyrkin Integrin-linked kinase
Integrin Linked Kinase Nvec jgi|Nemve1|31638|gw.2.85.1integrin-linked kinase [Danio rerio] >gi|339918... 1.00E-135 Ank (3x), trykin Integrin-linked kinase
Integrin Linked Kinase Acropora Contig5477integrin-linked kinase [Gallus gallus] >gi|1050... 1.00E-148 Ank (3x), trykin Integrin-linked kinase
FAK Acropora Contig1043 FAK 0FAK Nvec jgi|Nemve1|60034|gw.13.293.1 FAK 0FAK Clytia CL7784Contig1 FAK 1.00E-51Parvin Hydra CL698Contig1 Alpha-parvin [Osmerus mordax] 1.00E-85Parvin Acropora Contig28157 Alpha-parvin [Osmerus mordax] 1.00E-77
Parvin Nvecjgi|Nemve1|207272|fgenesh1_pg.scaffold_78000074 Alpha-parvin [Osmerus mordax] 1.00E-55
Paxillin Hydra CL1132Contig1dbj|BAA18998.1|,paxillin gamma [Homo sapiens] 1.00E-120
Paxillin Acropora Contig8199ref|NP_963882.1|,paxillin [Danio rerio] >gi|41350255|gb|AAS00452 1.00E-125
Paxillin Mbrevjgi|Monbr1|19246|estExt_Genewise1.C_20560
ref|NP_002850.2|,paxillin isoform 2 [Homo sapiens] 1.00E-42
Paxillin Nvec jgi|Nemve1|104924|e_gw.75.150.1 paxillin [Culex quinquefasciatus] 1.00E-122Paxillin Clytia SA0AAB113YJ11CTG paxillin, isoform B [Drosophila melanogaster] 1.00E-129
c-Src Acropora Contig2876dbj|BAG70102.1|,c-src tyrosine kinase [Homo sapiens] 1.00E-140
c-Src Clytia SA0AAB19YJ19RM1dbj|BAG70102.1|,c-src tyrosine kinase [Homo sapiens] 1.00E-78
PINCH Acropora Contig17667 gb|ABS17667.1|,PINCH-1 [Xenopus laevis], 1.00E-117PINCH Hydra CL5059Contig1 PINCH 1.00E-81
PINCH Nvecjgi|Nemve1|160257|estExt_gwp.C_120473
gb|EDL42131.1|,"LIM and senescent cell antigen-like domains 1 1.00E-122
ILKAK Hydra CL1236Contig1integrin-linked kinase-associated protein phosp... 7.00E-75 PP2C
Integrin-linked kinase associated Phosphatase
ILKAK Nvec jgi|Nemve1|110119|e_gw.98.154.1ref|NP_075832.1| integrin-linked kinase-associated serine/threon… 2.00E-88 PP2C
integrin-linked kinase-associated serine/threonin...
ILKAK Mbrevjgi|Monbr1|36991|estExt_fgenesh1_pg.C_90170
Integrin-linked kinase-associated serine/threonin... 6.00E-53 PP2C
Integrin-linked kinase-associated serine/threonin...
Disintegrin Containing Hydra gb|DN813922.2 No good hits DISIN Disintegrin containingDisintegrin Containing Hydra gb|DN811041.2 hemicentin1 DISIN, TSP1 Disintegrin containing
Appendix B JCUSMART survey of the cnidarian adhesome
181
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
Disintegrin Containing Nvec jgi|Nemve1|5599|gw.3050.2.1kinesin [Leishmania infantum JPCM5] >gi|1340... 2.00E-16 DISIN (wobbly) DISIN containing
ADAM Nvec jgi|Nemve1|20904|gw.66.19.1a disintegrin and metalloprotease domain 10b [D... 1.00E-167 Reprolysin, DISIN ADAM
ADAM Nvecjgi|Nemve1|175837|estExt_gwp.C_4720015
a disintegrin and metalloprotease domain 10b [D... 1.00E-124
pep_M12B_propep, Reprolysin, DISIN ADAM
ADAM Nvec jgi|Nemve1|5671|gw.3057.2.1a disintegrin and metalloprotease domain 10b [D... 2.00E-56 No domains ADAM
ADAM Clytia SA0AAA13YF04RM1A disintegrin and metalloprotease domain 13 [Xeno... 7.00E-65 reprolysin, DISIN, acr ADAM
ADAM Mbrevjgi|Monbr1|22131|fgenesh2_pg.scaffold_2000338
a disintegrin and metalloprotease domain 17 pre... 8.00E-46 Reprolysin, DISIN ADAM
ADAM Hydra gb|CN552903.1a disintegrin and metalloprotease domain 17 pre... 2.00E-37 reprolysin ADAM
reprolysin: The members of this family are enzymes that cleave peptides. These proteases require zinc for catalysis. Members of this family are also known as adamalysins. Most members of this family are snake venom endopeptidases, but there are also some mammalian proteins such as P78325 and fertilin Q28472. Fertilin and closely related proteins appear to not have some active site residues and may not be active enzymes.
ADAM Mbrevjgi|Monbr1|37642|estExt_fgenesh1_pg.C_150079
a disintegrin and metalloproteinase domain 17a ... 2.00E-08 DISIN (wobbly), GRAN_2 (wobbly) ADAM
ADAM Hydra CL6661Contig1a disintegrin and metalloproteinase domain 24 (... 6.00E-14 reprolysin, DISIN ADAM
ADAM Clytia SA0AAB61YH16CTGadam (a disintegrin and metalloprotease [Aed... 1.00E-64 reprolysin, DISIN, acr ADAM
ADAM Hydra CL970Contig1ADAM metallopeptidase domain 10 [Gallus gallus]... 3.00E-77 reprolysin, DISIN ADAM
ADAM Mbrevjgi|Monbr1|29860|fgenesh2_pg.scaffold_39000061
ADAM metallopeptidase domain 10 [Pongo abeli... 6.00E-50
pep_M12B_propep, Reprolysin, DISIN ADAM
ADAM Mbrevjgi|Monbr1|31368|estExt_fgenesh2_pg.C_30574
ADAM metallopeptidase domain 10 [Pongo abeli... 2.00E-46
pep_M12B_propep, Reprolysin, DISIN ADAM
ADAM Nvec jgi|Nemve1|91360|e_gw.27.264.1ADAM metallopeptidase domain 10, isoform CRA_a [H... 4.00E-45 Reprolysin, DISIN, acr ADAM
ADAM Hydra gb|DN814600.2ADAM metallopeptidase domain 12 [Bos taurus]... 4.00E-36 DISIN, acr ADAM acr = ADAM_CR = ADAM cystein rich region
ADAM Clytia SA0AAA4YP11CTGADAM metallopeptidase domain 12 [Xenopus (Si... 3.00E-56 DISIN, acr ADAM
ADAM Nvecjgi|Nemve1|212032|fgenesh1_pg.scaffold_148000031
ADAM metallopeptidase domain 8 precursor [Homo ... 4.00E-45 DISIN, acr ADAM
ADAM Clytia SA0AAB70YK05CTGADAM metallopeptidase domain 9 [Rattus norve... 6.00E-37 reprolysin, DISIN ADAM
ADAM Nvec jgi|Nemve1|126434|e_gw.232.25.1ADAM metallopeptidase with thrombospondin type 1 ... 4.00E-39 Reprolysin ADAM
ADAM Hydra gb|CV659930.1ADAM metallopeptidase with thrombospondin type 1 ... 2.00E-29 reprolysin, acr (wobbly) ADAM
ADAM Nvec jgi|Nemve1|16735|gw.148.22.1ADAM33 [Mus musculus] >gi|123229989|emb|CAM18043.... 9.00E-38 DISIN, acr ADAM
ADAM Nvecjgi|Nemve1|200723|fgenesh1_pg.scaffold_23000088 ADAM33 protein [Homo sapiens] 3.00E-15 Reprolysin, DISIN, acr ADAM
ADAM Mbrevjgi|Monbr1|23347|fgenesh2_pg.scaffold_4000090 No good hits Reprolysin, DISIN, SH3 ADAM Novel SH3 domain?
ADAM Mbrevjgi|Monbr1|22277|fgenesh2_pg.scaffold_2000484
tace, isoform A [Drosophila melanogaster] >gi|4... 2.00E-54
pep_M12B_propep, Reprolysin, DISIN ADAM
ADAM Mbrevjgi|Monbr1|24567|fgenesh2_pg.scaffold_7000115
tumor necrosis factor alpha converting enzyme [Ga... 1.00E-34
Ribosomal_L9_N, Reprolysin, DISIN ADAM Tace/ADAM17
ADAM Mbrevjgi|Monbr1|36614|estExt_fgenesh1_pg.C_60325
tumor necrosis factor-alpha-converting enzyme [Su... 1.00E-16
DISIN (wobbly), DEFSN_4 (wobbly) ADAM Tace/ADAM17
ADAM Mbrevjgi|Monbr1|9808|fgenesh1_pg.scaffold_17000081
tumor necrosis factor-alpha-converting enzyme mut... 7.00E-18 No domains ADAM Tace/ADAM17
ADAM Hydra gb|DN240294.2a disintegrin and metallopeptidase domain 34 [M... 4.00E-19 DISIN ADAM - putative
ADAM Hydra CL2583Contig1ADAM metallopeptidase domain 10 [Sus scrofa]... 8.00E-27 DISIN (wobbly) ADAM - putative
ADAM Hydra gb|CV985877.1ADAM metallopeptidase domain 33 isoform beta pr... 2.00E-11 DISIN ADAM - putative
ADAM Hydra gb|CN631642.1disintegrin and metalloprotease domain 33 [Mus mu... 1.00E-09 DISIN, TSP1 ADAM - putative
ADAM Hydra CL8785Contig1MIND-MELD [Drosophila melanogaster] or ADAM 7.00E-12 DISIN ADAM - putative mind meld is an ADAM
ADAM Nvec jgi|Nemve1|40207|gw.95.86.1tace, isoform A [Drosophila melanogaster] >gi|4... 2.33E-156 Reprolysin, DISIN ADAM17 homologue tace is ADAM17
ADAM Nvec jgi|Nemve1|6627|gw.3466.2.1a disintegrin and metalloprotease domain 10b [D... 7.00E-22 Pep_M12B_propep (wobbly) M12B peptidase family
Appendix B JCUSMART survey of the cnidarian adhesome
182
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
ADAM Nvecjgi|Nemve1|198740|fgenesh1_pg.scaffold_11000152
a disintegrin and metalloproteinase domain 9... 6.00E-15 Pep_M12B_propep M12B peptidase family
ADAM_TS Nvec jgi|Nemve1|127746|e_gw.242.21.1metalloprotease disintegrin 16 with thrombospond... 7.00E-34 acr (wobbly), TSP1 (wobbly) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|212944|fgenesh1_pg.scaffold_166000015
ref|NP_001074489.1| a disintegrin-like and metalloprotease (repr… 2.00E-80
acr, TSP1, ADAM_spacer(wobbly), TSP1 (4x wobbly), SLG (wobbly) ADAM_TS
ADAM_TS Hydra gb|CX054604.1 ADAMTS20 A short isoform [Mus musculus] 1.00E-42 ADAM_spacer ADAM_TSADAM_TS Nvec jgi|Nemve1|139926|e_gw.497.1.1 ADAMTS6 variant 2 [Homo sapiens] 2.00E-66 ADAM_spacer, TSP1 (4x), PLAC ADAM_TSADAM_TS Nvec jgi|Nemve1|99266|e_gw.52.22.1 ADAMTS6 variant 2 [Homo sapiens] 1.00E-102 ADAM_spacer, TSP1 (4x), PLAC ADAM_TS
ADAM_TS Nvecjgi|Nemve1|240559|estExt_fgenesh1_pg.C_310043
ADAM metallopeptidase with thrombospondin type ... 8.00E-51
Pep_M12B_propep, ADAM_spacer, TSP1 (3x) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|246793|estExt_fgenesh1_pg.C_2420026
ADAM metallopeptidase with thrombospondin type ... 1.00E-63
pep_M12B_propep, Reprolysin, acr (wobbly), TSP1 (3x), FN3 ADAM_TS FN3 domain is 1E-05… interesting
ADAM_TS Nvec jgi|Nemve1|87270|e_gw.15.9.1ADAM metallopeptidase with thrombospondin type ... 1.00E-157
pep_M12B_propep, Reprolysin, acr (wobbly), TSP1 (4x) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|212947|fgenesh1_pg.scaffold_166000018
gb|EAW95600.1| ADAM metallopeptidase with thrombospondin type 1 ... 1.00E-75
pep_M12B_propep, Reprolysin, acr (wobbly), TSP1, ADAM_spacer (wobbly), TSP1 (3x) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|246133|estExt_fgenesh1_pg.C_1990008
ref|NP_001074489.1| a disintegrin-like and metalloprotease (repr… 4.00E-82
pep_M12B_propep, Reprolysin, acr (wobbly), TSP1, ADAM_spacer (wobbly), TSP1 (4x), SLG (wobbly) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|246132|estExt_fgenesh1_pg.C_1990007
ref|NP_922932.2| ADAM metallopeptidase with thrombospondin type … 1.00E-104
pep_M12B_propep, Reprolysin, TSP1, acr (wobbly), ADAM_spacer (wobbly), TSP1 (3x) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|222694|fgenesh1_pg.scaffold_1855000001
gb|EAW68907.1| ADAM metallopeptidase with thrombospondin type 1 … 1.00E-51 Reprolysin, acr, TSP1 ADAM_TS
ADAM_TS Nvecjgi|Nemve1|215897|fgenesh1_pg.scaffold_242000019
gb|EAW99151.1| ADAM metallopeptidase with thrombospondin type 1 … 9.00E-58 Reprolysin, acr, TSP1 ADAM_TS
ADAM_TS Nvec jgi|Nemve1|101427|e_gw.60.18.1ref|NP_112217.2| ADAM metallopeptidase with thrombospondin type … 1.00E-125 Reprolysin, acr, TSP1 ADAM_TS
ADAM_TS Nvecjgi|Nemve1|212948|fgenesh1_pg.scaffold_166000019
gb|EDL11544.1| a disintegrin-like and metallopeptidase (reprolys… 1.00E-54 Reprolysin, acr, TSP1 (2x) ADAM_TS
ADAM_TS Nvec jgi|Nemve1|30434|gw.314.48.1gb|AAQ94616.1| COMPase precursor [Homo sapiens] 0 Reprolysin, acr, TSP1 (4x) ADAM_TS
ADAM_TS Nvecjgi|Nemve1|201879|fgenesh1_pg.scaffold_31000039
ref|NP_001074870.1| a disintegrin-like and metalloprotease (repr… 8.00E-81
Reprolysin, acr, TSP1, ADAM_spacer, TSP1 (2x) ADAM_TS
ADAM_TS Clytia IL0ABA21YG19RM1A disintegrin-like and metallopeptidase (reprolys... 4.00E-42 Reprolysin, TSP1 (wobbly), MAM ADAM_TS
ADAM_TS Nvecjgi|Nemve1|215900|fgenesh1_pg.scaffold_242000022
ref|NP_922932.2| ADAM metallopeptidase with thrombospondin type … 3.00E-76 Reprolysin, TSP1, ADAM_spacer ADAM_TS
ADAM_TS Nvec jgi|Nemve1|101342|e_gw.60.83.1ref|NP_001029049.2| ADAM metallopeptidase with thrombospondin ty… 6.00E-21 TSP (3x) ADAM_TS - putative
ADAM_TS Nvec jgi|Nemve1|115857|e_gw.134.16.1 ADAMTS3 protein [Homo sapiens] 1.00E-20 TSP1 ADAM_TS - putative
ADAM_TS Clytia SA0AAB75YP11RM1ADAM metallopeptidase with thrombospondin type 1 ... 1.00E-15 TSP1 (2x), TSP1 (wobbly) ADAM_TS - putative
ADAM_TS Clytia SA0AAB108YD12CTG ADAMTS-like 3 [Homo sapiens] 1.00E-19 TSP1 (3x) ADAM_TS - putativeADAM_TS SA0AAB75YH20RM1 ADAMTS9 1.00E-15 TSP1 (3x) ADAM_TS - putative
ADAM_TS Nvec jgi|Nemve1|5633|gw.2032.1.1ref|NP_001074489.1| a disintegrin-like and metalloprotease (repr… 1.00E-16 TSP1 (wobbly) ADAM_TS - putative
ADAM_TS Hydra CL1752Contig1ADAM metallopeptidase with thrombospondin type ... 4.00E-10 TSP1, MAM ADAM_TS - putative
ADAM_TS Nvecjgi|Nemve1|224190|fgenesh1_pg.scaffold_4120000002
ref|NP_780523.2| a disintegrin-like and metalloprotease (reproly… 3.00E-27 GON ADAM_TS GON
ADAM_TS Nvec jgi|Nemve1|95408|e_gw.39.117.1ref|NP_891550.1| ADAM metallopeptidase with thrombospondin type … 0
Reprolysin, acr, TSP1, ADAM_spacer, TSP1 (13x), GON ADAM_TS GON
ADAM_TS Hydra gb|CN632985.1 ADAMTS9 protein [Homo sapiens] 7.00E-23 TSP1, GON ADAM_TS GON ref|XP_002166940.1| PREDICTED: similar to abnormal GONad develop... 1.00E-81
FG-GAP containing Nvec jgi|Nemve1|103100|e_gw.67.19.1 Bardet-Biedl syndrome 7 [Mus musculus] 0 FG-GAP (wobbly), BB1(wobbly)Bardet-Biedl syndrome 7 [Mus musculus]
FG-GAP containing Hydra CL337Contig1 embryonic-1 [Hydra vulgaris] 1.00E-126 FG-GAP (3x wobbly) Embryonic-1
Embryonic-1 = SP, one wobbly FG-GAP repeat in its C-terminal. BLAST is hitting an almost perfect match along the full length of the contig (267aa). Embryonic is 340aa.
FG-GAP containing Hydra CL1Contig798 embryonic-1 [Hydra vulgaris] 1.00E-133 FG-GAP (4x wobbly) Embryonic-1
FG-GAP containing Hydra CL8462Contig1ASPIC/UnbV domain-containing protein [Opitut... 2.00E-16 FG-GAP (3x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|235159|estExt_fgenesh1_pm.C_1000002
Bardet-Biedl syndrome 2 [Mus musculus] >gi|2045... 0 FG-GAP (3x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|161101|estExt_gwp.C_180226
DEX1 (DEFECTIVE IN EXINE FORMATION 1) [Arabidop... 2.00E-46 FG-GAP (5x wobbly) FG-GAP containing
FG-GAP containing Clytia SA0AAA22YK19RM1FG-GAP repeat protein [bacterium Ellin514] >g... 4.00E-11 FG-GAP (5x wobbly) FG-GAP containing
Appendix B JCUSMART survey of the cnidarian adhesome
183
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
FG-GAP containing Nvecjgi|Nemve1|245375|estExt_fgenesh1_pg.C_1570033
FG-GAP repeat-containing protein [Arabidopsis t... 9.00E-44 FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|200317|fgenesh1_pg.scaffold_21000002
integrin alpha FG-GAP repeat containing 1 [P... 1.00E-103 FG-GAP (5x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|191884|estExt_GenewiseH_1.C_2190096
integrin alpha FG-GAP repeat containing 2 [D... 2.00E-82 FG-GAP (3x wobbly) FG-GAP containing
FG-GAP containing Hydra CL4337Contig1integrin alpha FG-GAP repeat containing 2 [D... 2.00E-27 No domains FG-GAP containing
FG-GAP containing Clytia IL0ABA24YM21RM1integrin alpha FG-GAP repeat containing 2 [Homo... 3.00E-60 FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Hydra CL6840Contig1integrin alpha FG-GAP repeat containing 2 [R... 2.00E-17 FG-GAP (wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|242072|estExt_fgenesh1_pg.C_560046 ITFG3 [Salmo salar] 2.00E-08 FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Hydra CL7613Contig1 No good hits FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|203954|fgenesh1_pg.scaffold_47000013 No good hits FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|225852|fgenesh1_pg.scaffold_13181000001 predicted proteins only FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Nvec jgi|Nemve1|52083|gw.92.151.1 predicted proteins only FG-GAP (3x wobbly) FG-GAP containing
FG-GAP containing Nvecjgi|Nemve1|225927|fgenesh1_pg.scaffold_14603000001
Rhs family protein [Vibrio vulnificus CMCP6] >g... 5.00E-22 FG-GAP (2x wobbly) FG-GAP containing
FG-GAP containing Clytia SA0AAB75YF22CTGFG-GAP repeat domain protein [Verrucomicrobi... 5.00E-47 FG-GAP (7x wobbly) Integrin alpha/FG-GAP containing
FG-GAP containing Clytia SA0AAB109YA16RM1integrin alpha FG-GAP repeat containing 1 [P... 2.00E-68 FG-GAP (5x wobbly) Integrin alpha/FG-GAP containing Often there are missing or weak FG-GAPs in alpha subunits
FG-GAP containing Mbrevjgi|Monbr1|30147|fgenesh2_pg.scaffold_44000012
FG-GAP repeat-containing protein [Nostoc pun... 1.00E-25 FG-GAP (14x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12516|fgenesh1_pg.scaffold_40000040 Fibronectin type III domain protein [Chloroh... 2.00E-69 FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|34687|estExt_fgenesh2_pg.C_460013 Fibronectin type III domain protein [Chloroh... 2.00E-65 FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|24745|fgenesh2_pg.scaffold_7000293
Integrins alpha chain [Anabaena variabilis ATCC... 1.00E-19 FG-GAP (13x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12733|fgenesh1_pg.scaffold_44000011
integrins alpha chain [Stigmatella aurantiaca... 7.00E-15 FG-GAP (19x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|25716|fgenesh2_pg.scaffold_11000116 No good hits FG-GAP
FG-GAP containing Mbrevjgi|Monbr1|30879|estExt_fgenesh2_pg.C_20417 No good hits FG-GAP (2x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|6487|fgenesh1_pg.scaffold_4000369 No good hits FG-GAP (2x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|11330|fgenesh1_pg.scaffold_28000048 No good hits FG-GAP (3x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12825|fgenesh1_pg.scaffold_46000035 predicted peptides only FG-GAP (13x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|7129|fgenesh1_pg.scaffold_6000201 predicted peptides only FG-GAP (13x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|39187|estExt_fgenesh1_pg.C_410059 predicted peptides only FG-GAP (13x wobbly), trykin
FG-GAP containing Mbrevjgi|Monbr1|11861|fgenesh1_pg.scaffold_32000092 predicted peptides only
FG-GAP (13x wobbly), tyrkin, tSNARE/MA (wobbly), CCP (wobbly), EGF
FG-GAP containing Mbrevjgi|Monbr1|12802|fgenesh1_pg.scaffold_46000012 predicted peptides only FG-GAP (14x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|28592|fgenesh2_pg.scaffold_27000068 predicted peptides only FG-GAP (14x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|11774|fgenesh1_pg.scaffold_32000005 predicted peptides only FG-GAP (14x wobbly), Tyrkin
FG-GAP containing Mbrevjgi|Monbr1|9157|fgenesh1_pg.scaffold_14000063 predicted peptides only FG-GAP (14x wobbly), Tyrkin
FG-GAP containing Mbrevjgi|Monbr1|9932|fgenesh1_pg.scaffold_18000039 predicted peptides only FG-GAP (14x wobbly), Tyrkin
FG-GAP containing Mbrevjgi|Monbr1|12189|fgenesh1_pg.scaffold_36000014 predicted peptides only FG-GAP (19x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|31515|estExt_fgenesh2_pg.C_40272 predicted peptides only FG-GAP (20x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|11601|fgenesh1_pg.scaffold_30000062 predicted peptides only FG-GAP (21x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|11908|fgenesh1_pg.scaffold_33000031 predicted peptides only FG-GAP (21x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|8458|fgenesh1_pg.scaffold_11000082 predicted peptides only FG-GAP (21x wobbly)
Appendix B JCUSMART survey of the cnidarian adhesome
184
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
FG-GAP containing Mbrevjgi|Monbr1|12961|fgenesh1_pg.scaffold_50000007 predicted peptides only FG-GAP (4x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30203|fgenesh2_pg.scaffold_45000043 predicted peptides only FG-GAP (4x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30259|fgenesh2_pg.scaffold_46000046 predicted peptides only FG-GAP (4x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|5660|fgenesh1_pg.scaffold_3000158 predicted peptides only FG-GAP (4x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12719|fgenesh1_pg.scaffold_43000063 predicted peptides only FG-GAP (5x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|27189|fgenesh2_pg.scaffold_18000008 predicted peptides only FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|27321|fgenesh2_pg.scaffold_18000140 predicted peptides only FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|28819|fgenesh2_pg.scaffold_29000031 predicted peptides only FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|38366|estExt_fgenesh1_pg.C_240008 predicted peptides only FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|9601|fgenesh1_pg.scaffold_16000070 predicted peptides only FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|10026|fgenesh1_pg.scaffold_18000133 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12404|fgenesh1_pg.scaffold_39000003 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12405|fgenesh1_pg.scaffold_39000004 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|12958|fgenesh1_pg.scaffold_50000004 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|13153|fgenesh1_pg.scaffold_108000003 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|27322|fgenesh2_pg.scaffold_18000141 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|28036|fgenesh2_pg.scaffold_23000065 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|29924|fgenesh2_pg.scaffold_40000049 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|29926|fgenesh2_pg.scaffold_40000051 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30223|fgenesh2_pg.scaffold_46000010 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30227|fgenesh2_pg.scaffold_46000014 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30400|fgenesh2_pg.scaffold_50000005 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30521|fgenesh2_pg.scaffold_55000006 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|30881|estExt_fgenesh2_pg.C_20421 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|31371|estExt_fgenesh2_pg.C_30580 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|33623|estExt_fgenesh2_pg.C_220027 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|33753|estExt_fgenesh2_pg.C_240012 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|37143|estExt_fgenesh1_pg.C_100227 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|37680|estExt_fgenesh1_pg.C_150162 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|37972|estExt_fgenesh1_pg.C_180127 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|39176|estExt_fgenesh1_pg.C_410033 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|5094|fgenesh1_pg.scaffold_2000410 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|8280|fgenesh1_pg.scaffold_10000141 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|8371|fgenesh1_pg.scaffold_10000232 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|9466|fgenesh1_pg.scaffold_15000156 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|9474|fgenesh1_pg.scaffold_15000164 predicted peptides only FG-GAP (7x wobbly)
Appendix B JCUSMART survey of the cnidarian adhesome
185
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
FG-GAP containing Mbrevjgi|Monbr1|9476|fgenesh1_pg.scaffold_15000166 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|9520|fgenesh1_pg.scaffold_15000210 predicted peptides only FG-GAP (7x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|27324|fgenesh2_pg.scaffold_18000143 predicted peptides only FG-GAP (9x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|10795|fgenesh1_pg.scaffold_24000009 predicted peptides only FG-GAP, FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|11321|fgenesh1_pg.scaffold_28000039 predicted peptides only FG-GAP, FG-GAP (6x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|39388|estExt_fgenesh1_pg.C_1080001 predicted peptides only FG-GAP(11x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|37922|estExt_fgenesh1_pg.C_180022 quinoprotein (ISS) [Ostreococcus tauri] 9.00E-16 FG-GAP (5x wobbly)
FG-GAP containing Mbrevjgi|Monbr1|26194|fgenesh2_pg.scaffold_13000102
vcbs [Stigmatella aurantiaca DW4/3-1] >gi|115... 6.00E-17 FG-GAP (7x wobbly)
Appendix B JCUSMART survey of the cnidarian adhesome
186
Lectins
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes C-type Secreted Clytia SA0AAA6YN21RM1
gb|ACN91267.1| mannose receptor C1-like protein [Danio rerio] e-06 CLECT (2x) Yes No C-lectin - Secreted
C-type Secreted Clytia SA0AAB8YD20RM1 hydra homolgue only CLECT (2x) Yes No C-lectin - Secreted
ref|XP_002155423.1| PREDICTED: similar to type II transmembrane ... 263 2.00E-68ref|XP_002153863.1| PREDICTED: type II transmembrane C-type lect... 253 1.00E-65ref|XP_002159076.1| PREDICTED: similar to type II transmembrane ... 252 4.00E-65
C-type Secreted Clytia IL0ABA28YD22RM1 three hydra homolgues only e-66 CLECT (2x) Yes No C-lectin - Secreted
ref|XP_002153863.1| PREDICTED: type II transmembrane C-type lect... 254 6.00E-66ref|XP_002155423.1| PREDICTED: similar to type II transmembrane ... 254 8.00E-66ref|XP_002159076.1| PREDICTED: similar to type II transmembrane ... 246 2.00E-63
C-type Secreted Clytia SA0AAA21YL24RM1 No good hits EGF, CLECT Yes No C-lectin - SecretedC-type Secreted Clytia SA0AAB66YG22RM1 mannose receptor, C type 1 e-43 TSP1, CLECT (4x) Yes No C-lectin - Secreted
blast hits all have multiple c-lectin domains but no TSP1 domain. The TSP1 domain hits at e-11 -pfam and e-17 SMART.
C-type Secreted Hydra CL5624Contig1 No good hits CLECT Yes No C-lectin - SecretedC-type Secreted Hydra gb|CO370876.1 tyrosine kinase receptor [Hydra vulgaris] 3.00E-35 CLECT Yes No C-lectin - SecretedC-type Secreted Hydra gb|DN813687.2 tyrosine kinase receptor [Hydra vulgaris] 3.00E-23 CLECT Yes No C-lectin - SecretedC-type Secreted Mbrev
jgi|Monbr1|10838|fgenesh1_pg.scaffold_24000052 No good hits Candida_ALS, CLECT (2x) Yes No C-lectin - Secreted
C-type Secreted Mbrev
jgi|Monbr1|26977|fgenesh2_pg.scaffold_16000165 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Mbrev
jgi|Monbr1|27090|fgenesh2_pg.scaffold_17000078 No good hits CLECT (2x) Yes No C-lectin - Secreted
C-type Secreted Mbrev
jgi|Monbr1|33819|estExt_fgenesh2_pg.C_250040 No good hits CLECT (3x) Yes No C-lectin - Secreted
C-type Secreted Mbrev
jgi|Monbr1|7558|fgenesh1_pg.scaffold_7000290 No good hits CLECT (4x), LamG (2x) Yes No C-lectin - Secreted
C-type Secreted Mbrev
jgi|Monbr1|30149|fgenesh2_pg.scaffold_44000014 No good hits
CLECT, int_alpha_rpt(2x very wobbly) Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|196946|fgenesh1_pg.scaffold_4000026 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|200110|fgenesh1_pg.scaffold_19000102 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|210420|fgenesh1_pg.scaffold_121000039 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|211778|fgenesh1_pg.scaffold_143000055 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|211783|fgenesh1_pg.scaffold_143000060 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|220577|fgenesh1_pg.scaffold_542000003 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|223756|fgenesh1_pg.scaffold_3283000001 No good hits CLECT Yes No C-lectin - Secreted
C-type Secreted Nvec jgi|Nemve1|83039|e_gw.7.243.1 No good hits CLECT Yes No C-lectin - SecretedC-type Secreted Nvec
jgi|Nemve1|212709|fgenesh1_pg.scaffold_161000025 matrilin 2 [Xenopus (Silurana) tropicalis] >gi|... 2.00E-22 CLECT (2x), VWA Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|196785|fgenesh1_pg.scaffold_3000106
gb|AAR24388.1| mannose receptor C1 [Sus scrofa] 1.00E-82
CLECT (3x), F5_F8_type_C, CLECT, LamG (wobbly), CLECT, Gal_lectin, CLECT (5x), disc_4, F5_F8_type_C, disc_4, F5_F8_type_C, SEA Yes No C-lectin - Secreted Unique Archtecture with SEA F5_F8_type_C and CLECT
C-type Secreted Nvec
jgi|Nemve1|199450|fgenesh1_pg.scaffold_15000042
CUB and Sushi multiple domains 3 isoform 2 [Hom... 1.00E-79
F5_F8_type_C, CCP (12x), CLECT Yes No C-lectin - Secreted CUB and Sushi has lots of (CUB,CCP) repeated
C-type Secreted Nvec
jgi|Nemve1|208381|fgenesh1_pg.scaffold_91000082 No good hits ntp (wobbly, CLECT (wobbly) Yes No C-lectin - Secreted
C-type Secreted Nvec
jgi|Nemve1|196789|fgenesh1_pg.scaffold_3000110
brevican [Danio rerio] >gi|134054440|emb|CAM... 6.00E-10
ntp (wobbly), EGF (wobbly), LINK_2, CLECT Yes No C-lectin - Secreted
C-type Soluble Clytia IL0ABA9YE18RM1 chondroitin sulfate proteoglycan 2 [Xenopus ... e-22 CLECT No No C-lectin - Soluble chondroitin sulfate proteoglycan 2 is big with multiple domainsC-type Soluble Clytia SA0AAB101YI01CTG hydra homolgue only e-21 CLECT No No C-lectin - Soluble ref|XP_002168680.1| PREDICTED: similar to predicted protein, par…C-type Soluble Clytia SA0AAB12YJ20RM1
DC-SIGN protein isoform B [Canis lupus familiaris] e-12 CLECT, egf No No C-lectin - Soluble ref|XP_002168680.1| PREDICTED: similar to predicted protein, par... 253 4.00E-65 hydra homologue
C-type Soluble Clytia SA0AAB3YK03CTG CLEC16A protein [Homo sapiens] e-92 No domains No No CLEC16A family - Soluble ref|XP_393990.2| PREDICTED: similar to CG12753-PA, isoform A [Ap... 369 1.00E-100C-type Soluble Hydra CL123Contig2 Cnidarian only CLECT No No C-lectin - Soluble ref|XP_001639701.1| predicted protein [Nematostella vectensis] >... 8.00E-50C-type Soluble Hydra CL9574Contig1 collectin-43 [Bos taurus] 3.00E-08 CLECT No No C-lectin - SolubleC-type Soluble Hydra CL8505Contig1
gb|AAA29218.2| tyrosine kinase receptor [Hydra vulgaris] 1.00E-20 CLECT No No C-lectin - Soluble
C-type Soluble Hydra gb|CV659831.1 tyrosine kinase receptor [Hydra vulgaris] 6.00E-48 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|CV985524.1 tyrosine kinase receptor [Hydra vulgaris] 1.00E-18 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|DN816073.2 tyrosine kinase receptor [Hydra vulgaris] 1.00E-23 CLECT No No C-lectin - Soluble
Appendix B JCUSMART survey of the cnidarian adhesome
187
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
C-type Soluble Hydra gb|DT611934.1 tyrosine kinase receptor [Hydra vulgaris] 1.00E-71 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|DT616206.1 tyrosine kinase receptor [Hydra vulgaris] 2.00E-56 CLECT No No C-lectin - SolubleC-type Soluble Hydra CL5450Contig1 No good hits CLECT (2x) No No C-lectin - SolubleC-type Soluble Hydra CL648Contig1 No good hits CLECT (2x) No No C-lectin - SolubleC-type Soluble Hydra gb|DN812128.2 No good hits CLECT, F5_F8_type_C No No C-lectin - SolubleC-type Soluble Hydra CL5387Contig1
C-type lectin domain family 16, member A [Mus m... 1.00E-41 No Domains No No CLEC16A family - Soluble
C-type Soluble Mbrev
jgi|Monbr1|35486|estExt_fgenesh1_pg.C_20250 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Mbrev
jgi|Monbr1|24761|fgenesh2_pg.scaffold_7000309 No good hits
CLECT (wobbly), Recep_L_domain (wobbly) No No C-lectin - Soluble
C-type Soluble Mbrev
jgi|Monbr1|17036|estExt_gwp_gw1.C_20126 gb|AAP78681.1| MBCTL2 [Monosiga brevicollis] 0 CCP (3x very wobbly), CLECT No No
gb|AAP78681.1| MBCTL2 [Monosiga brevicollis] same domain structure also
C-type Soluble Nvec jgi|Nemve1|17962|gw.655.3.1 ACAN protein [Homo sapiens] 2.00E-20 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|45368|gw.367.41.1 ACAN protein [Homo sapiens] 2.00E-20 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|149561|e_gw.3035.1.1 aggrecan isoform 1 precursor [Homo sapiens] 9.00E-17 CLECT No No C-lectin - SolubleC-type Soluble Nvec
jgi|Nemve1|239025|estExt_fgenesh1_pg.C_110046 aggrecan isoform 2 precursor [Homo sapiens] 5.00E-15 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|7634|gw.4914.1.1
Brevican [Homo sapiens] >gi|20380804|gb|AAH27971.... 3.00E-15 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|154303|e_gw.7191.2.1 brevican core protein 8.00E-22 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|108202|e_gw.89.172.1 brevican isoform 1 [Rattus norvegicus] >gi|1... 3.00E-19 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|83478|e_gw.7.7.1
Brevican soluble core protein precursor [Xenopus ... 5.00E-12 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|88150|e_gw.17.229.1 C-type lectin [Pocillopora damicornis] 9.00E-24 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|87968|e_gw.17.235.1 C-type lectin [Pocillopora damicornis] 1.00E-22 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|6023|gw.37.6.1 C-type lectin 1 [Anguilla japonica] 3.00E-13 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|103399|e_gw.68.85.1
chondroitin sulfate proteoglycan 2 [Xenopus laevis] 5.00E-15 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|138199|e_gw.425.7.1
dbj|BAA95671.1| C-type lectin [Cyprinus carpio] 1.00E-12 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|85784|e_gw.12.21.1 endocytic receptor Endo180 [Homo sapiens] 8.00E-10 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|129425|e_gw.266.73.1 gb|AAA87847.1| brevican core protein 1.00E-21 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|137796|e_gw.413.1.1 gb|AAA87847.1| brevican core protein 1.00E-21 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|118816|e_gw.156.96.1 gb|ABD16187.1| c-type lectin [Danio rerio] 1.00E-10 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|102786|e_gw.66.179.1
gb|ACJ64661.1| hypothetical protein A043-D8 [Acropora millepora] 1.00E-18 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|154334|e_gw.7241.1.1
gb|ACN53515.1| C-type lectin [Pocillopora damicornis] 1.00E-09 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|113743|e_gw.120.98.1
mannose receptor C type 1 precursor [Homo sapie... 6.00E-13 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|66344|gw.3.677.1 mannose receptor C1-like protein [Danio rerio] 3.00E-15 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|149329|e_gw.2934.3.1
mannose receptor, C type 2, isoform CRA_a [Mus mu... 2.00E-09 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|88266|e_gw.18.164.1
neurocan [Mus musculus] >gi|40675766|gb|AAH6511... 8.00E-19 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|118814|e_gw.156.101.1
neurocan [Rattus norvegicus] >gi|1709256|sp|P55... 7.00E-16 CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|207088|fgenesh1_pg.scaffold_76000072 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|211781|fgenesh1_pg.scaffold_143000058 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|212201|fgenesh1_pg.scaffold_151000025 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|218066|fgenesh1_pg.scaffold_327000008 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|229557|fgenesh1_pm.scaffold_85000002 No good hits CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|29214|gw.76.113.1 No good hits CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|64326|gw.15.244.1 No good hits CLECT No No C-lectin - Soluble
Appendix B JCUSMART survey of the cnidarian adhesome
188
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
C-type Soluble Nvec
jgi|Nemve1|221388|fgenesh1_pg.scaffold_748000003
ref|NP_001075276.1| Fc fragment of IgE, low affinity II, recepto… 1.00E-12 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|138198|e_gw.425.36.1
ref|NP_001138689.1| Fc fragment of IgE, low affinity II, recepto… 1.00E-13 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|119405|e_gw.161.1.1
regenerating islet-derived 2 [Mus musculus] >gi... 5.00E-11 CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|145810|e_gw.1517.2.1 No good hits CLECT (wobbly) No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|78993|e_gw.1.634.1 No good hits CLECT (wobbly) No No C-lectin - SolubleC-type Soluble Nvec
jgi|Nemve1|203353|fgenesh1_pg.scaffold_42000068 No good hits CLECT, I-set, ntp No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|203358|fgenesh1_pg.scaffold_42000073 No good hits CLECT, ntp No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|210423|fgenesh1_pg.scaffold_121000042 No good hits EGF, CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|210418|fgenesh1_pg.scaffold_121000037 No good hits EGF, LINK, CLECT No No C-lectin - Soluble
C-type Soluble Nvec
jgi|Nemve1|210422|fgenesh1_pg.scaffold_121000041
chondroitin sulfate proteoglycan 2 variant V3 [Xe... 3.00E-13 LINK, CLECT No No C-lectin - Soluble
C-type Soluble Nvec jgi|Nemve1|84933|e_gw.10.237.1 protein CLEC16A, putative [Ixodes scapularis] 0 No domains No No CLEC16A family - SolubleCollectin Clytia SA0AAA11YD17CTG No good hits Collagen, CLECT No No Collectin - putative
Collectin Nvecjgi|Nemve1|199302|fgenesh1_pg.scaffold_14000067
hypothetical protein A044-C2 [Acropora millepora] 5.00E-36 Collagen, CLECT Yes No Collectin - putative Collectins have a collagen and CLECT domain. Start and stop codons present
Collectin-Like Hydra CL1Contig283hypothetical protein A044-C2 [Acropora millepora] 1.00E-69 Collagen, Gal_lectin No No Collectin like gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora]
Collectin-Like Hydra CL5890Contig1hypothetical protein A044-C2 [Acropora millepora] 1.00E-33 Collagen, Gal_lectin No No Collectin like gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora]
Collectin-Like Hydra CL684Contig1hypothetical protein A044-C2 [Acropora millepora] 8.00E-51 Collagen, Gal_lectin No No Collectin like collagen, Gal_lectin is not found in other organisms
Collectin-Like Nvecjgi|Nemve1|232015|fgsh_est.C_scaffold_14000009
hypothetical protein A044-C2 [Acropora millepora] 7.00E-81 Collagen, Gal_lectin Yes No Collectin like start and stop codons present
Collectin-Like Clytia IL0ABA6YM11RM1hypothetical protein A044-C2 [Acropora millepora] 3.00E-40 Collagen, Gal_lectin Yes No Collectin like
IL0ABA6YM11RM1 gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora] 169 3.00E-40IL0ABA6YM11RM1 ref|XP_002160660.1| PREDICTED: similar to predicted protein isof... 167 9.00E-40IL0ABA6YM11RM1 ref|XP_001639343.1| predicted protein [Nematostella vectensis] >... 160 1.00E-37. Starts with an M.
Collectin-Like Clytia IL0ABA19YN17RM1Hypothetical protein A044-C2 [Acropora millepora] 3.00E-28 Collagen, Gal_lectin Yes No Collectin like
A044-C2 is part of contig C_mge-A048-G3-post22-T which has no good blast hits and domains: SP, Collagen, Gal_lectin and 1 predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM.
Collectin-Like Nvecjgi|Nemve1|220309|fgenesh1_pg.scaffold_499000008
hypothetical protein A044-C2 [Acropora millepora] 4.00E-11
Collagen (2x), Gal_lectin (wobbly) No No Collectin like - putative start and stop present
Collectin-Like Nvecjgi|Nemve1|232014|fgsh_est.C_scaffold_14000008
hypothetical protein A032-H1 [Acropora millepora] 5.00E-62 Collagen, Gal_lectin Yes 1 Collectin like
start and stop present predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM
Collectin-Like Clytia SA0AAB24YI18RM1hypothetical protein A032-H1 [Acropora millepora] 9.00E-64 Collagen, Gal_lectin Yes 1 Collectin like
ref|XP_002157988.1| PREDICTED: similar to predicted protein isof... 262 3.00E-68ref|XP_002158019.1| PREDICTED: similar to predicted protein isof... 262 3.00E-68gb|ACJ64659.1| hypothetical protein A032-H1 [Acropora millepora] 247 9.00E-64ref|XP_001639420.1| predicted protein [Nematostella vectensis] >... 242 2.00E-62 predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM
Collectin-Like Acropora Contig31112hypothetical protein A044-C2 [Acropora millepora] 2.00E-93 Collagen, Gal_lectin Yes No Collectin like
Collectin-Like Acropora Contig12324hypothetical protein A032-H1 [Acropora millepora] 3.00E-93 Collagen, Gal_lectin Yes No Collectin like
Collectin-Like Acropora Contig8056hypothetical protein A044-C2 [Acropora millepora] 5.00E-37 Collagen, Gal_lectin No No Collectin like
Collectin-Like Acropora Contig4833hypothetical protein A044-C2 [Acropora millepora] 5.00E-11 Collagen, Gal_lectin Yes No Collectin like
Collectin-Like Acropora Contig4828hypothetical protein A043-H7 [Acropora millepora] 1.00E-86 Collagen, Gal_lectin No No Collectin like
C-type Transmembrane Clytia SA0AAA8YD10CTG
ref|NP_055545.1| malectin [Homo sapiens] >gi|2495712|sp|Q14165.1… e-64 No domains Yes 1 Malectin
SA0AAA8YD10CTG ref|XP_002168415.1| PREDICTED: similar to predicted protein [Hyd... 301 4.00E-80SA0AAA8YD10CTG ref|XP_001639476.1| predicted protein [Nematostella vectensis] >... 268 4.00E-70. No domains is consistent with the blast hit. Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation
C-type Transmembrane Clytia SA0AAB55YA06CTG hydra homolgue only Ig, CLECT (2x) Yes 1 C-type Lectin - lectin/Ig
ref|XP_002160934.1| PREDICTED: similar to predicted protein [Hyd... 315 5.00E-84 this hit contains 2 CLEC, TM.
C-type Transmembrane Hydra gb|CN774438.1 No good hits CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra gb|DN812863.2 tyrosine kinase receptor [Hydra vulgaris] 5.00E-27 CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra CL8965Contig1 No good hits CLECT No 3 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra CL1143Contig1
malectin [Homo sapiens] >gi|2495712|sp|Q14165.1... 2.00E-57 No Domains No No Malectin
Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation
Appendix B JCUSMART survey of the cnidarian adhesome
189
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
C-type Transmembrane Mbrev
jgi|Monbr1|33817|estExt_fgenesh2_pg.C_250037 MBCTL1 [Monosiga brevicollis] 3.00E-14 CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present
C-type Transmembrane Mbrev
jgi|Monbr1|34706|estExt_fgenesh2_pg.C_460051 MBCTL1 [Monosiga brevicollis] 1.00E-177 CLECT (2x) Yes 1 AAP78680 MBCTL1 MBCTL1 has 3 lectin domains however this may have been an edited model
C-type Transmembrane Nvec
jgi|Nemve1|210164|fgenesh1_pg.scaffold_117000056 aggrecan isoform 1 precursor [Homo sapiens] 2.00E-16 ntp (wobbly, CLECT (wobbly) No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present
C-type Transmembrane Nvec
jgi|Nemve1|211915|fgenesh1_pg.scaffold_146000014
gb|ABW80963.1| acidic repeat protein [Treponema paraluiscuniculi] 1.00E-55
Astacin, TSP1, CLECT, TSP1 (2x) No 2 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present
C-type Transmembrane Nvec
jgi|Nemve1|221181|fgenesh1_pg.scaffold_685000004 cyclase family protein [Roseovarius sp. 217] ... 1.00E-15 CLECT (wobbly), cyclase No 2 C-type Lectin - TM
Can not be confirmed that these are Type II or Type III. Start and stop codons present Unique architecture CLECT, TM, Cyclase, TM
C-type Transmembrane Nvec
jgi|Nemve1|248367|estExt_fgenesh1_pg.C_5290004
gb|ABK78718.1| dendritic cell immunoactivating receptor [Anas pl… 1.00E-07 CLECT Yes 1 C-type Lectin - TM Type I
C-type Transmembrane Nvec
jgi|Nemve1|197952|fgenesh1_pg.scaffold_7000235 No good hits CLECT Yes 1 C-type Lectin - TM Type I
C-type Transmembrane Nvec
jgi|Nemve1|238062|estExt_fgenesh1_pg.C_30109
gb|AAR24388.1| mannose receptor C1 [Sus scrofa] 1.00E-72
CLECT (3x), F5_F8_type_C, CLECT, LamG (wobbly), CLECT (5x), disc4, F5_F8_type_C (2x), SEA (wobbly) Yes 1 C-type Lectin - TM Type I Unique Architecture
C-type Transmembrane Nvec
jgi|Nemve1|199676|fgenesh1_pg.scaffold_16000126 Pod-EPPT [Podocoryne carnea] 8.00E-11 CLECT (wobbly), ntp (wobbly) Yes 1 C-type Lectin - TM Type I
C-type Transmembrane Nvec
jgi|Nemve1|204386|fgenesh1_pg.scaffold_50000070
PTX (wobbly), CLECT, ntp (wobbly) Yes 1 C-type Lectin - TM Type I
C-type Transmembrane Nvec
jgi|Nemve1|198924|fgenesh1_pg.scaffold_12000147 No good hits CLECT (2x) Yes 2 C-type Lectin - TM Type I probably only single pass
C-type Transmembrane Nvec
jgi|Nemve1|179566|estExt_GenewiseH_1.C_130133
ref|NP_055545.1| malectin [Homo sapiens] >gi|2495712|sp|Q14165.1… 1.00E-53 No domains Yes No Malectin
malectin has no domains. Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation
Fucolectin Nvecjgi|Nemve1|216294|fgenesh1_pg.scaffold_256000002 Fucolectin-4 precursor [Esox lucius] 2.00E-06 CLECT, ftp No 2 Fucolectin - Transmembrane
There are only 2 transmembrane fucolectins… Drosophila- furrowed (CCP repeats, one CLECT, 1 ftp) and Tachylectin-4 (SP, ftp) which recognizes bacterial lipopolysaccharide, probably through binding to fucose-like sugars. Start and stop codons are presnt. Probable artifact due to lack of EST support and SP, highly repetative TM region, short scaffold.
Fucolectin Nvecjgi|Nemve1|214591|fgenesh1_pg.scaffold_204000015 FBP32 precursor [Morone chrysops] 2.00E-31 ftp Yes No Fucolectin - Secreted (FBP32 like)
Fucolectin Nvecjgi|Nemve1|221173|fgenesh1_pg.scaffold_683000006 Fucolectin-4 precursor [Esox lucius] 8.00E-28 ftp Yes No Fucolectin - Secreted
Fucolectin Nvecjgi|Nemve1|221640|fgenesh1_pg.scaffold_887000005 No good hits F5_F8_type_C, ftp (wobbly) Yes No Fucolectin - Secreted
Fucolectin Nvec jgi|Nemve1|144857|e_gw.1185.2.1 bryohealin precursor [Bryopsis plumosa] 8.00E-18 ftp No No Fucolectin - Soluble
Fucolectin Nvec jgi|Nemve1|100075|e_gw.55.26.1 FBP32 precursor [Morone saxatilis] 1.00E-41 ftp No No Fucolectin - Solubleeel-Fucolectin Tachylectin-4 Pentaxrin-1 Domain, ftp domain is the SMART version of F5_F8_type_C/FA58C
Fucolectin Nvec jgi|Nemve1|123063|e_gw.196.12.1 FBP32II precursor [Morone chrysops] 3.00E-51 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|11782|gw.196.4.1 FBP32II precursor [Morone chrysops] 9.00E-38 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|106318|e_gw.81.40.1 fucolectin [Fundulus heteroclitus] 2.00E-27 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|8429|gw.9884.1.1 Fucolectin-4 precursor [Esox lucius] 7.00E-32 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|130395|e_gw.279.36.1 Fucolectin-4 precursor [Esox lucius] 5.00E-31 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|17374|gw.224.24.1 Fucolectin-4 precursor [Esox lucius] 3.00E-30 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|113390|e_gw.117.83.1 Fucolectin-4 precursor [Esox lucius] 3.00E-29 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|101196|e_gw.60.139.1 Fucolectin-4 precursor [Esox lucius] 2.00E-26 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|16092|gw.174.11.1 Fucolectin-4 precursor [Esox lucius] 4.00E-24 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|28280|gw.196.56.1 Fucolectin-4 precursor [Esox lucius] 6.00E-23 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|101150|e_gw.59.65.1 Fucolectin-4 precursor [Esox lucius] 5.00E-18 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|155896|e_gw.9311.2.1 Fucolectin-4 precursor [Esox lucius] 4.00E-17 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|6868|gw.741.1.1 Fucolectin-4 precursor [Esox lucius] 2.00E-13 ftp No No Fucolectin - Soluble
Fucolectin Nvecjgi|Nemve1|217467|fgenesh1_pg.scaffold_298000014 Fucolectin-4 precursor [Esox lucius] 7.00E-07 ftp No No Fucolectin - Soluble
Fucolectin Nvec jgi|Nemve1|9509|gw.10015.2.1 No good hits ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|123139|e_gw.196.46.1 FBP32 precursor [Morone saxatilis] 3.00E-47 ftp (2x) No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|112201|e_gw.110.50.1 fucose binding lectin [Dicentrarchus labrax] 5.00E-43 ftp (2x) No No Fucolectin - Soluble
Fucolectin Nvecjgi|Nemve1|239599|estExt_fgenesh1_pg.C_170022
furrowed [Aedes aegypti] >gi|108875029|gb|EA... 3.00E-12 Ricin_B_lectin (wobbly), ftp No No Fucolectin - Soluble
Fucolectin Nvec jgi|Nemve1|65775|gw.61.259.1 No good hits ftp No No Fucolectin - SolubleFucolectin Hydra CL3322Contig1 No good hits ftp Yes No Fucolectin - SecretedFucolectin Acropora Contig2805 BTB, ftp FucolectinFucolectin Acropora Contig8409 ftp FucolectinFucolectin Acropora run002_427609 ftp FucolectinFucolectin Acropora Contig11806 ftp FucolectinFucolectin Acropora Amil_c11587 ftp FucolectinFucolectin Acropora run002_434733 ftp FucolectinFucolectin Acropora run001daytona_1706338 ftp (2x) Fucolectin
Appendix B JCUSMART survey of the cnidarian adhesome
190
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
Fucolectin Acropora run001daytona_1732288 ftp FucolectinFucolectin Acropora Amil_c56240 ftp FucolectinFucolectin Acropora run001daytona_1712753 ftp FucolectinFucolectin Acropora run002_436148 ftp FucolectinFucolectin Acropora Contig18100 ftp, disc4 FucolectinFucolectin Acropora run001daytona_1711122 ftp FucolectinFucolectin Acropora run001daytona_1719357 ftp FucolectinFucolectin Acropora run001daytona_1723531 ftp FucolectinFucolectin Acropora run001daytona_1714209 ftp FucolectinFucolectin Acropora run001daytona_1704584 ftp FucolectinFucolectin Acropora Contig2564 ricin_B_lectin(wobbly),ftp FucolectinFucolectin Acropora Amil_c24802 ftp FucolectinFucolectin Acropora Contig25156 ftp FucolectinFucolectin Acropora Amil_c83456 ftp Fucolectin
legume like Hydra CL4270Contig1mannose-binding endoplasmic reticulum-golgi inter... 3.00E-75 Lectin_leg-like Yes 1
mannose-binding endoplasmic reticulum-golgi inter...
legume like Hydra CL641Contig1Vesicular integral-membrane protein VIP36 precurs... 1.00E-84 Lectin_leg-like No No
Vesicular integral-membrane protein VIP36 precurs...
legume like Mbrevjgi|Monbr1|33100|estExt_fgenesh2_pg.C_160036
ref|XP_001654670.1| vesicular mannose-binding lectin [Aedes aegy… 1.00E-66 Lectin_leg-like Yes 1 vesicular mannose-binding lectin
legume like Mbrevjgi|Monbr1|34633|estExt_fgenesh2_pg.C_430019 vesicular mannose-binding lectin [Aedes aegy... 1.00E-52 Lectin_leg-like Yes 1 vesicular mannose-binding lectin
legume like Nvec jgi|Nemve1|129670|e_gw.268.1.1gb|EDM14702.1| lectin, mannose-binding, 1, isoform CRA_a [Rattus… 1.00E-102 Lectin_leg-like No 1 lectin, mannose-binding
legume like Nvec jgi|Nemve1|82673|e_gw.6.366.1 vesicular mannose-binding lectin [Aedes aegy... 2.00E-90 Lectin_leg-like Yes 1 vesicular mannose-binding lectinGal-lectin Hydra CL1084Contig1 No good hits Gal_lectin Yes 1Gal-lectin Hydra CL1096Contig1 No good hits Gal_lectin No NoGal-lectin Hydra CL2279Contig1 No good hits Gal_lectin Yes 1Gal-lectin Hydra CL3738Contig1 No good hits Gal_lectin No NoGal-lectin Hydra gb|CO537930.1 No good hits Gal_lectin No NoGal-lectin Hydra gb|DN245375.2 No good hits Gal_lectin No NoGal-lectin Hydra gb|DT614072.1 No good hits Gal_lectin Yes NoGal-lectin Hydra CL1172Contig1 No good hits Gal_lectin (2x) No No
Gal-lectin Hydra CL320Contig1rhamnose-binding lectin OLL [Spirinchus lanceola... 5.00E-29 Gal_lectin (2x) Yes No
Gal-lectin Hydra CL3363Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-27 Gal_lectin (2x) Yes 1
Gal-lectin Hydra CL385Contig2 24 kDa egg lectin [Oncorhynchus tshawytscha] 4.00E-31 Gal_lectin (2x) No NoGal-lectin Hydra CL385Contig3 24 kDa egg lectin [Oncorhynchus tshawytscha] 2.00E-30 Gal_lectin (2x) No NoGal-lectin Hydra CL385Contig1 rhamnose binding lectin [Tribolodon brandtii] 3.00E-34 Gal_lectin (3x) No No
Gal-lectin Hydra CL9067Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 1.00E-31 Gal_lectin (3x) No No
Gal-lectin Hydra CL1523Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-30 Gal_lectin (4x) No No
Gal-lectin Hydra CL2416Contig1rhamnose binding lectin STL3 [Oncorhynchus m... 4.00E-26 Gal_lectin (4x) No No
Gal-lectin Hydra CL55Contig1rhamnose binding lectin STL3 [Oncorhynchus m... 7.00E-30 Gal_lectin (4x) No No
Gal-lectin Hydra CL5403Contig1 No good hits Gal_lectin (wobbly) No No
Gal-lectin Hydra CL6176Contig1nematocyst outer wall antigen precursor [Clytia h... 2.00E-38
Gal_lectin (wobbly), keratin_B2 (wobbly) No No
Gal-lectin Nvecjgi|Nemve1|241976|estExt_fgenesh1_pg.C_540065 No good hits CD20 (wobbly), Gal_lectin
Gal-lectin Nvecjgi|Nemve1|214497|fgenesh1_pg.scaffold_202000001 depsiphilin [Cooperia oncophora] 1.00E-10 Gal_lectin
Gal-lectin Nvecjgi|Nemve1|216053|fgenesh1_pg.scaffold_247000022 depsiphilin [Cooperia oncophora] 4.00E-07 Gal_lectin
Gal-lectin Nvecjgi|Nemve1|232013|fgsh_est.C_scaffold_14000007
hypothetical protein A043-H7 [Acropora millepora] 5.00E-40 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|18799|gw.247.36.1 lectomedin 1 alpha-like [Ciona intestinalis] 1.00E-12 Gal_lectinGal-lectin Nvec jgi|Nemve1|102384|e_gw.64.13.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|106512|e_gw.82.91.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111827|e_gw.108.32.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111847|e_gw.108.96.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111952|e_gw.108.39.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|118227|e_gw.152.81.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|124712|e_gw.213.49.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|124735|e_gw.213.92.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142147|e_gw.645.22.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142154|e_gw.645.34.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142157|e_gw.645.18.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142164|e_gw.645.13.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142165|e_gw.645.31.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142168|e_gw.645.15.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|148507|e_gw.2440.4.1 No good hits Gal_lectin
Gal-lectin Nvecjgi|Nemve1|205905|fgenesh1_pg.scaffold_64000028 No good hits Gal_lectin
Gal-lectin Nvecjgi|Nemve1|216054|fgenesh1_pg.scaffold_247000023 No good hits Gal_lectin
Appendix B JCUSMART survey of the cnidarian adhesome
191
Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes
Gal-lectin Nvecjgi|Nemve1|246049|estExt_fgenesh1_pg.C_1940004 No good hits Gal_lectin
Gal-lectin Nvec jgi|Nemve1|128256|e_gw.247.49.1 novel rhamnose binding lectin [Danio rerio] 9.00E-09 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|87157|e_gw.15.283.1rhamnose-binding lectin precursor [Branchiostoma ... 3.00E-16 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|142145|e_gw.645.12.1rhamnose-binding lectin precursor [Branchiostoma ... 3.00E-15 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|148434|e_gw.2408.1.1rhamnose-binding lectin precursor [Branchiostoma ... 3.00E-15 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|146503|e_gw.1697.9.1rhamnose-binding lectin precursor [Branchiostoma ... 2.00E-14 Gal_lectin
Gal-lectin Nvec jgi|Nemve1|82364|e_gw.5.561.1 skin mucus lectin [Leiognathus nuchalis] 1.00E-08 Gal_lectin
Gal-lectin Nvecjgi|Nemve1|209268|fgenesh1_pg.scaffold_103000043
rhamnose-binding lectin OLL [Spirinchus lanceola... 3.00E-13 Gal_lectin (2x wobbly)
Gal-lectin Nvec jgi|Nemve1|106614|e_gw.82.111.1 No good hits Gal_lectin (2x)Gal-lectin Nvec jgi|Nemve1|140909|e_gw.552.7.1 No good hits Gal_lectin (2x)
Gal-lectin Nvecjgi|Nemve1|205904|fgenesh1_pg.scaffold_64000027
Rhamnose-binding lectin [Salmo salar] >gi|20... 2.00E-15 Gal_lectin (2x)
Gal-lectin Nvec jgi|Nemve1|174671|estExt_gwp.C_3180014rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-30 Gal_lectin (2x)
Gal-lectin Nvec jgi|Nemve1|122916|e_gw.194.48.1 rhamnose binding lectin [Tribolodon brandtii] 7.00E-37 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|142158|e_gw.645.36.1 rhamnose binding lectin [Tribolodon brandtii] 2.00E-36 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|111951|e_gw.108.118.1 rhamnose binding lectin [Tribolodon brandtii] 2.00E-34 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|131398|e_gw.294.64.1 rhamnose binding lectin [Tribolodon brandtii] 3.00E-21 Gal_lectin (3x)
Gal-lectin Nvecjgi|Nemve1|237832|estExt_fgenesh1_pg.C_10304 rhamnose binding lectin [Tribolodon brandtii] 1.00E-14 Gal_lectin (4x)
Gal-lectin Nvecjgi|Nemve1|212656|fgenesh1_pg.scaffold_160000021
hypothetical protein A044-C2 [Acropora millepora] 2.00E-14 Gal_lectin (wobbly)
Gal-lectin Nvecjgi|Nemve1|222353|fgenesh1_pg.scaffold_1557000001 No good hits Gal_lectin (wobbly)
Gal-lectin Nvecjgi|Nemve1|212847|fgenesh1_pg.scaffold_164000008
gb|AAX09934.1| ganglioside M2 activator-like protein [Aurelia au… 1.00E-22
Gal_lectin (wobbly), pgtp_13 (wobbly)
Gal-lectin Mbrevjgi|Monbr1|12732|fgenesh1_pg.scaffold_44000010 No good hits EGF (2x), Gal_lectin No 1
Gal-lectin Mbrevjgi|Monbr1|27352|fgenesh2_pg.scaffold_19000006
Beta-galactosidase precursor, putative, expressed... 2.00E-18 Gal_lectin No No
Gal-lectin Clytia IL0ABA5YA20RM1 novel rhamnose binding lectin [Danio rerio] e-10 Gal_lectin No No
ref|XP_002161306.1| PREDICTED: similar to novel rhamnose binding... 198 2.00E-49 this has 2xGal_lec. The Rhaminose binding lectin has one Gal_lec domain and is very short… poor hit indicates this may be something different but length is similar (this starts with an M)
Gal-lectin Clytia SA0AAB36YH22RM1 hydra homolgue only Wobbly Gal_lectin No 1 ref|XP_002169231.1| PREDICTED: similar to LOC733382 protein [Hyd... 151 1.00E-34
Gal-lectin Clytia SA0AAB73YD10CTG Cnidarian homolgues only Gal_lectin Yes No
ref|XP_002169260.1| PREDICTED: similar to predicted protein, par... 218 3.00E-55ref|XP_002159989.1| PREDICTED: similar to predicted protein [Hyd... 218 4.00E-55ref|XP_002160043.1| PREDICTED: similar to predicted protein [Hyd... 153 1.00E-35ref|XP_001639419.1| predicted protein [Nematostella vectensis] >... 130 1.00E-28gb|ACJ64660.1| hypothetical protein A043-H7 [Acropora millepora] 119 3.00E-25
Gal-lectin Clytia IL0ABA4YP02RM1gb|ABY71251.1| nematocyst outer wall antigen precursor [Clytia h... 0 Gal_lectin Yes No gb|ABY71251.1| nematocyst outer wall antigen precursor [Clytia h...
haemolytic Clytia IL0ABA8YL09RM1 CELIII 1.00E-93 2x ricin CELIII
haemolytic Clytia IL0ABA2YE22RM1 CELIII 1.00E-92 2xricin CELIII
has same domain structure and a good blast - heamolytic lectin gb|ACJ64657.1| hypothetical protein A049-E7 [Acropora millepora] 319 4.00E-85gb|ACJ64656.1| hypothetical protein A036-E7 [Acropora millepora] 305 6.00E-81
haemolytic Acropora A036-E7 A036-E7 0 2xricin CELIIIhaemolytic Acropora A049-E7 A049-E7 0 2xricin CELIIIhaemolytic Acropora Contig20384 CELIII 1.00E-131 2xricin CELIII
legume like Acropora Contig6923ref|NP_446338.1| lectin, mannose-binding, 1 [Rattus norvegicus] 1.00E-96 Lectin_leg-like
legume like Acropora run002_224851 vesicular mannose-binding lectin [Aedes aegy... 1.00E-72 Lectin_leg-likelegume like Acropora Contig768 ref|XP_001843193.1| VIP36 1.00E-85 Lectin_leg-like
Appendix B JCUSMART survey of the cnidarian adhesome
192
LRR Adhesion
Sub Family Organism Sequence Id BLAST E-value HMMPFAM SP TM Conclusion Notes
LRR-dsl Nematostella jgi|Nemve1|200004|fgenesh1_pg.scaffold_18000128leucine rich repeat containing 15 [Rattus norve... 6.00E-22 lrrnt (wobbly), LRR (8x 7wobbly), GCC2_GCC3, dsl Yes 7 LRR-dsl
LRR-EGF Clytia SA0AAA15YN12CTG noLRR (3x wobbly), EGF (3x wobbly), ringv (wobbly), EGF (wobbly) No 1 LRR-EGF
LRR-EGF Nematostella jgi|Nemve1|203583|fgenesh1_pg.scaffold_44000028 uromodulin 2.00E-14 lrrct, EGF (3x) No 1 LRR-EGF
LRR-EGF Nematostella jgi|Nemve1|199637|fgenesh1_pg.scaffold_16000087slit-like 2 [Homo sapiens] >gi|74748436|sp|Q6EM... 2.00E-22 LRR (9x 8wobbly), lrrct, EGF (4x) Yes 1 LRR-EGF
LRR-EGF Nematostella SA0AAB6YN08RM1 no good hits LRR (6x wobbly), lrrct (wobbly), EGF Yes 1 LRR-EGF
LRR-EGF Nematostella jgi|Nemve1|208146|fgenesh1_pg.scaffold_89000008 microneme protein 4 [Eimeria tenella] 1.00E-40 lrrct (wobbly), EGF (5x) Yes 1 LRR-EGFVILL/gel domains now called GEL by SMART - Gelsolin/severin/villin homology domain
LRR-EGF Clytia jgi|Nemve1|197001|fgenesh1_pg.scaffold_4000081 no good hits lrrnt, LRR (5x), EGF, GCC2_GCC3 (all wobbly) Yes No LRR-EGF
LRR-FN3 Monosiga jgi|Monbr1|31954|estExt_fgenesh2_pg.C_60352Leukocyte-antigen-related-like, isoform B [Dros... 2.00E-22 lrrnt, LRR (10x wobbly), lrrct (wobbly), FN3 (5x) Yes 1 LRR-FN3
LRR-FN3 Nematostella jgi|Nemve1|248399|estExt_fgenesh1_pg.C_5430006ref|NP_001094549.1| protein tyrosine phosphatase, receptor type,… 2.00E-48
F-box (2x wobbly), LRR (3x wobbly), Pentaxin, LamG (wobbly), FBG, PTX, 109ultra, f5_f5_type_C (2x wobbly), MAM (wobbly), LamG (2x wobbly), VirB8 (wobbly), FN3 (9x),TM, Ion_trans_2, Yes 3 LRR-FN3
4260-4282, 4328-4350, 4508-4525 = TM regions. The first 2 overlap with the Ion_trans domain. May not be a surface protein because it has an F-box.
LRR-IG Nematostella jgi|Nemve1|247910|estExt_fgenesh1_pg.C_3760020gb|EDL91410.1| leucine-rich repeats and immunoglobulin-like doma… 6.00E-32
lrrnt, LRR (7x 6wobbly), lrrct, IG, I-set, EGF (wobbly) Yes No LRR-IG
LRR-IG Nematostella jgi|Nemve1|238858|estExt_fgenesh1_pg.C_90051ref|NP_700356.2| leucine-rich repeats and immunoglobulin-like do… 1.00E-131
lrrnt, LRR (10x 6wobbly), lrrct, IG, I-set (2x), ChitinBD_3 (wobbly) Yes 1 LRIG3
LRR-LDLa Monosiga jgi|Monbr1|38718|estExt_fgenesh1_pg.C_300050 no good hits LRR (3x wobbly), LDLa, tyrkin No 1 LRR-LDLa
LRR-LDLa Monosiga jgi|Monbr1|29577|fgenesh2_pg.scaffold_36000018 Epha4a protein [Danio rerio] 5.00E-24 FBG (wobbly), LRR (wobbly), LDLa, tyrkin Yes 1 LRR-LDLadomain structure is very different to Ephrin recept 4a. The fibrinogen C-term may be false hit making this an kinase.
LRR-LDLa Monosiga jgi|Monbr1|37789|estExt_fgenesh1_pg.C_160136 no good hits LRR (2x), LDLa, SKG6 (all wobbly) Yes 1 LRR-LDLa
LRR-LDLa Monosiga jgi|Monbr1|34608|estExt_fgenesh2_pg.C_420022zeta-chain associated protein kinase 70kDa isof... 3.00E-35 LRR (3x wobbly), LDLa (wobbly), tyrkin yes 1 LRR-LDLa
LRR-LDLa Monosiga jgi|Monbr1|28676|fgenesh2_pg.scaffold_28000035 no good hits LRR, LDLa, tyrkin Yes 1 LRR-LDLa
LRR-LDLa Monosiga jgi|Monbr1|33342|estExt_fgenesh2_pg.C_180096Nef associated protein 1 [Homo sapiens] >gi|152... 3.00E-40 lrrnt (wobbly), LDLa, Pkinase_tyr, UPF0066 yes 1 LRR-LDLa TMH is in the SP
LRR-LDLa Monosiga jgi|Monbr1|12385|fgenesh1_pg.scaffold_38000060 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 2 LRR-LDLa only 1 TMH is real.LRR-LDLa Monosiga jgi|Monbr1|32039|estExt_fgenesh2_pg.C_70171 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 2 LRR-LDLa only 1 TMH is real.
LRR-LDLa Monosiga jgi|Monbr1|34526|estExt_fgenesh2_pg.C_390046gb|EEB18061.1| tyrosine-protein kinase transmembrane receptor RO… 2.00E-32 LRR (3x wobbly), LDLa, tyrkin,Pkinase_tyr Yes 2 LRR-LDLa only 1 TMH is real.
LRR-LDLa Monosiga jgi|Monbr1|7318|fgenesh1_pg.scaffold_7000050 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 3 LRR-LDLa one is in the sp
LRR-LDLa Monosiga jgi|Monbr1|33520|estExt_fgenesh2_pg.C_200100ref|ZP_03154554.1| WD-40 repeat protein [Cyanothece sp. PCC 7822… 1.00E-48
WD40 (7x 1wobbly), DUF1042, ADK (wobbly), LRR (wobbly), LDLa, tyrkin, No No LRR-LDLa
LRR-LDLa Monosiga jgi|Monbr1|25751|fgenesh2_pg.scaffold_11000151 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin (wobbly) Yes No LRR-LDLa
LRR-SUSHI Nematostella jgi|Nemve1|197996|fgenesh1_pg.scaffold_8000037 no good hitsMAM (wobbly), CCP (wobbly), SR, LRR (4x), EGF (2x wobbly) Yes 5 LRR-SUSHI
LRR-SUSHI / LRR-dsl Monosiga jgi|Monbr1|33936|estExt_fgenesh2_pg.C_270059 no good hits LRR (6x 3wobbly), CCP (4x wobbly), dsl, SKG6 Yes 1 LRR-SUSHI / LRR-dsl
dsl- Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates -pfam
Scribbled Nematostella jgi|Nemve1|10117|gw.267.1.1 Scrib protein [Mus musculus] 1.00E-122 LRR (13x wobbly), PDZ (4x) No No scribbledScribbled has a role in cell polarity pathways and is associated with Fat-1.
Scribbled Acropora Contig8961scribbled homolog [Danio rerio] >gi|71000206... 1.00E-150 LRR (15x wobbly), PDZ (4x) No No Scribbled
VWA-LRR Clytia SA0AAB78YB05CTG no good hits VWA (wobbly), lrrnt (wobbly) Yes No VWA-LRRLRR-IG Acropora Amil_c39609 gb|AAI38423.1|,Lrig2 protein [Mus musculus] 1.00E-14 LRRCT, I-set LRR-IGLRR-IG Acropora run002_428190 no good hits LRRCT, I-set LRR-IGLRR-EGF Acropora Amil_c47067 no good hits LRR(3x), EGF (2x) LRR-EGF
LRR-IG Acropora Contig16713sp|Q6P1C6.1|LRIG3_MOUSE RecName: Full=Leucine-rich repeats and i… 1.00E-109 LRRNT, LRR (14x wobbly), lrrct, Ig, I-set (2x) Yes 1 LRIG3
LRR-IG Acropora Contig24535 gb|EDL91410.1| LRIG 1.00E-51 LRR (12x), Ig (2x) LRR-IG
Appendix B JCUSMART survey of the cnidarian adhesome
193
Class B GPCR
Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
7tm_2 Only Acropora Amil_c32588brain-specific angiogenesis inhibitor 3 [Mus mu... 1.00E-11 7tm_2 7tm_2
7tm_2 Only Acropora Amil_c47079class B secretin-like G-protein coupled receptor ... 3.00E-17 7tm_2 7tm_2
7tm_2 Only Acropora Amil_c53870latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-22 7tm_2 7tm_2
7tm_2 Only Acropora Amil_c5902G protein-coupled receptor 64 [Mus musculus] 6.00E-08 7tm_2 7tm_2
7tm_2 Only Acropora Amil_c68305G protein-coupled receptor 133 [Danio rerio]... 1.00E-37 7tm_2 7tm_2
7tm_2 Only Acropora Contig10827 Bai3 protein [Mus musculus] 2.00E-32 7tm_2 7tm_2
7tm_2 Only Acropora Contig11360G protein-coupled receptor 133 [Bos taurus] ... 9.00E-24 7tm_2 7tm_2
7tm_2 Only Acropora Contig13559cadherin EGF LAG seven-pass G-type receptor 1 p... 3.00E-26 7tm_2 7tm_2
7tm_2 Only Acropora Contig15714 GPR133 protein [Homo sapiens] 8.00E-19 7tm_2 7tm_2
7tm_2 Only Acropora Contig1586G protein-coupled receptor 133 [Bos taurus] ... 2.00E-38 7tm_2 7tm_2
7tm_2 Only Acropora Contig17422Latrophilin receptor protein 2 [Brugia malay... 1.00E-13 7tm_2 7tm_2
7tm_2 Only Acropora Contig19559G-protein coupled receptor GPR133 [Homo sapiens] 3.00E-49 7tm_2 7tm_2
7tm_2 Only Acropora Contig19686G protein-coupled receptor 157 [Danio rerio]... 6.00E-23 7tm_2 7tm_2
7tm_2 Only Acropora Contig19849latrophilin 3, isoform CRA_d [Rattus norvegicus] 3.00E-33 7tm_2 7tm_2
7tm_2 Only Acropora Contig5114 latrophilin 3, isoform CRA_d [Homo sapiens] 2.00E-22 7tm_2 7tm_2
7tm_2 Only Acropora Contig7929G protein-coupled receptor 112 [Mus musculus] >g... 6.00E-25 7tm_2 7tm_2
7tm_2 Only Acropora run001daytona_1711571latrophilin-like protein 2 [Haemonchus contortus]... 7.00E-41 7tm_2 7tm_2
7tm_2 Only Acropora Contig22262G protein-coupled receptor 157 [Danio rerio]... 8.00E-43 7tm_2(wobbly) 7tm_2
7tm_2 Only Acropora Contig26829G protein-coupled receptor 157 [Danio rerio]... 2.00E-45 7tm_2(wobbly) 7tm_2
7tm_2 Only Acropora Contig7015cAMP receptor TasA [Polysphondylium pallidum] 5.00E-14 7tm_2(wobbly) 7tm_2
7tm_2 Only Acropora run001daytona_1717054brain-specific angiogenesis inhibitor 2 [Mus mus... 7.00E-13 7tm_2(wobbly) 7tm_2
7tm_2 Only Acropora run001daytona_1725222 latrophilin 3 precursor [Homo sapiens] 7.00E-11 7tm_2(wobbly) 7tm_2
7tm_2 Only Acropora run001daytona_4304G-protein coupled receptor MtH2, putative [Ixodes... 2.00E-10 7tm_2(wobbly) 7tm_2
7tm_2 Only Clytia SA0AAB90YJ23RM1 latrophilin 2, isoform CRA_a [Homo sapiens] 1.00E-30 7tm_2 7tm_2
7tm_2 Only Clytia SA0AAB153YN24CTGlatrophilin 3, isoform CRA_h [Rattus norvegicus] 2.00E-25 7tm_2 7tm_2
7tm_2 Only Clytia SA0AAB37YC22RM1G protein-coupled receptor 157 [Danio rerio]... 4.00E-28 Dicty_CAR (wobbly), 7tm_2 7tm_2
7tm_2 Only Clytia SA0AAB111YC11CTG latrophilin 3 precursor [Homo sapiens] 3.00E-10 Dicty_CAR (wobbly), 7tm_2 7tm_2
7tm_2 Only Hydra gb|DT607625.1G protein-coupled receptor 133 [Bos taurus] ... 3.00E-15 7tm_2 7tm_2
7tm_2 Only Hydra CL2460Contig1latrophilin 3, isoform CRA_h [Rattus norvegicus] 1.00E-14 7tm_2 7tm_2
7tm_2 Only Hydra gb|CX054457.1G protein-coupled receptor 133 [Danio rerio]... 2.00E-12 7tm_2 (wobbly) 7tm_2
7tm_2 Only Hydra CL8507Contig1G-protein coupled receptor, putative [Ixodes scap... 2.00E-10 7tm_2 (wobbly) 7tm_2
7tm_2 Only Mbrevjgi|Monbr1|33227|estExt_fgenesh2_pg.C_170070
cAMP receptor cAR [Dictyostelium aureostipes] 6.00E-19 Dicty_CAR/7tm_2 (wobbly) 7tm_2
Appendix B JCUSMART survey of the cnidarian adhesome
194
Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
7tm_2 Only Nvecjgi|Nemve1|202568|fgenesh1_pg.scaffold_36000045
G protein-coupled receptor 157 [Danio rerio]... 2.00E-45 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|133486|e_gw.317.3.1
latrophilin 1, isoform CRA_a [Rattus norvegicus] 1.00E-44 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|35189|gw.75.111.1G protein-coupled receptor 157 [Danio rerio]... 1.00E-44 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|145494|e_gw.1440.2.1
G-protein coupled receptor GPR133 [Homo sapiens] 8.00E-41 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|36088|gw.29.89.1G protein-coupled receptor 133 [Bos taurus] ... 2.00E-39 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|79743|e_gw.2.248.1
latrophilin 3, isoform CRA_d [Rattus norvegicus] 9.00E-34 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|81203|e_gw.4.198.1
latrophilin 3, isoform CRA_d [Rattus norvegicus] 1.00E-31 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|83594|e_gw.8.330.1
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 9.00E-30 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|53186|gw.299.70.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-29 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|86208|e_gw.13.340.1
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 8.00E-27 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|139162|e_gw.463.5.1 latrophilin 1, isoform CRA_d [Homo sapiens] 1.00E-26 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|44457|gw.7163.2.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-26 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|61965|gw.13.327.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 2.00E-26 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|102060|e_gw.63.184.1
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-24 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|62515|gw.36.181.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 6.00E-24 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|42930|gw.418.37.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 2.00E-20 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|187681|estExt_GenewiseH_1.C_1050036
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 4.00E-20 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|200012|fgenesh1_pg.scaffold_19000004
G protein-coupled receptor 112 [Mus musculus] >g... 1.00E-17 7tm_2 7tm_2 Group III - Has unseen FA5/8 domains
7tm_2 Only Nvecjgi|Nemve1|112360|e_gw.111.101.1
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 5.00E-17 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|43482|gw.166.57.1latrophilin 2 [Homo sapiens] >gi|56204774|emb|CA... 5.00E-17 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|218198|fgenesh1_pg.scaffold_334000025
G protein-coupled receptor 133 [Bos taurus] ... 3.00E-14 7tm_2 7tm_2
7tm_2 Only Nvec jgi|Nemve1|53347|gw.155.43.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-13 7tm_2 7tm_2
7tm_2 Only Nvecjgi|Nemve1|242602|estExt_fgenesh1_pg.C_670060
G protein-coupled receptor 157 [Danio rerio]... 1.00E-42 7tm_2 (wobbly) 7tm_2
7tm_2 Only Nvecjgi|Nemve1|131939|e_gw.302.47.1
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-19 7tm_2 (wobbly) 7tm_2
7tm_2 Only Nvecjgi|Nemve1|215652|fgenesh1_pg.scaffold_234000007
G protein-coupled receptor 133 [Bos taurus] ... 3.00E-13 7tm_2 (wobbly) 7tm_2
7tm_2 Only Nvecjgi|Nemve1|131887|e_gw.302.38.1 predicted peptides only 7tm_2 (wobbly) 7tm_2
7tm_2 Only Nvecjgi|Nemve1|198777|fgenesh1_pg.scaffold_11000189 predicted peptides only 7tm_2 (wobbly) 7tm_2
7tm_2 Only Nvecjgi|Nemve1|211774|fgenesh1_pg.scaffold_143000051 No good hits 7tm_2 (wobbly) 7tm_2
CLECT-GPS-7tm_2 Mbrev
jgi|Monbr1|37365|estExt_fgenesh1_pg.C_120196 MB7TM1 [Monosiga brevicollis] 2.00E-08 CLECT (wobbly), GPS, 7tm_2
Group V -Adhesion homologues in C.elegans (2) - PFAM
Appendix B JCUSMART survey of the cnidarian adhesome
195
Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes CLECT-GPS-7tm_3 Nvec
jgi|Nemve1|201898|fgenesh1_pg.scaffold_31000058
gb|EEB17331.1| class B secretin-like G-protein coupled receptor … 9.00E-45 CLECT, GPS, 7tm_2
Group V -Adhesion homologues in C.elegans (2) - PFAM
CLECT-GPS-7tm_4 Nvec
jgi|Nemve1|204814|fgenesh1_pg.scaffold_54000011 latrophilin 3, isoform CRA_b [Homo sapiens] 2.00E-48 CLECT, GPS, 7tm_2
Group V -Adhesion homologues in C.elegans (2) - PFAM
CLECT-GPS-7tm_5 Nvec
jgi|Nemve1|211772|fgenesh1_pg.scaffold_143000049 latrophilin 2, isoform CRA_a [Homo sapiens] 6.00E-29 CLECT, GPS, 7tm_2
Group V -Adhesion homologues in C.elegans (2) - PFAM
Frizzled Clytia SA0AAB17YK01RM1 frizzled 2 [Hydra magnipapillata] 1.00E-122 Frizzled/7tm_2 Frizzled
Frizzled Clytia IL0ABA3YD02RM1frizzled-8 [Xenopus laevis] >gi|3869266|gb|A... 2.00E-22 Fz
Frizzled - putative There are described Frizzled proteins in Hydra.
Frizzled Clytia SA0AAB36YE16RM17-transmembrane receptor frizzled-1 [Xenopus... 8.00E-20 Fz, Gal_lectin(wobbly)
Frizzled - putative
Frizzled Clytia IL0ABA4YO10RM1frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 2.00E-19 Fz, Gal_lectin(wobbly)
Frizzled - putative
Frizzled Hydra CL4401Contig1 frizzled 2 [Hydra magnipapillata] 1.00E-101 Fz Frizzled
Frizzled Hydra CL6396Contig1 frizzled receptor [Hydra vulgaris] 1.00E-127 Fz Frizzlednext best hit frizzled [Aedes aegypti] >gi|108875685|gb|EA... 6.00E-07
Frizzled Hydra CL10411Contig1 frizzled receptor [Hydra vulgaris] 7.00E-60 No domains Frizzled
Frizzled Hydra gb|CX835366.2PREDICTED: Frizzled4/9/10 [Hydra magnipapill... 3.00E-59 Fz (wobbly) Frizzled
Frizzled Hydra CL2039Contig1 Fzd7 protein [Mus musculus] 6.00E-25 FzFrizzled - putative
Frizzled Hydra CL5686Contig1frizzled homolog 8 [Rattus norvegicus] >gi|1... 5.00E-16 Fz, gal_lectin (wobbly)
Frizzled - putative
Frizzled Hydra gb|CN560484.1 frizzled 2 [Hydra magnipapillata] 1.00E-33 No domainsFrizzled - putative
Frizzled Nvecjgi|Nemve1|171640|estExt_gwp.C_1830043
7-transmembrane receptor frizzled-1 [Xenopus... 1.00E-180 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|168924|estExt_gwp.C_1170009
frizzled homolog 10 [Xenopus (Silurana) trop... 1.00E-155 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|139208|e_gw.466.3.1
frizzled homolog 4 [Gallus gallus] >gi|17433062... 1.00E-137 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|183962|estExt_GenewiseH_1.C_530042
frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 0 Fz, Frizzled Frizzled
PREDICTED: similar to More Of MS family memb... 1.00E-107
GPCR125 Acropora Contig5283 Gpr125 protein [Mus musculus] 5.00E-75 LRRCT, Ig, HormR_1, GPS, 7tm_2 GPCR 125
GPCR125 Nvecjgi|Nemve1|242046|estExt_fgenesh1_pg.C_550116 Gpr125 protein [Mus musculus] 3.00E-69
LRR (5x 3wobbly), lrrct1, IG, Horm_R1 (wobbly), 7tm_2 GPCR 125
VLGR1 Acropora Contig33900G protein-coupled receptor 98 [Danio rerio] ... 1.00E-142 Calx-beta, GPS, 7tm_2
VLGR1/GPCR 98 - c-term
GPS-7tm_2 Acropora Contig13413 Bai3 protein [Mus musculus] 2.00E-40 GPS, 7tm_2 GPS-7tm_2GPS-7tm_2 Acropora Contig14869 GPR133 protein [Homo sapiens] 2.00E-55 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora Contig17113latrophilin 1, isoform CRA_a [Rattus norvegicus] 1.00E-24 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora Contig3639G protein-coupled receptor 133 [Bos taurus] ... 7.00E-45 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora Contig5847latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-38 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora Contig8177G protein-coupled receptor 133 [Homo sapiens] >... 1.00E-57 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora run001daytona_697863 Lphn3 protein [Mus musculus] 6.00E-65 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Acropora run001daytona_1134667EGF, latrophilin and seven transmembrane dom... 3.00E-21 GPS, 7tm_2(wobbly) GPS-7tm_2
GPS-7tm_2 Clytia SA0AAA8YB23RM1G-protein coupled receptor 112 [Homo sapiens] >... 3.00E-49 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Clytia SA0AAB111YO10CTGG protein-coupled receptor 133 [Homo sapiens] >... 4.00E-12 GPS, 7tm_2 (wobbly) GPS-7tm_2
GPS-7tm_2 Hydra CL9076Contig1gb|EEB17331.1| class B secretin-like G-protein coupled receptor … 2.00E-18 GPS, 7tm_2 (wobbly) GPS-7tm_2
Appendix B JCUSMART survey of the cnidarian adhesome
196
Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
GPS-7tm_2 Mbrev jgi|Monbr1|3267|gw1.2.830.1latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-25 GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2
GPS-7tm_2 Mbrevjgi|Monbr1|6049|fgenesh1_pg.scaffold_3000547 No good hits GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2
GPS-7tm_2 Mbrevjgi|Monbr1|38087|estExt_fgenesh1_pg.C_200011 MB7TM1 [Monosiga brevicollis] 0 PMT (wobbly), GPS, 7tm_2 MB7TM1 7tm_2 may be a 7TM_GPCR_str
VLGR1 Nvecjgi|Nemve1|242264|estExt_fgenesh1_pg.C_600022
G protein-coupled receptor 98 [Danio rerio] ... 0
Calx-beta (2x), LamG, calx-beta (8x), IG (wobbly), calx-beta (9x), GPS, 7tm_2 VLGR1/GPCR98 SPU_027371 neural development
GPS-7tm_2 Nvecjgi|Nemve1|125574|e_gw.221.20.1
latrophilin 1 [Bos taurus] >gi|46576871|sp|O... 3.00E-67 GPS (wobbly), 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|78835|e_gw.1.138.1
G-protein coupled receptor GPR133 [Homo sapiens] 4.00E-53 GPS (wobbly), 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|90006|e_gw.23.202.1
latrophilin 2, isoform CRA_a [Rattus norvegicus] ... 5.00E-28 GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2
GPS-7tm_2 Nvec jgi|Nemve1|13642|gw.297.15.1G protein-coupled receptor 133 [Bos taurus] ... 5.00E-49 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|146118|e_gw.1586.3.1 flamingo 1 [Gallus gallus] 2.00E-40 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvec jgi|Nemve1|20971|gw.105.20.1G protein-coupled receptor 133 [Bos taurus] ... 8.00E-58 GPS, 7tm_2 GPS-7tm_2 7tm_2 may be a 7TM_GPCR_Sru 0.045
GPS-7tm_2 Nvecjgi|Nemve1|211777|fgenesh1_pg.scaffold_143000054 latrophilin 2, isoform CRA_a [Homo sapiens] 9.00E-31 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|217885|fgenesh1_pg.scaffold_317000008
latrophilin 1 [Rattus norvegicus] >gi|2239297|g... 2.00E-54 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|237703|estExt_fgenesh1_pg.C_10031
latrophilin-like protein 2 [Haemonchus contortus]... 2.00E-14 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvec jgi|Nemve1|24490|gw.39.39.1latrophilin 2 splice variant baabe [Bos taurus] 2.00E-71 GPS, 7tm_2 GPS-7tm_2 Group I on Phylogenetics
GPS-7tm_2 Nvec jgi|Nemve1|25584|gw.39.51.1 latrophilin 3, isoform CRA_c [Homo sapiens] 2.00E-53 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|98237|e_gw.48.80.1
latrophilin-like protein 2 [Haemonchus contortus]... 2.00E-35 GPS, 7tm_2 GPS-7tm_2
GPS-7tm_2 Nvecjgi|Nemve1|241064|estExt_fgenesh1_pg.C_390006 latrophilin 2, isoform CRA_a [Homo sapiens] 1.00E-13 GPS, 7tm_2 (with a GPS in one loop) GPS-7tm_2
Novel Acropora Contig18752G-protein coupled receptor 112 [Homo sapiens] >... 5.00E-50
CA (wobbly), IG, SEA (wobbly), HormR1 (wobbly), GPS, 7tm_2 Novel
Novel Acropora Contig9410latrophilin 2 splice variant baaae [Bos taurus] 3.00E-32 SEA, HormR_1, GPS, 7tm_2 Novel
Novel Mbrevjgi|Monbr1|34543|estExt_fgenesh2_pg.C_400022
gb|ABB84827.1| epidermal growth factor domain-containing protein… 3.00E-23
EGF (3x), LamG (wobbly), EGF, TSP1, OPT (wobbly)/ (GPS (wobbly), 7tm_2 (wobbly)) Adhesion Novel
Novel Nvecjgi|Nemve1|217955|fgenesh1_pg.scaffold_319000019 GPR133 protein [Homo sapiens] 2.00E-58 PA14 (2x wobbly), GPS, 7tm_2 Adhesion Novel
Novel Nvecjgi|Nemve1|199785|fgenesh1_pg.scaffold_17000074
G protein-coupled receptor 112 [Mus musculus] >g... 4.00E-59
disc_4 (3x), IG (wobbly), FN3 (wobbly), FN3,GPS, 7tm_2 Novel
Novel Nvecjgi|Nemve1|238736|estExt_fgenesh1_pg.C_80029
G protein-coupled receptor 126 [Gallus gallu... 3.00E-47
DUF885, SLG (wobbly), GCC2_GCC3, igc2_5, HormR_1 (wobbly), GPS, 7tm_2 Novel
NvX Group Nvecjgi|Nemve1|218502|fgenesh1_pg.scaffold_351000013
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-21 SO_2 (wobbly), 7tm_2 No SO (NvX)
NvX Group Nvecjgi|Nemve1|210434|fgenesh1_pg.scaffold_121000053
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 3.00E-26 SO_2, 7tm_2 No SO (NvX)
NvX Group Nvecjgi|Nemve1|211490|fgenesh1_pg.scaffold_139000007
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-20 SO_2, 7tm_2 No SO (NvX)
NvX Group Nvecjgi|Nemve1|212781|fgenesh1_pg.scaffold_162000044
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 3.00E-27 SO_2, 7tm_2 Yes SO (NvX)
NvX Group Nvecjgi|Nemve1|215376|fgenesh1_pg.scaffold_226000026
class B secretin-like G-protein coupled receptor ... 6.00E-38
SO_2, c8 (wobbly), doub1_11 (wobbly), 7tm_2 No SO (NvX)
NvX Group Nvecjgi|Nemve1|206150|fgenesh1_pg.scaffold_66000090
gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-32 Collagen, SO_2, 7tm_2 No SO (NvX) Novel
Appendix B JCUSMART survey of the cnidarian adhesome
197
Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Smoothened Acropora Contig8091smoothened homolog [Rattus norvegicus] >gi|6226... 1.00E-133 Fz,Frizzled,7tm_2 smoothened
Like frizzled but operates in the hedgehog pathway
Smoothened Clytia SA0AAB54YE06CTGsmoothened [Xenopus laevis] >gi|13194566|gb|... 2.00E-43 Frizzled Smoothened
Like frizzled but operates in the hedgehog pathway
Smoothened Nvecjgi|Nemve1|208236|fgenesh1_pg.scaffold_90000004
smoothened homolog (Drosophila) [Mus musculus] 1.00E-135 Fz, Frizzled Smoothened
Like frizzled but operates in the hedgehog pathway
Smoothened Nvec jgi|Nemve1|92220|e_gw.29.7.1smoothened [Homo sapiens] >gi|6226142|sp|Q99835... 2.00E-92 Fz, Frizzled Smoothened
Like frizzled but operates in the hedgehog pathway
Appendix B JCUSMART survey of the cnidarian adhesome
198
Immunoglobulin
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion NotesCOLIG Acropora Contig6942 Collagen, I-setCOLIG Acropora Contig657 Collagen, I-set (2x), IgCOLIG Acropora Contig16723 Collagen, I-setCOLIG Acropora Contig27548 Collagen, I-set (2x), IgCOLIG Acropora Contig24993 Collagen, igc2 (2x)COLIG Acropora Contig11455 Collagen, I-set (2x), SushiCOLIG Acropora Contig21270 Collagen, igc2 (2x)COLIG Acropora Amil_c21796 Collagen, I-setCOLIG Acropora Contig10772 Collagen, I-setCOLIG Nvec jgi|Nemve1|207840|fgenesh1_pg.scaffold_85000017 predicted peptides only Collagen (wobbly), IG (3x) Yes No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|208243|fgenesh1_pg.scaffold_90000011 predicted peptides only Collagen (wobbly), igc2 (2x), I-set, igc2, ultra No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|221269|fgenesh1_pg.scaffold_709000005 No good hits Collagen, igc2 (2x) No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|209947|fgenesh1_pg.scaffold_114000019 predicted peptides only Collagen, igc2 (2x), IG Yes No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|200560|fgenesh1_pg.scaffold_22000082 predicted peptides only Collagen, igc2, I-set, igc2, I-set, igc2 No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.
DCC Hydra CL1Contig813neogenin 1 [Danio rerio] >gi|23428357|gb|AAK330... 2.00E-13 igc2_5 (2x) Yes No Neogenin putative partial
DCC Nvec jgi|Nemve1|105427|e_gw.77.120.1 neogenin variant 1b [Xenopus borealis] 3.00E-43 igc2, I-set (2x) Yes No Neogenin partial (Putative)
DS-CAM Clytia SA0AAB54YD20RM1gb|AAZ85125.1| Down Syndrome adhesion molecule splice variant 3.... 9.00E-25 igc2_5, IG, FN3 (3x) No No DS-CAM partial
DS-CAM Hydra CL2309Contig1Down Syndrome adhesion molecule splice variant 3.... 3.00E-20 IG, FN3 (3x) No 2 DS-CAM partial
DS-CAM Nvec jgi|Nemve1|104479|e_gw.73.146.1 down syndrome cell adhesion molecule [Aedes ... 2.00E-33 FN3 (2x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|101511|e_gw.61.65.1 down syndrome cell adhesion molecule [Aedes ... 2.00E-27 FN3 (2x) No No DS-CAM partial (putative)
DS-CAM Nvec jgi|Nemve1|2043|gw.439.10.1 Down syndrome cell adhesion molecule [Rattus no... 8.00E-27 FN3 (2x) No No DS-CAM partial (putative)
DS-CAM Nvec jgi|Nemve1|108927|e_gw.92.51.1gb|AAL99986.1|AF487348_1 Down syndrome cell adhesion molecule-li… 9.00E-30 FN3 (2x) No No DS-CAM partial (putative)
DS-CAM Nvec jgi|Nemve1|120490|e_gw.171.38.1gb|AAL99986.1|AF487348_1 Down syndrome cell adhesion molecule-li… 1.00E-29 FN3 (2x) No No DS-CAM partial (putative)
DS-CAM Nvec jgi|Nemve1|224185|fgenesh1_pg.scaffold_4113000001 Down syndrome cell adhesion molecule [Xenopu... 4.00E-23 FN3 (3x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|127554|e_gw.239.11.1 Down syndrome cell adhesion molecule-like... 2.00E-42 FN3 (3x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|43898|gw.239.46.1 DSCAM splice variant 4.12 [Drosophila yakuba] 2.00E-28 igc2 (wobbly) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|224982|fgenesh1_pg.scaffold_6945000001 dscam [Drosophila mojavensis] >gi|193911131|... 1.00E-16 igc2 (wobbly), madsneu2 (wobbly) No No DS-CAM partial (putative)
F11/Contactin Hydra CL2585Contig1 contactin [Drosophila melanogaster] >gi|5597662... 2.00E-31 igc2_5, IG (wobbly), FN3 (2x wobbly) No No contactin putative partialF11/Contactin Nvec 199756|fgenesh1_pg.scaffold_17000045 Contactin 6 [Mus] 3.00E-96 IG (6x), FN3 (4x) Contactin This gene is a very close match to Contactin but was filtered Ig and Adhesion Clytia SA0AAA18YH11RM1 predicted proteins only CCP, CCP (wobbly), HYR, GCC2_GCC3 (2x), IG No No IG-CCPIg and Adhesion Clytia SA0AAB9YG10RM1 Bcam protein [Xenopus tropicalis] 2.00E-09 IG, I-set, IG (2x), EGF No No Ig-EGFIg and Adhesion Clytia SA0AAB66YD22CTG predicted proteins only Wap (wobbly), Kazal_3 (wobbly), IG (wobbly), KU, Kazal_3, FOLN (wobbly), Kazal_3Yes No Ig-KazalIg and Adhesion Clytia IL0ABA11YM18RM1 predicted proteins only I-set, IG, IG (wobbly), LDLa Yes No Ig-LDLaIg and Adhesion Clytia SA0AAB26YD21RM1 No good hits IG, igc2_5, TSP1, igc2_5 Yes 1 IG-TSP1Ig and Adhesion Clytia IL0ABA15YJ23RM1 No good hits igc2_5 (2x), TSP1 No 1 Ig-TSP1Ig and Adhesion Clytia SA0AAA1YF21RM1 roundabout 1 [Aedes aegypti] >gi|108879336|g... 2.00E-14 IG (2x), TSP1 (wobbly) No No Ig-TSP1Ig and Adhesion Clytia SA0AAA8YP06RM1 No good hits IG, igc2_5, TSP1, igc2_5 Yes No Ig-TSP1Ig and Adhesion Clytia SA0AAA8YI05RM1 No good hits IG (2x), GCC2_GCC3, VWA No No IG-VWAIg and Adhesion Hydra CL2877Contig1 No good hits IG (wobbly), TSP1 (wobbly), igc2_5 No No Ig-TSP1Ig and Adhesion Hydra CL1208Contig1 SPEG complex locus [Bos taurus] 5.00E-50 I-set, serkin_6 No 3 SPEG striated preferentially expressed geneIg and Adhesion Nvec jgi|Nemve1|198435|fgenesh1_pg.scaffold_10000027 No good hits KR, IG (wobbly), F5_F8_type_C (wobbly), spt (wobbly) Ig- KRIg and Adhesion Nvec jgi|Nemve1|248172|estExt_fgenesh1_pg.C_4390008 predicted peptides only CUB, igc2 No 2 Ig-CUBIg and Adhesion Nvec jgi|Nemve1|213715|fgenesh1_pg.scaffold_183000001 No good hits IG, CUB Ig-CUBIg and Adhesion Nvec jgi|Nemve1|246222|estExt_fgenesh1_pg.C_2050018 No good hits igv1 (wobbly), igc2 (wobbly), CUB Ig-CUBIg and Adhesion Nvec jgi|Nemve1|209643|fgenesh1_pg.scaffold_109000021 predicted peptides only F5_F8_type_C, EGF (4x), CCP (2x), HYR, EGF, CCP, GCC2_GCC3, IG (wobbly)Yes No Ig-EGF, CCPIg and Adhesion Nvec jgi|Nemve1|202086|fgenesh1_pg.scaffold_32000098 perlecan (heparan sulfate proteoglycan 2) [Mus m... 8.00E-23 F5_F8_type_C, I-set (2x), IG No 1 Ig-F5_F8_type_CIg and Adhesion Nvec jgi|Nemve1|241670|estExt_fgenesh1_pg.C_490046 echinonectin [Lytechinus variegatus] 4.00E-32 F5_F8_type_C, IG, PAN_1 Yes No Ig-F5_F8_type_CIg and Adhesion Nvec jgi|Nemve1|215547|fgenesh1_pg.scaffold_230000039 No good hits IG (3x), LDLa, IG (3x) Yes No Ig-LDLaIg and Adhesion Nvec jgi|Nemve1|220325|fgenesh1_pg.scaffold_502000002 predicted peptides only TPR (8x 5wobbly), I-set No No Ig-TPRIg and Adhesion Nvec jgi|Nemve1|203987|fgenesh1_pg.scaffold_47000046 No good hits IG, TY Yes 2 Ig-TYIg and Adhesion Nvec jgi|Nemve1|197457|fgenesh1_pg.scaffold_5000263 predicted peptides only WAP (3x), IG (2x), zp No 1 Ig-WAPIg and FN3 containing Acropora Amil_c7199 no good hits Ig, I-set, FN3 (2x) No 2 Ig-FN3
Ig and FN3 containing Acropora Contig10504 neuroglian 7.00E-66 Ig (6x), FN3 (wobbly) Yes No Ig-FN3ref|XP_001637406.1| predicted protein [Nematostella vectensis] >… 1E-130
Ig and FN3 containing Acropora Contig10617 no good hits I-set, FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig11811 sidekick 2 [Rattus norvegicus] >gi|149054713... 0 I-set (2x), Ig, FN3 (13x) No 1 Ig-FN3Ig and FN3 containing Acropora Contig16551 Ret proto-oncogene [Xenopus laevis] 9.00E-78 Ig, I-set, Ig, FN3, tyrkin Yes No Ig-FN3
Ig and FN3 containing Acropora Contig1699roundabout 1 [Danio rerio] >gi|13509385|gb|AAK2... 2.00E-53 Ig (2x), I-set, Ig (2x), FN3 No 4 Ig-FN3
Ig and FN3 containing Acropora Contig212 neural cell adhesion molecule 1 [Danio rerio] >... 7.00E-19 Ig, I-set, Ig, FN3 (4x wobbly) Yes No Ig-FN3Ig and FN3 containing Acropora Contig22827 retII [Homo sapiens] 1.00E-64 I-set, Ig, FN3 (4x), tyrkin No 1 Ig-FN3Ig and FN3 containing Acropora Contig2812 no good hits Ig (wobbly), FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig31261 Ncam1 protein [Danio rerio] 5.00E-37 Ig (5x), FN3 No No Ig-FN3
Ig and FN3 containing Acropora Contig5333gb|AAN32614.1|AF304305_1 Down syndrome cell adhesion molecule li… 4.00E-85 Ig, FN3 (4x), Ig, FN3 No 1 Ig-FN3
Ig and FN3 containing Acropora Contig6272 no good hits Ig (wobbly), FN3 No No Ig-FN3
Ig and FN3 containing Acropora Contig6343ref|NP_523604.2| Leukocyte-antigen-related-like, isoform A [Dros… 1.00E-89 Ig, I-set (2x), FN3 (4x) Yes No Ig-FN3
Ig and FN3 containing Acropora Contig8183 no good hits Ig (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Acropora Contig9170 no good hits I-set Yes No Ig-FN3Ig and FN3 containing Acropora Contig9397 no good hits I-set, FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig942 Ncam1 protein [Danio rerio] 1.00E-33 Ig (4x), FN3 Yes No Ig-FN3Ig and FN3 containing Acropora run001daytona_1717233 no good hits Ig (wobbly), FN3 No No Ig-FN3
Appendix B JCUSMART survey of the cnidarian adhesome
199
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion Notes
Ig and FN3 containing Acropora run002_426847 kalirin, RhoGEF kinase isoform 3 [Homo sapiens]... 5.00E-16 Ig (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Acropora run002_428308 no good hits Ig (3x), FN3 No No Ig-FN3Ig and FN3 containing Clytia SA0AAB49YE16CTG No good hits IG (wobbly), FN3 (2x) No No Ig-FN3Ig and FN3 containing Hydra CL574Contig1 No good hits igc2_5 (wobbly), FN3_2 (wobbly) No 1 Ig-FN3Ig and FN3 containing Hydra CL3251Contig1 No good hits IG (3x 2wobbly), FN3 No No Ig-FN3Ig and FN3 containing Hydra gb|CV464365.1 nephrin [Mus musculus] 6.00E-12 igc2 (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Hydra CL6367Contig1 myosin light chain kinase [synthetic construct] 2.00E-49 Serkin (wobbly), I-set, FN3 (wobbly) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|243882|estExt_fgenesh1_pg.C_1010020 No good hits IG (2x), FN3 (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|213889|fgenesh1_pg.scaffold_186000034 predicted peptides only IG (2x), FN3 (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|241554|estExt_fgenesh1_pg.C_470053 mesoglein variant 1 [Aurelia aurita] 5.00E-30 IG (2x), FN3, zp Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|199622|fgenesh1_pg.scaffold_16000072 No good hits IG, FN3 (wobbly), IG, SEFIR (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216320|fgenesh1_pg.scaffold_257000003 No good hits igc2 (wobbly), FN3 (wobbly) No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|240082|estExt_fgenesh1_pg.C_240078 neural cell adhesion molecule [Gallus gallus] 1.00E-21 igc2, FN3 No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|201959|fgenesh1_pg.scaffold_31000119 predicted peptides only igc2, FN3 (2x 1wobbly) No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218534|fgenesh1_pg.scaffold_353000001 predicted peptides only igc2, IG, igc2, FN3 No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|219947|fgenesh1_pg.scaffold_458000005 predicted peptides only IG (2x 1wobbly), FN3 (wobbly) No 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216378|fgenesh1_pg.scaffold_259000002 No good hits IG, FN3 Yes 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|241594|estExt_fgenesh1_pg.C_470130 No good hits igc2, FN3 Yes 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|201955|fgenesh1_pg.scaffold_31000115 predicted peptides only IG (wobbly), IG (3x), IG (wobbly), IG, madsneu2, FN3No 3 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|202226|fgenesh1_pg.scaffold_33000070 predicted peptides only igc2, FN3 (2x), MFS No 10 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|225380|fgenesh1_pg.scaffold_8390000001 predicted peptides only I-set, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|204514|fgenesh1_pg.scaffold_51000096 titin, isoform CRA_e [Homo sapiens] 1.00E-28 IG (2x wobbly), FN3 (3x 2wobbly) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216592|fgenesh1_pg.scaffold_265000021 predicted peptides only IG (2x), FN3 (2x) No No Ig-FN3
Ig and FN3 containing Nvec jgi|Nemve1|85090|e_gw.10.263.1sidekick homolog 2 (chicken), isoform CRA_a [Homo... 3.00E-38 IG, FN3 No No Ig-FN3
Ig and FN3 containing Nvec jgi|Nemve1|217293|fgenesh1_pg.scaffold_291000027 No good hits IG, FN3 (2x 1wobbly) Yes No Ig-FN3
Ig and FN3 containing Nvec jgi|Nemve1|201938|fgenesh1_pg.scaffold_31000098neural cell adhesion molecule 1, isoform CRA_a [M... 9.00E-13 igc2, FN3 No No Ig-FN3
Ig and FN3 containing Nvec jgi|Nemve1|216597|fgenesh1_pg.scaffold_265000026 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218290|fgenesh1_pg.scaffold_340000001 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218823|fgenesh1_pg.scaffold_370000001 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|202038|fgenesh1_pg.scaffold_32000050 predicted peptides only V-set (wobbly), IG, FN3 No No Ig-FN3Ig_LON Acropora Amil_c2655 limbic system-associated membrane protein [Ratt… 8.00E-13 Ig (3x) Yes 1Ig_LON Acropora Contig18527 No good hits Ig Yes 1 Ig-LON subgroup likeIg_LON Clytia SA0AAB35YA21RM1 fibroblast growth factor receptor A [Nematostella... 6.00E-20 IG (3x 1wobbly) Yes 0 Ig-LON subgroup Not an FGFR part of IgLON subgroupIg_LON Clytia SA0AAB22YF24RM1 No hits Found IG (wobbly) Yes 0 Ig-LON subgroup like
Ig_LON Hydra CL1538Contig1 neural cell adhesion molecule 1 [Felis catus] 8.00E-10 IG (2x) No 1 Ig-LON subgroupThis can not be an N-CAM as there areno FN3 domains before the TM. Must be an IgLON subgroup
Ig_LON Hydra CL2516Contig1 No good hits IG (wobbly), IG Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|210858|fgenesh1_pg.scaffold_128000042 No good hits IG (3x 2wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246644|estExt_fgenesh1_pg.C_2300034 No good hits IG (3x 2wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246645|estExt_fgenesh1_pg.C_2300038 No good hits IG (3x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246329|estExt_fgenesh1_pg.C_2110042 predicted peptides only IG (3x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|214858|fgenesh1_pg.scaffold_211000043 predicted peptides only IG (wobbly), igc2, IG (wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|222990|fgenesh1_pg.scaffold_2139000001 predicted peptides only igc2 (2x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|204525|fgenesh1_pg.scaffold_51000107 fibroblast growth factor receptor B [Nematostella... 1.00E-29 igc2 (2x), IG Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|183662|estExt_GenewiseH_1.C_500040 No good hits IG (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|220356|fgenesh1_pg.scaffold_506000010 No good hits igc2 Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|198407|fgenesh1_pg.scaffold_9000196 No good hits igc2 (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|205973|fgenesh1_pg.scaffold_65000008 No good hits igc2 (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|242464|estExt_fgenesh1_pg.C_640060 predicted peptides only igc2 (wobbly), IG (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|207612|fgenesh1_pg.scaffold_82000061 No good hits IG (wobbly), igc2 No 2 Ig-LON subgroup likeL1-Like Acropora Contig7988 neuroglian 7.00E-77 Ig (4x), FN3 (5x) No No L1L1-Like Acropora Contig20212 neuroglian 1.00E-69 Ig (3x), FN3 (5x) No 1 L1L1-Like Nvec jgi|Nemve1|240518|estExt_fgenesh1_pg.C_300161 neurofascin [Danio rerio] >gi|224383688|gb|A... 5.00E-82 IG (2x), igc2 (2x), I-set (wobbly), FN3 (5x 3 wobbly)No 2 neurofascinL1-Like Nvec jgi|Nemve1|87913|e_gw.17.200.1 hBRAVO/Nr-CAM precursor [Homo sapiens] 9.00E-29 FN3 (2x) No No Nr-CAM like (putative) fragmented contactin 6 fgenesh1_pg.scaffold_17000045
MALT-1 Clytia SA0AAA20YJ09CTGref|NP_694508.1| mucosa associated lymphoid tissue lymphoma tran… 3.00E-49 Peptidase_C14 No No MALT1
MALT 1 appears to be only in vertebrates, C.elegans, Dictostellium.
MALT-1 Nvec jgi|Nemve1|244962|estExt_fgenesh1_pg.C_1400022mucosa associated lymphoid tissue lymphoma transl… gb|EAW63079.1| 2.00E-52 igc2 (2x), Peptidase_C14 No No MALT1
MALT 1 appears to be only in vertebrates, C.elegans, Dictostellium.
MALT-1 Nvec jgi|Nemve1|211581|fgenesh1_pg.scaffold_140000023mucosa associated lymphoid tissue lymphoma transl… gb|EDM14684.1| 2.00E-68 igc2, IG, Peptidase_C14 No No MALT1
MALT 1 appears to be only in vertebrates, C.elegans, Dictostellium.
MALT-1 Acropora Contig6524ref|NP_694508.1|,mucosa associated lymphoid tissue lymphoma tran 1.00E-43 MALT1
MALT-1 Acropora Contig26161ref|NP_694508.1|,mucosa associated lymphoid tissue lymphoma tran 1.00E-48 MALT1
N-CAM Acropora Contig259immunoglobulin superfamily, member 9 [Mus muscu... 6.00E-43 Ig, (4x), FN3 (2x) Yes 1 N-CAM (putative)
N-CAM Acropora Contig3409 Ncam1 protein [Danio rerio] 2.00E-36 Ig (4x), FN3 No 1 N-CAM (putative)N-CAM Acropora Contig2418 neural cell adhesion molecule 1 [Bos taurus] 1.00E-42 Ig (5x), FN3 (2x) Yes 1 N-CAM (putative)N-CAM Nvec jgi|Nemve1|241555|estExt_fgenesh1_pg.C_470054 neural cell adhesion molecule 1 [Danio rerio] >... 9.00E-31 IG (2x), igc2 (wobbly), FN3 (2x 1wobbly) No 1 N-CAM (putative)N-CAM Nvec jgi|Nemve1|236748|estExt_fgenesh1_kg.C_470007 neural cell adhesion molecule 2 [Danio rerio] >... 2.00E-36 IG (3x), igc2 (wobbly), FN3 (2x) Yes 1 N-CAM (putative) This is N-CAM according to the KOG based approachN-CAM Nvec jgi|Nemve1|241557|estExt_fgenesh1_pg.C_470057 ncam2 [Danio rerio] 4.00E-34 IG, igc2, IG, igc2, igv1_8 (wobbly), FN3 (2x) Yes No N-CAM (putative)N-CAM Nvec jgi|Nemve1|185528|estExt_GenewiseH_1.C_710203 protogenin homolog a (Gallus gallus) [Danio ... 1.00E-75 igc2, IG (2x), igc2, FN3 (2x) Yes No protogeninRepeat PTP Acropora Contig12034 XPTP-D protein [Xenopus laevis] 1.00E-154 Ig, I-set, Ig (2x), FN3 (4x), PTP (2x) Yes 1 PTPRepeat PTP Nvec jgi|Nemve1|210515|fgenesh1_pg.scaffold_123000015 predicted peptides only MAM, IG (3x), igc2 (wobbly) Yes No PTP N-term putative partialRepeat PTP Nvec jgi|Nemve1|88020|e_gw.17.7.1 XPTP-D protein [Xenopus laevis] 0 igc2, I-set, igc2, FN3 (8x), Y_phosphatase, PTPc_3 No 1 PTP-D proteinToll Like TIR Hydra CL6112Contig1 Toll-receptor-related 1 [Hydra magnipapillata] 1.00E-102 TIR HyTRR1Toll Like TIR Hydra CL6921Contig1 Toll-receptor-related 2 [Hydra magnipapillata] 1.00E-58 TIR HyTRR2Toll Like TIR Hydra CL9285Contig1 myeloid differentiation primary response factor 8... 1.00E-16 Death (wobbly), TIR (wobbly) MyD88
Appendix B JCUSMART survey of the cnidarian adhesome
200
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion Notes
Toll Like TIR Hydra CL9986Contig1 myeloid differentiation primary response gene 88... 4.00E-07 Death (wobbly), TIR (wobbly) MyD88Toll Like TIR Mbrev jgi|Monbr1|30936|estExt_fgenesh2_pg.C_20536 No good hits PfkB, TIR (wobbly) PfkB family includes carbohyrdate and pyriamidine kinases.Toll Like TIR Mbrev jgi|Monbr1|27923|fgenesh2_pg.scaffold_22000108 predicted proteins only TUDOR_7 (wobbly), TIR (wobbly), TUDOR_7(wobbly)Toll Like TIR Nvec jgi|Nemve1|82163|e_gw.5.187.1 myeloid differentiation factor 88 [Larimichthys c... 5.00E-37 Death, TIR MyD88Toll Like TIR Nvec jgi|Nemve1|196737|fgenesh1_pg.scaffold_3000058 TIR1 [Acropora millepora] 2.00E-25 IG, igc2, IG (2x), TIR NvIL-1R
Toll Like TIR Nvec jgi|Nemve1|116780|e_gw.141.61.1 TIR1 [Acropora millepora] 3.00E-20 TIR NvIL-1RThis sequence is N-term truncated and also contains Ig domains (Miller et al 2007 Genome biology)
Toll Like TIR Nvec jgi|Nemve1|204009|fgenesh1_pg.scaffold_47000068 Toll9 [Culex quinquefasciatus] >gi|167864481... 6.00E-18 igc2, IG, TIR NvIL-1RToll Like TIR Nvec jgi|Nemve1|211916|fgenesh1_pg.scaffold_146000015 predicted proteins only TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|16632|gw.367.25.1 TIR1 [Acropora millepora] 2.00E-19 TIR TRRToll Like TIR Nvec jgi|Nemve1|91199|e_gw.26.221.1 Toll-like receptor (AGAP012385-PA) [Anopheles g... 3.00E-35 TIR TRRToll Like TIR Nvec jgi|Nemve1|57985|gw.141.125.1 Toll-receptor-related 2 [Hydra magnipapillata] 7.00E-17 TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|56601|gw.152.108.1 Toll-receptor-related 2 [Hydra magnipapillata] 4.00E-12 TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|217512|fgenesh1_pg.scaffold_300000025 predicted proteins only SAM (wobbly), TIR (wobbly)Toll Like TIR Nvec jgi|Nemve1|223246|fgenesh1_pg.scaffold_2476000001 predicted proteins only SAM (wobbly), TIR (wobbly)Toll Like TIR Nvec jgi|Nemve1|240728|estExt_fgenesh1_pg.C_330073 predicted proteins only Arm (3x 2wobbly), TIR(wobbly)Toll Like TIR Acropora Contig6496 LRR (2x), lrrct, TIRToll Like TIR Acropora run002_428851 TIRToll Like TIR Acropora Contig1450 TIRToll Like TIR Acropora run001daytona_1723038 TIRToll Like TIR Acropora Amil_c81406 TIRToll Like TIR Acropora Contig18494 Death, TIR MyD88Toll Like TIR Acropora Contig13468 TIR (wobbly)Toll Like TIR Acropora Contig33214 TIR (wobbly)Toll Like TIR Acropora Contig5811 TIRToll Like TIR Acropora Contig7691 TIR
Appendix B JCUSMART survey of the cnidarian adhesome
201
Extracellular Matrix
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion NotesCollagen Cltyia SA0AAB17YN15CTG collagen-like protein [Bacillus cereus Q1] >... 2.00E-28 Collagen CollagenCollagen Cltyia SA0AAA20YH02CTG collagen [Hydra vulgaris] 1.00E-23 VWA, Mucin (wobbly), Fasciclin (wobbly) Collagen -Hydra
Collagen Cltyia SA0AAA15YD01RM1gb|EDL92062.1| procollagen, type VI, alpha 3 (predicted), isofor… 8.00E-19 Collagen (5x) Collagen 6A3
Collagen Cltyia IL0ABA3YP01RM1 collagen [Hydra vulgaris] 8.00E-87 WAP, VWA Collagen - HydraCollagen Hydra CL2056Contig1 collagen, type XII, alpha 1 [Gallus gallus] >gi... 1.00E-23 VWA CollagenCollagen Hydra CL4008Contig1 collagen [Hydra vulgaris] 1.00E-22 VWA Collagen -HydraCollagen Hydra CL6422Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 0 Collagen (wobbly), VWA, Collagen, VWA Collagen - HydraCollagen Hydra CL3504Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 1.00E-158 Collagen (4x) Collagen - HydraCollagen Hydra CL602Contig2 gb|ABG80452.1| collagen [Hydra vulgaris] 1.00E-87 Collagen (3x) Collagen - HydraCollagen Hydra CL9498Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 5.00E-50 Collagen (3x) Collagen - Hydra
Collagen Monosigajgi|Monbr1|11132|fgenesh1_pg.scaffold_26000095 collagen [Hydra vulgaris] 8.00E-39
Collagen (6x), VWA, Collagen (3x), VWA, Collagen (3x), VWA, Collagen (19x) Collagen - Hydra
Collagen Nvec jgi|Nemve1|137069|e_gw.389.18.1 collagen alpha 2(I) chain precursor [Sorangi... 4.00E-22 Collagen (wobbly) CollagenCollagen Nvec jgi|Nemve1|79543|e_gw.1.96.1 collagen type VI alpha 5 [Homo sapiens] 3.00E-30 VWA (2x) Collagen
Collagen Nvecjgi|Nemve1|215413|fgenesh1_pg.scaffold_227000024 collagen type VI alpha 6 [Homo sapiens] >gi|... 5.00E-25 WAP (wobbly), VWA (4x) Collagen
Collagen Nvec jgi|Nemve1|106478|e_gw.82.69.1 collagen type XII alpha-1 precursor 1.00E-25 VWA CollagenCollagen Nvec jgi|Nemve1|828|gw.658.2.1 collagen-like protein [Hydra vulgaris] 1.00E-33 VWA CollagenCollagen Nvec jgi|Nemve1|133516|e_gw.318.15.1 collagen, type XII, alpha 1 [Gallus gallus] >gi... 2.00E-31 VWA Collagen
Collagen Nvecjgi|Nemve1|198700|fgenesh1_pg.scaffold_11000112
collagen, type XII, alpha 1, isoform CRA_c [Homo ... 4.00E-21 VWA Collagen
Collagen Nvec jgi|Nemve1|105672|e_gw.78.76.1 collagen, type XXII, alpha 1 [Gallus gallus] 2.00E-22 VWA CollagenCollagen Nvec jgi|Nemve1|88931|e_gw.19.298.1 Col protein [Suberites domuncula] 3.00E-35 Collagen Collagen
Collagen Nvecjgi|Nemve1|188642|estExt_GenewiseH_1.C_1270035 Col protein [Suberites domuncula] 5.00E-23 Collagen (2x) Collagen
Collagen Nvec jgi|Nemve1|128410|e_gw.250.80.1 Col3a1 protein [Bacillus cereus G9241] >gi|47... 4.00E-39 Collagen (2x) Collagen
Collagen Nvecjgi|Nemve1|205490|fgenesh1_pg.scaffold_60000013 collagen type XI alpha-2 [Danio rerio] >gi|1... 3.00E-23 TSPN, Collagen (5x) Collagen 11A2
Collagen 4 Cltyia IL0ABA11YI22RM1 collagen [Hydra vulgaris] 2.00E-70 VWA, Collagen (6x), C4 (2x) C4 - Hydra
Collagen 4 Hydra CL136Contig1type IV collagen alpha 1 chain precursor [Hydra v... 0 Collagen (22x), C4 (2x) C4 - Col4a1
Collagen 4 Hydra CL602Contig1 Col4a6 protein [Mus musculus] 5.00E-80 Collagen (2x), C4 (2x) C4 - Col4a6
Collagen 4 Nvec jgi|Nemve1|127140|e_gw.235.32.1alpha-1 type IV collagen >gi|119629514|gb|EAX0910... 1.00E-94 C4 (2x) C4 - Col4a1
Collagen 4 Nvec jgi|Nemve1|127157|e_gw.235.48.1 alpha2(IV)-like collagen [Strongylocentrotus pu... 2.00E-20 C4 C4
Collagen 4 Nvec jgi|Nemve1|16003|gw.235.37.1sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 6.00E-76 C4 (2x) C4 - Col4a2
Collagen 4 Nvec jgi|Nemve1|148660|e_gw.2548.1.1 3 alpha procollagen [Strongylocentrotus purpura... 2.00E-28 Collagen (3x), C4 C4 - Col3a
Collagen 4 Nvecjgi|Nemve1|221793|fgenesh1_pg.scaffold_1002000001
sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 2.00E-28 Collagen (4x), C4 C4 - Col4a2
Collagen 4 Nvecjgi|Nemve1|157742|estExt_gwp.C_10237
sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 6.00E-76 EGF (10x) C4 - Col4a2
Collagen-triple Helix Cltyia SA0AAA1YC09RM1 collagen triple helix repeat protein [Clostri... 3.00E-14 Collagen (5x) CTHDCCollagen-triple Helix Hydra gb|DN811540.2
ref|YP_142550.1| collagen triple helix repeat containing protein… 8.00E-18 Collagen (3x) CTHDC
Contactin-like Nvec jgi|Nemve1|60863|gw.3.462.1 UNCoordinated family member (unc-89) [Caenor... 2.00E-35 I-set, igc2, IG, I-set (wobbly) contactin?
Contactin-like Nvec jgi|Nemve1|80685|e_gw.3.464.1 UNCoordinated family member (unc-89) [Caenor... 4.00E-34 IG, I-set, IG contactin?Fibrilin Cltyia SA0AAB91YK06RM1 Fibrillin -1 6.00E-31 EGF (6x 1wobbly) FibrillinFibrilin Cltyia SA0AAB39YH05CTG Fibrillin -1 1.00E-24 EGF (5x), SEA (wobbly), EGF (wobbly) FibrillinFibrilin Cltyia SA0AAB122YL04RM1 Fibrillin -2 5.00E-44 EGF (9x) Fibrillin BLAST vs swissprot: Full=Fibrillin-2 [human] 5E-44
Fibrilin Cltyia IL0ABA1YF08RM1 fibrillin [Podocoryna carnea] 0EGF, TB, EGF (7x 1wobbly), TB, EGF (5x), TB, EGF (6x) Fibrillin
TB- This cysteine rich repeat is found in TGF binding protein and fibrillin - PFAM
Fibrilin Cltyia SA0AAB4YB12RM1sp|O08746.1|MATN2_MOUSE RecName: Full=Matrilin-2; Flags: Prec... 4.00E-58 CUB, LDLa (3x), EGF (9x), trypsin Fibrillin hits to fribrillin at 1E-58
Fibrilin Hydra CL5183Contig1 fibrillin [Oikopleura dioica] >gi|18029271|g... 4.00E-36 EGF (6x) FibrillinFibrilin Hydra CL1329Contig1 fibrillin 1 [Rattus norvegicus] >gi|4959650|gb|... 5.00E-29 EGF (10x 5wobbly) Fibrillin?Fibrilin Hydra gb|DN603738.2 fibrillin 1 [Bos taurus] >gi|1706768|sp|P98133.... 6.00E-23 EGF (3x)Fibrilin Hydra CL4709Contig1 fibrillin 4 [Danio rerio] 9.00E-22 EGF (5x)Fibrilin Nvec jgi|Nemve1|212|gw.113.1.1 fibrillin [Podocoryna carnea] 0 EGF (14x) FibrillinFibrilin Nvec jgi|Nemve1|22881|gw.113.27.1 fibrillin 2 [Mus musculus] 9.00E-89 EGF (8x) FibrillinFibrilin Nvec jgi|Nemve1|11770|gw.113.6.1 fibrillin 2 [Rattus norvegicus] >gi|4959652|gb|... 5.00E-82 EGF (8x) FibrillinFibrilin Nvec jgi|Nemve1|70073|gw.28.267.1 fibrillin 3, isoform CRA_a [Homo sapiens] 2.00E-83 EGF (12x) Fibrillin
Fibrilin Nvecjgi|Nemve1|221487|fgenesh1_pg.scaffold_791000001 Fibrillin-1 4.00E-82 EGF (16x) Fibrillin
Fibrilin Nvec jgi|Nemve1|32913|gw.1.126.1 Fibrillin-1 4.00E-69 EGF (10x) FibrillinFibrilin Nvec jgi|Nemve1|61301|gw.1.495.1 Fibrillin-1 6.00E-68 EGF (10x) FibrillinFibrilin Nvec jgi|Nemve1|80370|e_gw.3.48.1 Fibrillin-2 1.00E-82 EGF (16x) FibrillinFibrilin Nvec jgi|Nemve1|108241|e_gw.89.14.1 Fibrillin-2 [mus] 6.00E-35 Laminin_EGF (5x) Fibrillin
Appendix B JCUSMART survey of the cnidarian adhesome
202
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrilin Nvecjgi|Nemve1|223762|fgenesh1_pg.scaffold_3290000001 Fibrillin-3 5.00E-68 EGF (12x) Fibrillin
Fibrilin Nvecjgi|Nemve1|163197|estExt_gwp.C_380029 Fibrillin-3 precursor, putative [Pediculus humanu... 6.00E-89 Cub (2x), EGF (15x), vwc (wobbly) Fibrillin
Fibrilin Nvec jgi|Nemve1|24372|gw.113.33.1 fibrillin 1 [Sus scrofa] >gi|13626617|sp|Q9T... 6.00E-39 EGF (3x) Fibrillin?
Fibrilin Nvecjgi|Nemve1|224395|fgenesh1_pg.scaffold_4621000001 fibrillin 2b [Danio rerio] >gi|184198736|gb|... 7.00E-39 EGF (3x) Fibrillin?
Fibrilin Nvec jgi|Nemve1|18668|gw.113.18.1 fibrillin 1 [Bos taurus] >gi|1706768|sp|P98133.... 3.00E-21 EGF (3x)Fibrilin Nvec jgi|Nemve1|112718|e_gw.113.12.1 fibrillin 1 precursor [Homo sapiens] 1.00E-31 EGF (2x)Fibrillar Collagens Cltyia SA0AAB139YM20RM1 No good hits COLFI (wobbly) COLFIFibrillar Collagens Cltyia SA0AAB3YD15CTG
gb|AAA48707.1| alpha-1 type XI collagen/pro-collagen alpha1 type V 2.00E-37 Collagen (3x), COLFI COLFI - Col11A1
Fibrillar Collagens Cltyia IL0ABA5YB21RM1 alpha 1 type I collagen [Oncorhynchus mykiss] 1.00E-64 Collagen(7x), COLFI COLFI - COl1A1Fibrillar Collagens Cltyia IL0ABA5YM21RM1 fibrillar collagen [Hydra vulgaris] 2.00E-85 Collagen(7x), COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA25YC24RM1 fibrillar collagen [Hydra vulgaris] 7.00E-83 Collagen(7x), COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA1YB18RM1 fibrillar collagen [Hydra vulgaris] 1.00E-73 Collagen (16x), COLFI COLFI - hydraFibrillar Collagens Cltyia SA0AAB109YA10RM1 fibrillar collagen [Hydra vulgaris] 2.00E-46 Collagen, COLFI COLFI - HydraFibrillar Collagens Cltyia SA0AAB121YB12RM1 fibrillar collagen [Hydra vulgaris] 7.00E-22 Collagen, COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA16YI03RM1 fibrillar collagen [Hydra vulgaris] 5.00E-14 Collagen (3x) COLFI - Hydra putativeFibrillar Collagens Cltyia SA0AAA9YB21RM1 fibrillar collagen [Hydra vulgaris] 9.00E-14 VWA COLFI - Hydra putativeFibrillar Collagens Cltyia IL0ABA1YF15RM1
gb|AAM77398.1|AF525468_1 fibrillar collagen precursor [Hydra vul... 6.00E-75 Collagen(7x), COLFI COLFI - Hydra
Fibrillar Collagens Hydra CL8418Contig1 alpha 1 type V collagen [Gallus gallus] >gi|675... 7.00E-17 Collagen (wobbly), COLFI COLFI - Col5a1Fibrillar Collagens Hydra CL208Contig1
gb|AAM77398.1|AF525468_1 fibrillar collagen precursor [Hydra vul... 0 Collagen (17x), COLFI COLFI - Hydra
Fibrillar Collagens Hydra CL16Contig3 gb|ABG80449.1| fibrillar collagen [Hydra vulgaris] 0 Collagen (17x), COLFI COLFI - HydraFibrillar Collagens Hydra gb|DN815934.2 gb|ABG80449.1| fibrillar collagen [Hydra vulgaris] 3.00E-06 Collagen (wobbly) COLFI - Hydra putativeFibrillar Collagens Hydra gb|DN811440.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 1.00E-108 VWA COLFI - HydraFibrillar Collagens Hydra gb|DN636048.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 2.00E-51 Collagen (2x) COLFI - HydraFibrillar Collagens Hydra gb|CN554418.1 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 1.00E-21 Collagen COLFI putativeFibrillar Collagens Hydra gb|CX832276.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 2.00E-18 Collagen (wobbly) COLFI putativeFibrillar Collagens Hydra CL2403Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 0 Collagen (10x) COLFI - HydraFibrillar Collagens Hydra CL2038Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 6.00E-85 COLFI COLFI - HydraFibrillar Collagens Hydra CL7128Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 3.00E-78 Collagen COLFI - HydraFibrillar Collagens Hydra CL4453Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 4.00E-74 Collagen (3x) COLFI - HydraFibrillar Collagens Monosiga
jgi|Monbr1|31893|estExt_fgenesh2_pg.C_60234 No good hits COLFI COLFI
Fibrillar Collagens Monosiga
jgi|Monbr1|31892|estExt_fgenesh2_pg.C_60233 No good hits COLFI, MIT (wobbly) COLFI
Fibrillar Collagens Nvec
jgi|Nemve1|184625|estExt_GenewiseH_1.C_600080 COL11A1 protein [Homo sapiens] 2.00E-41 COLFI COLFI
Fibrillar Collagens Nvec jgi|Nemve1|80481|e_gw.3.268.1 collagen, type II, alpha 1 [Xenopus laevis] ... 5.00E-55 COLFI COLFIFibrillar Collagens Nvec
jgi|Nemve1|177989|estExt_GenewiseH_1.C_50144 fibrillar collagen [Lethenteron japonicum] 4.00E-47 COLFI COLFI
Fibrillar Collagens Nvec
jgi|Nemve1|226013|fgenesh1_pg.scaffold_16382000001 GCN1 general control of amino-acid synthesis 1-... 1.00E-12 COLFI (wobbly) COLFI
Fibrillar Collagens Nvec
jgi|Nemve1|240904|estExt_fgenesh1_pg.C_360101
procollagen, type XXVII, alpha 1, isoform CRA_a [... 4.00E-52 Collagen (12x), COLFI COLFI - Col17A1
Fibrillar Collagens Nvec jgi|Nemve1|80945|e_gw.3.433.1 alpha 2 type I collagen [Canis lupus familia... 7.00E-43 Collagen (6x), COLFI COLFI - Col2a1A
Appendix B JCUSMART survey of the cnidarian adhesome
203
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrillar Collagens Nvec
jgi|Nemve1|205652|fgenesh1_pg.scaffold_61000084 Col2a1a protein [Danio rerio] 4.00E-24 Collagen (16x), COLFI COLFI - Col2a1A
Fibrillar Collagens Nvec
jgi|Nemve1|240903|estExt_fgenesh1_pg.C_360100 collagen, type V, alpha 3 [Mus musculus] >gi|73... 8.00E-30 Collagen (15x), COLFI COLFI - Col5A3
Fibrillar Collagens Nvec jgi|Nemve1|182|gw.3.2.1 fibrillar collagen [Podocoryne carnea] 2.00E-08 Collagen (6x) COLFI putativeFibrillar Collagens Nvec
jgi|Nemve1|204742|fgenesh1_pg.scaffold_53000081 collagen, type I, alpha 2 [Rattus norvegicus] >... 1.00E-53 WAP, Collagen (16x), COLFI COLFI -Col1A2
Fibrinogen Domain Cltyia IL0ABA19YP18RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 2.00E-39 Conotoxin (wobbly), EGF (wobbly), FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB16YA15RM1 microfibrillar-associated protein 4 type I [Perca... 2.00E-26 FBG FBGFibrinogen Domain Cltyia SA0AAB31YC18RM1
angiopoietin 1 [Danio rerio] >gi|148725450|emb|C... 4.00E-24 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB30YI23RM1
Angiopoietin 1 [Danio rerio] >gi|190339926|gb|AAI... 1.00E-22 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB6YB01CTG
angiopoietin 1, isoform CRA_a [Rattus norvegicus]... 3.00E-24 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB1YF10RM1
angiopoietin 2 [Danio rerio] >gi|15077796|gb|AA... 3.00E-33 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB2YN22RM1
angiopoietin 2 [Danio rerio] >gi|15077796|gb|AA... 8.00E-30 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB44YF22CTG
angiopoietin 4 [Homo sapiens] >gi|17433288|sp|Q... 5.00E-32 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB30YC22RM1 angiopoietin-1 [Bos taurus] 2.00E-21 FBG FBG - angiopoietinFibrinogen Domain Cltyia SA0AAB14YK05RM1
angiopoietin-like protein 4 [Bos taurus] >gi|1884... 4.00E-23 FBG FBG - angiopoietin
Fibrinogen Domain Cltyia SA0AAB40YC16RM1
fibrinogen C domain containing 1 [Mus musculus]... 5.00E-35 FBG FBG - FCDC
Fibrinogen Domain Cltyia SA0AAB121YJ19CTG fibrinogen C domain containing 1 [Rattus nor... 9.00E-37 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB25YB20CTG fibrinogen C domain containing 1 [Rattus nor... 3.00E-36 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB151YO02CTG fibrinogen C domain containing 1 [Rattus nor... 1.00E-33 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB10YA11RM1 fibrinogen C domain containing 1 [Xenopus (S... 2.00E-34 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAA22YK09CTG fibrinogen-like 1 [Bos taurus] >gi|122140308... 2.00E-33 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB108YC15CTG fibrinogen-like 1 precursor [Homo sapiens] >gi|... 9.00E-33 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB6YL01RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 6.00E-43 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAA5YL04RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 2.00E-41 gth (wobbly), FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB3YK12RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 3.00E-41 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB19YK18CTG fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-39 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB16YN22RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-38 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB2YG24RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-31 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB41YK15RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 3.00E-30 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB18YN18RM1
Fcn3-A protein [Xenopus laevis] >gi|213625378|gb|... 7.00E-41 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAB103YH02RM1
ficolin 1 precursor [Homo sapiens] >gi|20455484... 7.00E-30 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAB119YM21RM1
ficolin B [Mus musculus] >gi|119370492|sp|O7049... 4.00E-38 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAB87YN23CTG ficolin-2 [Xenopus laevis] >gi|55154000|gb|A... 9.00E-32 FBG FBG - FicolinFibrinogen Domain Cltyia SA0AAB28YI15RM1
gb|EAW88140.1| ficolin (collagen/fibrinogen domain containing) 1… 4.00E-39 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAA19YE16RM1
ref|NP_112638.2| ficolin (collagen/fibrinogen domain containing)… 9.00E-48 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAB36YJ19RM1
ref|NP_112638.2| ficolin (collagen/fibrinogen domain containing)… 9.00E-19 FBG FBG - Ficolin
Fibrinogen Domain Cltyia SA0AAB40YO07CTG
sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico… 7.00E-24 FBG FBG - Ficolin
Appendix B JCUSMART survey of the cnidarian adhesome
204
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrinogen Domain Cltyia SA0AAB20YN11RM1
tenascin C [Rattus norvegicus] >gi|183013175|gb... 1.00E-42 FBG FBG - Tenascin C
Fibrinogen Domain Cltyia SA0AAB19YC15RM1
tenascin R [Gallus gallus] >gi|61216379|sp|Q005... 5.00E-44 EGF (wobbly), FBG FBG - Tenascin R
Fibrinogen Domain Cltyia SA0AAA17YC03RM1
tenascin R [Rattus norvegicus] >gi|61216102|sp|... 3.00E-33 FBG FBG - Tenascin R
Fibrinogen Domain Cltyia SA0AAB71YF03RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 3.00E-44 EGF (wobbly), FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA24YI07RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 2.00E-36 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB36YN13RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 1.00E-35 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA5YE04RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 7.00E-33 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA3YN21RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 1.00E-28 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB6YH05CTG tenascin R, isoform CRA_b [Rattus norvegicus] 2.00E-34 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB127YB20CTG TNR protein [Homo sapiens] 4.00E-30 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB57YG08CTG tenascin-W [Gallus gallus] 4.00E-29 FBG FBG - Tenascin WFibrinogen Domain Cltyia SA0AAB9YJ17RM1 FIBCD1 protein [Homo sapiens] 1.00E-33 FBG FBG DCFibrinogen Domain Hydra CL3068Contig1 No good hits FBG (wobbly) FBGFibrinogen Domain Hydra CL4995Contig1 predicted peptides only FBG (wobbly) FBGFibrinogen Domain Monosiga
jgi|Monbr1|37116|estExt_fgenesh1_pg.C_100171 No good hits EGF, FBG, EGF (2x) (all wobbly) FBG
Fibrinogen Domain Monosiga
jgi|Monbr1|25354|fgenesh2_pg.scaffold_10000004 No good hits FBG FBG
Fibrinogen Domain Monosiga
jgi|Monbr1|24183|fgenesh2_pg.scaffold_6000092 predicted peptides only FBG FBG
Fibrinogen Domain Nvec
jgi|Nemve1|213673|fgenesh1_pg.scaffold_181000014 adaptor-related protein complex 3, mu 1 subu... 6.00E-10 FBG FBG
Fibrinogen Domain Nvec jgi|Nemve1|104480|e_gw.73.98.1 angiopoietin-1 [Culex quinquefasciatus] >gi|... 6.00E-19 FBG FBGFibrinogen Domain Nvec
jgi|Nemve1|210726|fgenesh1_pg.scaffold_126000052 egg protein [Galaxea fascicularis] 2.00E-36 FBG (wobbly) FBG
Fibrinogen Domain Nvec jgi|Nemve1|124495|e_gw.211.54.1
gb|EAW88140.1| ficolin (collagen/fibrinogen domain containing) 1... 4.00E-19 FBG FBG
Fibrinogen Domain Nvec
jgi|Nemve1|200589|fgenesh1_pg.scaffold_22000111 No good hits EGF (wobbly), FBG FBG
Fibrinogen Domain Nvec jgi|Nemve1|65269|gw.16.345.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|66974|gw.38.279.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|84873|e_gw.10.325.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|92024|e_gw.29.228.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|95763|e_gw.40.258.1 No good hits FBG FBGFibrinogen Domain Nvec
jgi|Nemve1|201999|fgenesh1_pg.scaffold_32000011 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|216620|fgenesh1_pg.scaffold_266000018 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|220900|fgenesh1_pg.scaffold_598000005 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|224116|fgenesh1_pg.scaffold_3959000001 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|224158|fgenesh1_pg.scaffold_4056000001 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|224198|fgenesh1_pg.scaffold_4141000001 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|239078|estExt_fgenesh1_pg.C_110150 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec
jgi|Nemve1|248548|estExt_fgenesh1_pg.C_6550005 No good hits FBG (wobbly) FBG
Fibrinogen Domain Nvec jgi|Nemve1|85018|e_gw.10.356.1 No good hits FBG (wobbly) FBG
Appendix B JCUSMART survey of the cnidarian adhesome
205
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrinogen Domain Nvec jgi|Nemve1|9022|gw.9208.1.1 No good hits FBG (wobbly) FBGFibrinogen Domain Nvec
jgi|Nemve1|218031|fgenesh1_pg.scaffold_325000006 No good hits FBG (wobbly), TSP1 FBG
Fibrinogen Domain Nvec
jgi|Nemve1|229259|fgenesh1_pm.scaffold_63000016 No hits found FBG FBG
Fibrinogen Domain Nvec jgi|Nemve1|150226|e_gw.3344.3.1 predicted peptides only FBG FBGFibrinogen Domain Nvec
jgi|Nemve1|200421|fgenesh1_pg.scaffold_21000106 predicted peptides only FBG FBG
Fibrinogen Domain Nvec
jgi|Nemve1|214950|fgenesh1_pg.scaffold_214000013 predicted peptides only FBG FBG
Fibrinogen Domain Nvec
jgi|Nemve1|214952|fgenesh1_pg.scaffold_214000015 predicted peptides only FBG FBG
Fibrinogen Domain Nvec
jgi|Nemve1|200362|fgenesh1_pg.scaffold_21000047
ref|YP_001803317.1| putative pathogenesis related protein [Cyano… 5.00E-43 FBG FBG
Fibrinogen Domain Nvec jgi|Nemve1|110903|e_gw.103.133.1 techylectin-5B [Culex quinquefasciatus] >gi|... 9.00E-25 FBG FBGFibrinogen Domain Nvec jgi|Nemve1|154536|e_gw.7411.1.1
tenascin R [Danio rerio] >gi|30909302|gb|AAP370... 1.00E-18 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|152370|e_gw.4892.4.1 tenascin XB [Homo sapiens] 1.00E-20 FBG FBG - Tenascin XBFibrinogen Domain Nvec jgi|Nemve1|101484|e_gw.61.218.1 tenascin Y variant [Gallus gallus] 9.00E-18 FBG FBG - Tenascin YFibrinogen Domain Nvec jgi|Nemve1|134301|e_gw.334.16.1 angiopoietin-like 1 [Xenopus (Silurana) trop... 3.00E-37 FBG FBG - angiopoietinFibrinogen Domain Nvec jgi|Nemve1|109792|e_gw.96.16.1
angiopoietin-like protein 4 [Bos taurus] >gi|1884... 3.00E-27 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|81906|e_gw.5.175.1
sp|O43827.1|ANGL7_HUMAN RecName: Full=Angiopoietin-related prote... 1.00E-44 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|87498|e_gw.16.346.1
sp|Q24K15.1|ANGP4_BOVIN RecName: Full=Angiopoietin-4; Short=ANG-.. 1.00E-33 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|53126|gw.155.41.1
sp|Q24K15.1|ANGP4_BOVIN RecName: Full=Angiopoietin-4; Short=ANG-... 4.00E-34 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|63444|gw.40.231.1
sp|Q5EA66.1|ANGL7_BOVIN RecName: Full=Angiopoietin-related prote... 7.00E-54 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|89408|e_gw.21.100.1
sp|Q9UKU9.1|ANGL2_HUMAN RecName: Full=Angiopoietin-related prote.. 9.00E-30 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|89534|e_gw.21.217.1
sp|Q9UKU9.1|ANGL2_HUMAN RecName: Full=Angiopoietin-related prote... 2.00E-32 FBG FBG - angiopoietin
Fibrinogen Domain Nvec jgi|Nemve1|150225|e_gw.3344.5.1
sp|Q9Y264.1|ANGP4_HUMAN RecName: Full=Angiopoietin-4; Short=A... 3.00E-25 FBG FBG - angiopoietin
Fibrinogen Domain Nvec
jgi|Nemve1|218034|fgenesh1_pg.scaffold_325000009 collagen, type XI, alpha 2 [Xenopus (Siluran... 1.00E-40 FBG FBG - Collagen
Fibrinogen Domain Nvec jgi|Nemve1|123220|e_gw.197.32.1 fibrinogen C domain containing 1 [Xenopus (S... 3.00E-60 FBG FBG - FCDCFibrinogen Domain Nvec
jgi|Nemve1|216157|fgenesh1_pg.scaffold_250000030 fibrinogen C domain containing 1 [Xenopus (S... 3.00E-57 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|153808|e_gw.6595.1.1 fibrinogen C domain containing 1 [Xenopus (S... 2.00E-26 FBG FBG - FCDCFibrinogen Domain Nvec jgi|Nemve1|35431|gw.12.187.1
sp|A2AV25.1|FBCD1_MOUSE RecName: Full=Fibrinogen C domain-contai... 4.00E-33 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|54314|gw.121.105.1
sp|A2AV25.1|FBCD1_MOUSE RecName: Full=Fibrinogen C domain-contai... 1.00E-20 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|142843|e_gw.709.6.1
sp|Q6AX44.1|FBCDA_XENLA RecName: Full=Fibrinogen C domain-con... 2.00E-54 FBG FBG - FCDC
Fibrinogen Domain Nvec
jgi|Nemve1|228530|fgenesh1_pm.scaffold_22000015
sp|Q6AX44.1|FBCDA_XENLA RecName: Full=Fibrinogen C domain-con... 8.00E-52 FBG FBG - FCDC
Fibrinogen Domain Nvec
jgi|Nemve1|228654|fgenesh1_pm.scaffold_29000006
sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-con... 1.00E-53 FBG FBG - FCDC
Fibrinogen Domain Nvec
jgi|Nemve1|219569|fgenesh1_pg.scaffold_421000004
sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-con... 6.00E-42 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|78851|e_gw.1.251.1
sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-contai... 1.00E-48 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|87541|e_gw.16.164.1
sp|Q95LU3.1|FBCD1_MACFA RecName: Full=Fibrinogen C domain-contai... 3.00E-57 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|92090|e_gw.29.208.1
sp|Q95LU3.1|FBCD1_MACFA RecName: Full=Fibrinogen C domain-contai... 2.00E-53 FBG FBG - FCDC
Fibrinogen Domain Nvec jgi|Nemve1|118635|e_gw.155.49.1 fibrinogen [Branchiostoma belcheri tsingtaunese] 4.00E-35 FBG FBG - fibrinogenFibrinogen Domain Nvec jgi|Nemve1|113410|e_gw.118.81.1 FCN protein [Xenopus laevis] 3.00E-42 FBG FBG - Ficolin
Appendix B JCUSMART survey of the cnidarian adhesome
206
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrinogen Domain Nvec jgi|Nemve1|113476|e_gw.118.82.1 FCN protein [Xenopus laevis] 7.00E-42 FBG FBG - FicolinFibrinogen Domain Nvec jgi|Nemve1|90597|e_gw.24.49.1
ficolin (collagen/fibrinogen domain containing)… ref|NP_112638.2| 3.00E-57 Collagen, FBG Ficolin same domain structure as Ficolin
Fibrinogen Domain Nvec
jgi|Nemve1|198298|fgenesh1_pg.scaffold_9000087 ficolin 2 isoform a precursor [Homo sapiens] >g... 3.00E-50 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|107528|e_gw.86.58.1
ficolin A [Mus musculus] >gi|13124179|sp|O70165... 2.00E-53 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|125975|e_gw.226.21.1 ficolin B [Bos taurus] >gi|75061130|sp|Q5I2E... 4.00E-39 FBG FBG - FicolinFibrinogen Domain Nvec jgi|Nemve1|95664|e_gw.40.235.1
sp|O00602.2|FCN1_HUMAN RecName: Full=Ficolin-1; AltName: Full=Fi... 1.00E-55 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|95732|e_gw.40.215.1
sp|O00602.2|FCN1_HUMAN RecName: Full=Ficolin-1; AltName: Full=Fi... 1.00E-55 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|95608|e_gw.40.234.1
sp|O00602.2|FCN1_HUMAN RecName: Full=Ficolin-1; AltName: Full=Fi... 3.00E-51 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|82053|e_gw.5.274.1
sp|O75636.2|FCN3_HUMAN RecName: Full=Ficolin-3; AltName: Full=Co... 6.00E-37 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|95788|e_gw.40.232.1
sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi... 4.00E-57 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|35755|gw.45.115.1
sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi... 9.00E-51 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|36375|gw.45.120.1
sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi… 5.00E-51 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|81419|e_gw.4.205.1
sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico... 3.00E-42 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|89338|e_gw.21.197.1
sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico... 3.00E-30 FBG FBG - Ficolin
Fibrinogen Domain Nvec jgi|Nemve1|129798|e_gw.269.68.1 Tenascin-R - BLASTp vs swissprot 2.00E-47 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|147942|e_gw.2188.3.1
gb|EAW90994.1| tenascin R (restrictin, janusin), isoform CRA_a [… 8.00E-36 FBG FBG - Tenascin R
Fibrinogen Domain Nvec
jgi|Nemve1|222877|fgenesh1_pg.scaffold_2028000001
sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; ... 2.00E-55 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92262|e_gw.29.206.1
sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt... 3.00E-59 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|79044|e_gw.1.562.1
sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt... 8.00E-50 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|89364|e_gw.21.171.1
sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt... 1.00E-34 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|48984|gw.421.11.1
sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt… 8.00E-24 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|138098|e_gw.422.22.1
sp|Q8BYI9.1|TENR_MOUSE RecName: Full=Tenascin-R; Short=TN-R; Alt... 1.00E-44 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|36094|gw.10.149.1
sp|Q8BYI9.1|TENR_MOUSE RecName: Full=Tenascin-R; Short=TN-R; Alt… 3.00E-53 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92082|e_gw.29.93.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt.. 2.00E-54 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|95828|e_gw.40.128.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 8.00E-58 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92258|e_gw.29.67.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 1.00E-56 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92265|e_gw.29.70.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 5.00E-54 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|80480|e_gw.3.276.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 5.00E-49 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92150|e_gw.29.212.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 6.00E-49 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|94623|e_gw.37.231.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 2.00E-56 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|92266|e_gw.29.62.1
sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 3.00E-50 FBG FBG - Tenascin R
Fibrinogen Domain Nvec jgi|Nemve1|140550|e_gw.527.4.1 tenascin R, isoform CRA_b [Rattus norvegicus] 2.00E-55 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|113867|e_gw.121.24.1 tenascin-R [Homo sapiens] 1.00E-54 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|113913|e_gw.121.63.1 tenascin-R [Homo sapiens] 1.00E-54 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|108972|e_gw.93.52.1 tenascin-R [Homo sapiens] 4.00E-53 FBG FBG - Tenascin RFibrinogen Domain Nvec
jgi|Nemve1|214077|fgenesh1_pg.scaffold_191000041 tenascin-R [Homo sapiens] 3.00E-51 FBG FBG - Tenascin R
Appendix B JCUSMART survey of the cnidarian adhesome
207
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Fibrinogen Domain Nvec jgi|Nemve1|107155|e_gw.85.168.1 tenascin-R [Homo sapiens] 2.00E-42 FBG FBG - Tenascin RFibropellin/Fibrosurfin Clytia IL0ABA10YE07RM1 fibrosurfin [Paracentrotus lividus] 3.00E-29 No domainsFibropellin/Fibrosurfin Clytia IL0ABA8YH17RM1 RecName: Full=Adhesive plaque matrix prote... 3.00E-34Fibropellin/Fibrosurfin Clytia SA0AAB50YK15CTG fibropellin Ib 7.00E-58Fibropellin/Fibrosurfin Clytia SA0AAB73YD11RM1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-51Fibropellin/Fibrosurfin Hydra gb|CX831322.2 fibropellin Ia 2.00E-18 No domainsFibropellin/Fibrosurfin Monosiga
jgi|Monbr1|25143|fgenesh2_pg.scaffold_9000077 fibrosurfin [Paracentrotus lividus] 7.00E-57 EGF (7x), FN3 (2x), Tyrkin
Fibropellin/Fibrosurfin Nvec jgi|Nemve1|103841|e_gw.70.1.1 fibropellin Ib 1.00E-114Fibropellin/Fibrosurfin Nvec jgi|Nemve1|104325|e_gw.72.146.1 fibrosurfin [Paracentrotus lividus] 2.00E-97Fibropellin/Fibrosurfin Nvec jgi|Nemve1|1059|gw.947.1.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-67Fibropellin/Fibrosurfin Nvec jgi|Nemve1|106413|e_gw.81.97.1 fibropellin III 1.00E-41Fibropellin/Fibrosurfin Nvec jgi|Nemve1|110299|e_gw.99.104.1 fibropellin Ib 2.00E-27Fibropellin/Fibrosurfin Nvec jgi|Nemve1|110621|e_gw.101.87.1
Notch homolog 2 (Drosophila), isoform CRA_b [Homo... 5.00E-42
Fibropellin/Fibrosurfin Nvec jgi|Nemve1|114572|e_gw.126.143.1 fibropellin Ia 2.00E-29Fibropellin/Fibrosurfin Nvec jgi|Nemve1|128516|e_gw.251.27.1 RecName: Full=Adhesive plaque matrix prote... 3.00E-67Fibropellin/Fibrosurfin Nvec jgi|Nemve1|130363|e_gw.278.1.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-82Fibropellin/Fibrosurfin Nvec jgi|Nemve1|136382|e_gw.374.52.1 fibropellin III 1.00E-42Fibropellin/Fibrosurfin Nvec
jgi|Nemve1|223067|fgenesh1_pg.scaffold_2231000001 fibropellin Ia 6.00E-42
Fibropellin/Fibrosurfin Nvec jgi|Nemve1|40287|gw.934.3.1 fibropellin Ia 2.00E-54Fibropellin/Fibrosurfin Nvec jgi|Nemve1|52068|gw.170.90.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-108Fibropellin/Fibrosurfin Nvec jgi|Nemve1|60721|gw.29.181.1
notch homolog 1b [Danio rerio] >gi|60418506|gb|... 7.00E-41
Fibropellin/Fibrosurfin Nvec jgi|Nemve1|61395|gw.24.212.1 RecName: Full=Fibropellin-1; AltName: Ful... 2.00E-49Fibropellin/Fibrosurfin Nvec jgi|Nemve1|84211|e_gw.9.242.1 RecName: Full=Fibropellin-1; AltName: Ful... 3.00E-68Fibropellin/Fibrosurfin Nvec jgi|Nemve1|84597|e_gw.9.264.1 fibropellin Ia 1.00E-58Fibropellin/Fibrosurfin Nvec jgi|Nemve1|87929|e_gw.17.277.1 fibropellin Ia 1.00E-93
Fibulin Cltyia SA0AAB11YG20RM1 Fbln2 protein [Danio rerio] 2.00E-32 EGF (5x) Fibulinref|XP_001625395.1| predicted protein [Nematostella vectensis] >... 2.00E-47
Fibulin Cltyia SA0AAB49YL02RM1 Fibulin 1 2.00E-26 EMI (wobbly), EGF (6x), Gal_lectin FibulinFibulin Cltyia SA0AAB131YO02CTG Fibulin 1 [Homo sapiens] 5.00E-30 EGF (4x) FibulinFibulin Hydra gb|CN774245.1 fibulin-6 [Homo sapiens] 2.00E-25 TSP1 (2x) hemicentin-1
Fibulin Nvecjgi|Nemve1|182469|estExt_GenewiseH_1.C_380136 fibulin 2 [Danio rerio] 1.00E-80 EGF (10x) Fibulin
Fibulin Nvec jgi|Nemve1|919|gw.1034.1.1 fibulin-6 [Homo sapiens] 2.00E-41 TSP1 (4x) fibulin
Fibulin Nvecjgi|Nemve1|216821|fgenesh1_pg.scaffold_273000013 fibulin-6 [Homo sapiens] 5.00E-85 EGF (12x) Fibulin
Fibulin Nvec jgi|Nemve1|110159|e_gw.98.68.1 fibulin-6 [Homo sapiens] 8.00E-87 TSP1 (6x) Fibulin
Fibulin Nvec jgi|Nemve1|12396|gw.273.23.1hemicentin 1, isoform CRA_c [Homo sapiens] / Fibulin-6 [Homo sapiens] 8.00E-56 I-set, TSP1 (3x) hemicentin
Fibulin Nvec jgi|Nemve1|384|gw.329.6.1hemicentin 2 [Mus musculus] >gi|123228129|emb|CA... 5.00E-47 IG, I-set, igc2, I-set, igc2 hemicentin
Fibulin Nvecjgi|Nemve1|224641|fgenesh1_pg.scaffold_5311000001 Hemicentin-1 [human] 4.00E-42 EGF (5x)
Hemicentin-1/Fibrillin-6
Fibulin Nvecjgi|Nemve1|205787|fgenesh1_pg.scaffold_62000106 hemicentin [Homo sapiens] 3.00E-46 IG, igc2 (4x), ig, igc2 hemicentin
Fibulin Nvecjgi|Nemve1|185165|estExt_GenewiseH_1.C_660169 hemicentin 1, isoform CRA_c [Homo sapiens] 3.00E-36 TSP1 (3x)
Appendix B JCUSMART survey of the cnidarian adhesome
208
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
FN2 Domain Containing Cltyia SA0AAB59YM08RM1 coagulation factor XII precursor 1.00E-08 FN2 (3x) FN2
FN2 domains!!! These are supported as E-06 - E-11. there is only one FN2 domain in Nvec (genome paper).
FN2 Domain Containing Hydra CL1445Contig3 predicted peptides only Endotoxin_N (wobbly), FN2 FN2FN2 Domain Containing Hydra CL1445Contig1 predicted peptides only FN2 (wobbly) FN2FN2 Domain Containing Nvec jgi|Nemve1|134613|e_gw.343.31.1 matrix metalloproteinase 9 [Equus caballus] ... 4.00E-24 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|92581|e_gw.30.41.1 vMMP-Lio1 [Liophis poecilogyrus] 1.00E-20 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|28616|gw.30.33.1 vMMP-Lio1 [Liophis poecilogyrus] 8.00E-18 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|92494|e_gw.30.37.1 vMMP-Lio1 [Liophis poecilogyrus] 9.00E-18 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|9233|gw.686.4.1 matrix metalloproteinase 2 isoform a preproprot... 1.00E-11 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|142575|e_gw.686.3.1 matrix metallopeptidase 9 (gelatinase B, 92k... 1.00E-09 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|130895|e_gw.287.17.1 matrix metallopeptidase 9 (gelatinase B, 92k... 3.00E-09 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|86166|e_gw.13.285.1
matrix metalloproteinase 9 [Notophthalmus virides... 3.00E-08 FN2 FN2
FN2 Domain Containing Nvec
jgi|Nemve1|201172|fgenesh1_pg.scaffold_26000101 No good hits PAN (2x), FN2 FN2
FN2 Domain Containing Nvec
jgi|Nemve1|201718|fgenesh1_pg.scaffold_30000069 predicted peptides only EGF, SLG (wobbly), disc (wobbly), FN2 FN2
FN2 Domain Containing Nvec
jgi|Nemve1|198667|fgenesh1_pg.scaffold_11000079 MAM domain containing 4 [Mus musculus] 9.00E-80 FN2, MAM (23x) MAMDC
FN3 Domain Containing Monosiga
jgi|Monbr1|6293|fgenesh1_pg.scaffold_4000175
gb|EEB11871.1| Fibronectin type-III domain-containing protein 3A… 2.00E-41 FN3 (7x) FN3 DC
FN3 Domain Containing Monosiga
jgi|Monbr1|32243|estExt_fgenesh2_pg.C_90007 fibronectin, type III domain-containing protein... 7.00E-75 No domains FN3DC
FN3 Domain Containing Nvec
jgi|Nemve1|243939|estExt_fgenesh1_pg.C_1020048 fibronectin type III domain containing 3B [Homo... 1.00E-118 FN3 (9x) FN3DC
Fras1 Cltyia SA0AAA25YK13CTG Fras1 1.00E-108 No domains Fras1Fras1 Hydra CL917Contig2 FRAS1 protein [Homo sapiens] 6.00E-18 vwc (3x) Fras1
Fras1 Nvec jgi|Nemve1|107201|e_gw.85.18.1FRAS1 related extracellular matrix protein 2, iso… gb|EAX08608.1| 0 NIbeta_1 (3x) Fras1
Fras1 Nvecjgi|Nemve1|207897|fgenesh1_pg.scaffold_85000074
ref|NP_001131133.1| Fras1 related extracellular matrix protein 2… 0 CA, Nibeta_1 (all wobbly)
Fras1 related ECM protein 2
HSPG2 Cltyia SA0AAB41YI05CTG heparan sulfate proteoglycan 2 [Gallus gallu... 1.00E-81 LamG, EGF (2x), LamG, EGF (2x), LamG HSPG2
HSPG2 Clytia SA0AAB56YE16RM1 heparan sulfate proteoglycan 2 [Gallus gallu... 5.00E-34 igc2_5, I-set No Noperlecan puative partial
HSPG2 Hydra CL8208Contig1 heparan sulfate proteoglycan 2 [Danio rerio]... 2.00E-22 I-set, IG No Noperlecan putative partial
HSPG2 Nvec jgi|Nemve1|21768|gw.196.36.1 Hspg2 protein [Mus musculus] 1.00E-136igc2, LamG_3, EGF (2x), LamG_3, EGF, EGF (wobbly), LamG_3 No No Perlecan/HSPG2
Insulin Receptor Like Monosiga
jgi|Monbr1|12183|fgenesh1_pg.scaffold_36000008 insulin receptor, putative [Ixodes scapularis] 7.00E-69 Recep_L_domain, FN3 (2x wobbly), tyrkin insulin receptor
Insulin Receptor Like Monosiga
jgi|Monbr1|7936|fgenesh1_pg.scaffold_9000066
insulin receptor (AGAP012424-PA) [Anopheles gam... 2.00E-46
EGF (6x), Recep_L_domain (wobbly), FN3 (2x 1wobbly), tyrkin insulin receptor like
Insulin Receptor Like Nvec
jgi|Nemve1|198971|fgenesh1_pg.scaffold_12000194
gb|AAI70430.1| Insulin receptor, beta-subunit [Xenopus laevis] >… 2.00E-75
Recep_L_domain, furin, Recep_L_domain, FN3 (3x) insulin receptor beta
Laminin Alpha Hydra gb|DT619742.1 laminin, alpha 5 [Mus musculus] >gi|12328398... 4.00E-28 EGF (3x) Laminin A putativeLaminin Alpha Hydra CL9494Contig1 laminin alpha-1, 2 chain, putative [Ixodes scapul... 9.00E-05 No domains Laminin A putativeLaminin Alpha Monosiga
jgi|Monbr1|6041|fgenesh1_pg.scaffold_3000539 laminin, alpha 1 precursor [Homo sapiens] >gi|2... 4.00E-14
Laminin_N, EGF (3x 2wobbly), PAN (wobbly) Laminin A putative
Laminin Alpha Nvec jgi|Nemve1|110910|e_gw.103.117.1 laminin A chain, putative [Aedes aegypti] >g... 0
Laminin_N, Laminin_EGF (15x), Laminin_B Laminin A
Laminin Alpha Nvec
jgi|Nemve1|248148|estExt_fgenesh1_pg.C_4300002 laminin subunit alpha [Culex quinquefasciatu... 1.00E-115
Laminin_N, Laminin_EGF (7x), Laminin_B, Laminin_EGF (6x) Laminin A
Laminin Alpha Nvec
jgi|Nemve1|208267|fgenesh1_pg.scaffold_90000035 laminin alpha 3 subunit isoform 3 [Homo sapi... 4.00E-64 Laminin_N, Laminin_EGF (11x) Laminin A
Laminin Alpha Nvec jgi|Nemve1|109158|e_gw.93.3.1 laminin subunit alpha [Culex quinquefasciatu... 1.00E-59 EGF (6x 1wobbly) Laminin ALaminin Alpha Nvec jgi|Nemve1|111045|e_gw.103.86.1 laminin, alpha 1 [Danio rerio] >gi|71370785|... 6.00E-51 Laminin_EGF (4x) Laminin A
Appendix B JCUSMART survey of the cnidarian adhesome
209
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Laminin Alpha Nvec jgi|Nemve1|108950|e_gw.93.116.1 laminin, alpha 5 [Mus musculus] >gi|12328398... 3.00E-45 Laminin_EGF (5x) Laminin ALaminin Alpha Nvec jgi|Nemve1|155465|e_gw.8623.2.1 laminin alpha-1, 2 chain [Culex quinquefasci... 2.00E-29 Laminin_EGF (5x) Laminin A putativeLaminin Alpha Nvec jgi|Nemve1|34098|gw.27.98.1 laminin, alpha 2, isoform CRA_a [Mus musculus] 3.00E-26 EGF (4x) Laminin A putativeLaminin Alpha Nvec jgi|Nemve1|155008|e_gw.8010.1.1 laminin alpha 1 [Danio rerio] 1.00E-22 EGF (2x 1wobbly) Laminin A putative
Laminin Beta Nvecjgi|Nemve1|187372|estExt_GenewiseH_1.C_1000105 laminin, beta 1 precursor [Homo sapiens] >gi|51... 0
Laminin_N, Laminin_EGF (13x), SPEC (wobbly)/HAMP (wobbly), MA (wobbly) Laminin B
Laminin Gamma Hydra gb|CX831146.2 LAMC1 protein [Homo sapiens] 9.00E-34 Laminin_N Laminin GLaminin Gamma Hydra CL5311Contig1 laminin gamma 1 chain [Aedes aegypti] >gi|10... 1.00E-39 EGF (2x) Laminin G putativeLaminin Gamma Hydra gb|CO538695.1 laminin gamma 1 [Gallus gallus] 1.00E-24 EGF (2x 1wobbly) Laminin G putativeLaminin Gamma Hydra gb|DT616193.1
laminin, beta 2 [Mus musculus] >gi|19913504|gb|... 1.00E-24 EGF (2x) Laminin G putative
Laminin Gamma Monosiga
jgi|Monbr1|24778|fgenesh2_pg.scaffold_8000012 laminin subunit gamma-3 [Culex quinquefascia... 2.00E-73
CUB, Kelch (wobbly), PSI (3x wobbly), EGF (4x), CUB, EGF (2x), Kelch (5x wobbly), PSI (4x wobbly), EGF (3x wobbly) Laminin G putative
Laminin Gamma Nvec jgi|Nemve1|119462|e_gw.162.15.1
laminin, gamma 1 precursor [Homo sapiens] >gi|2... 0
Laminin_N, Laminin_EGF (11x), tSNARE (wobbly), MA (wobbly), hr1 (wobbly) Laminin G
Laminin Gamma Nvec
jgi|Nemve1|245481|estExt_fgenesh1_pg.C_1620025
sp|Q61292.1|LAMB2_MOUSE RecName: Full=Laminin subunit beta-2; Al… 1.00E-107
Laminin_N, Laminin_EGF (9x), Laminin_B (wobbly), Laminin_EGF, MA (wobbly) Laminin G
Laminin Gamma Nvec
jgi|Nemve1|245480|estExt_fgenesh1_pg.C_1620024 laminin, beta 2 [Gallus gallus] >gi|2708707|gb|... 1.00E-94
Laminin_N, Laminin_EGF (7x), Laminin_B, Laminin_EGF (3x), MA (wobbly) Laminin G
Laminin Gamma Nvec
jgi|Nemve1|221570|fgenesh1_pg.scaffold_839000001 laminin beta-2 chain [Aedes aegypti] >gi|108... 6.00E-50 Laminin_EGF (10x) Laminin G
MEGF Cltyia IL0ABA9YP03RM1 MEGF6 [homo sapien] 1.00E-51 EMI (wobbly), furin (wobbly), EGF (7x) MEGFMEGF Cltyia SA0AAB125YO15CTG MEGF6 [rat] 3.00E-50 EGF (7x), CCP MEGF
MEGF Cltyia SA0AAA2YG05RM1sp|Q7Z7M0.2|MEGF8_HUMAN RecName: Full=Multiple epidermal growth … 8.00E-33 EGF (4x 3wobbly) MEGF
MEGF Monosigajgi|Monbr1|38323|estExt_fgenesh1_pg.C_230054
multiple EGF-like-domains 8 [Homo sapiens] >gi|... 4.00E-54
CUB, EGF (2x), Kelch (5x wobbly), EGF (wobbly), ftp (wobbly), PSI (5x 4wobbly), EGF (18x), CUB, EGF (2x), KELCH (3x 2wobbly), PSI (3x 2wobbly), EGF (4x), lim (wobbly) MEGF
MEGF Nvec jgi|Nemve1|97756|e_gw.47.6.1 MEGF8 [Homo sapiens] 0
CUB, EGF (wobbly), Kelch (5x wobbly), PSI (5x wobbly), EGF (4x 2wobbly), CUB, EGF (3x wobbly), Kelch (7x wobbly), PSI, P_2, PSI (2x), EGF (4x) MEGF
MEGF Nvec jgi|Nemve1|20469|gw.71.15.1sp|Q80T91.3|MEG11_MOUSE RecName: Full=Multiple epidermal growth … 1.00E-120 EGF (14x) MEGF
MEGF Nvec jgi|Nemve1|104003|e_gw.71.71.1 multiple EGF-like-domains 10 [Xenopus (Silur... 2.00E-47 EGF (5x wobbly) MEGF?Minicollagen Cltyia IL0ABA3YI18RM1_p minicollagen 1 [Clytia hemisphaerica] 0.0003 Collagen (wobbly) Minicollagen
Minicollagen Cltyia SA0AAB59YD11RM1ref|XP_002163332.1| PREDICTED: hypothetical protein [Hydra magni… 8.00E-22 Collagen (wobbly), GRP (wobbly) Minicollagen
GRP is a rare domain that is found in plants (not in combination), there are only 4 in metazoa. Only blast hit is PREDICTED: hypothetical protein [Hydra magni... 8.00E-22 ref|XP_002163332.1|
Minicollagen Hydra CL1Contig600 PREDICTED: similar to minicollagen-15 [Hydra... 1.00E-34 Collagen (wobbly) Minicollagen minicollagen similar region is interupted
Minicollagen Hydra CL1Contig103pir||C41132 collagen-related protein 3 precursor - Hydra magnipa...sphaerica] 8.00E-12 GRP (wobbly), Collagen Minicollagen
Minicollagen Hydra CL1Contig299nematoblast-specific protein nb001 [Hydra oligactis] 2.00E-10 Collagen Minicollagen
Minicollagen Hydra CL1Contig531gb|ABR19841.1| minicollagen-15 [Hydra vulgaris] >gi|193792440|gb… 3.00E-10 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL334Contig2 hypothetical protein A032-H1 [Acropora millepora] 4.00E-10 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein.
Minicollagen Hydra CL1Contig549gb|ABR19841.1| minicollagen-15 [Hydra vulgaris] >gi|193792440|gb… 5.00E-09 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig295minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 1.00E-08 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig296collagen-related protein 2 - Hydra magnipapillata (f... 2.00E-08 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig300collagen-related protein 2 - Hydra magnipapillata (f... 2.00E-08 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL334Contig1 hypothetical protein A032-H1 [Acropora millepora] 9.00E-08 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein. Minicollagen Hydra CL334Contig3 hypothetical protein A032-H1 [Acropora millepora] 9.00E-08 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein.
Minicollagen Hydra CL1Contig323collagen-related protein 2 - Hydra magnipapillata (f... 5.00E-06 Collagen (wobbly) Minicollagen
Minicollagen Hydra gb|CD680901.1collagen-related protein 2 - Hydra magnipapillata (f... 8.00E-06 Collagen (wobbly) Minicollagen
Appendix B JCUSMART survey of the cnidarian adhesome
210
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Minicollagen Hydra CL1Contig495collagen-related protein 2 - Hydra magnipapillata (f... 0.0001 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig97collagen-related protein 3 precursor - Hydra magnipa... 0.0006 Collagen Minicollagen
Minicollagen Hydra CL1Contig638minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.049 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig500minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.093 Collagen (wobbly) Minicollagen
Minicollagen Hydra CL1Contig572minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.17 No domains Minicollagen
Minicollagen Hydra CL1Contig287collagen-related protein 4 - Hydra magnipapillata (f... 0.43 Collagen Minicollagen
Minicollagen Hydra CL1Contig83ref|XP_002163332.1| PREDICTED: hypothetical protein [Hydra magni… 3.00E-88 Collagen (wobbly), GRP Minicollagen
Minicollagen Nvecjgi|Nemve1|238353|estExt_fgenesh1_pg.C_50097 mini-collagen [Acropora donei] 0.029 Collagen Minicollagen
Minicollagen Nvecjgi|Nemve1|211803|fgenesh1_pg.scaffold_144000020 mini-collagen [Acropora donei] 0.096 Collagen Minicollagen
Minicollagen Nvec jgi|Nemve1|81941|e_gw.5.7.1 mini-collagen [Acropora donei] 0.28 Collagen (wobbly) MinicollagenNetrin Hydra gb|DT613962.1 ntn1b [Danio rerio] 4.00E-16 Laminin_N netrin-like
Netrin Nvec jgi|Nemve1|118526|e_gw.154.10.1 netrin [Nematostella vectensis] 1.00E-155 Laminin_N, Laminin_EGF (3x), c345c Netrin [Nvec]c345c - Netrin C-terminal Domainhe domain is also found in cobra venom factor and in complement factors C3, C4 and C5
Notch Nvec jgi|Nemve1|41337|gw.2075.6.1 Notch 2 [Takifugu rubripes] 5.00E-73Other Clytia IL0ABA17YC15RM1 papilin, isoform A [Drosophila melanogaster] >g... 6.00E-48 TSP1, ADAM_spacer (wobbly) Papilin related
Other Hydra CL84Contig2emb|CAJ80765.2| thrombospondin type 1 repeat-containing protein … 0 TSP1 (8x)
thrombospondin type 1 repeat-containing protein 2 precursor [Hydra magnipapillata]
Other Hydra CL1487Contig2gb|AAD43811.1|AF159157_1 head-activator binding protein precurso… 1.00E-130 FN3
head-activator binding protein precursor [Hydra vulgaris]
Other Nvecjgi|Nemve1|193237|estExt_GenewiseH_1.C_2820017 nidogen [Aedes aegypti] >gi|108876881|gb|EAT... 5.00E-72 EGF (12x ?wobbly), LY (4x) Nidogen
Other Nvec jgi|Nemve1|87211|e_gw.15.190.1sp|Q61982.1|NOTC3_MOUSE RecName: Full=Neurogenic locus notch … 8.00E-52 EGF (10x)
Notch3 homolgue_N terminal
Other Nvecjgi|Nemve1|238669|estExt_fgenesh1_pg.C_70144 periostin isoform 2 [Danio rerio] >gi|42627706|... 7.00E-50 Fasciclin (3x) periostin
Other Nvecjgi|Nemve1|199904|fgenesh1_pg.scaffold_18000028 usherin isoform B [Homo sapiens] 0
Laminin_N, Laminin_EGF (10x), FN3 (4x), LamG (2x), FN3 (11x) Usherin
Other Nvecjgi|Nemve1|199903|fgenesh1_pg.scaffold_18000027 usherin isoform B [Homo sapiens] 1.00E-118 LamG, Laminin_N, laminin_EGF (5x) Usherin
Other Nvec jgi|Nemve1|59990|gw.18.225.1 usherin isoform B [Homo sapiens] 0 FN3 (16x) Usherin?
Other Nvec jgi|Nemve1|20140|gw.66.6.1rabconnectin-3 beta isoform 2 [Homo sapiens] >g... 0 WD40 (8x) Rabconnectin synaptic transport scaffold for Rab3 GEP and GAP
Other Nvecjgi|Nemve1|240569|estExt_fgenesh1_pg.C_310074
sortilin-related receptor, LDLR class A repeats... ref|NP_035566.2| 0 vps10, LY (3x), LDLa (12x), FN3 (3x) Sortilin lysosomal trafficking
Other Nvecjgi|Nemve1|206807|fgenesh1_pg.scaffold_74000003 sidekick 1 [Gallus gallus] >gi|82242600|sp|Q8AV... 8.00E-67 FN3 (13x) sidekick?
Other Nvecjgi|Nemve1|209051|fgenesh1_pg.scaffold_100000057
stabilin 2 precursor [Homo sapiens] >gi|1455595... 1.00E-159
EGF (4x wobbly), Fasciclin (2x), EGF (6x), Fasciclin (2x) stabilin engulfment
Other Nvec jgi|Nemve1|134716|e_gw.344.35.1 sorting nexin 2 [Gallus gallus] >gi|60098595... 1.00E-137 PX, vps5 sorting nexin endosomal trafficking
Other Nvecjgi|Nemve1|204357|fgenesh1_pg.scaffold_50000041 sidekick 1 [Gallus gallus] >gi|82242600|sp|Q8AV... 2.00E-55
Pan (wobbly), FN3, SEA (wobbly), FN3 (11x) Sidekick?
Other Nvecjgi|Nemve1|242037|estExt_fgenesh1_pg.C_550105
sidekick homolog 1 (chicken), isoform CRA_a [Homo... 1.00E-42 FN3 (9x), Ion_trans_2 sidekick?
Other Nvec jgi|Nemve1|35615|gw.11.151.1 bone morphogenetic protein gb|AAC41710.1| 4.00E-44 CUB (2x) BMP1 like
Other Nvec jgi|Nemve1|36325|gw.11.185.1bone morphogenetic protein 1 [Branchiostoma flori... 1.00E-31 CUB (2x) BMP1 like
PAN-FBG Nvecjgi|Nemve1|206268|fgenesh1_pg.scaffold_67000113 GP2 THP-like protein [Montipora capitata] 5.00E-17 PAN (wobbly), EGF PAN
PAN-FBG Nvecjgi|Nemve1|239485|estExt_fgenesh1_pg.C_150108 No good hits PAN (wobbly), COLFI (wobbly) PAN-COLFI
PAN-FBG Nvecjgi|Nemve1|220640|fgenesh1_pg.scaffold_549000011 No good hits PAN (wobbly), FBG PAN-FBG
PAN-FBG Nvecjgi|Nemve1|221450|fgenesh1_pg.scaffold_771000004 No good hits PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|240565|estExt_fgenesh1_pg.C_310061 No good hits PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|210066|fgenesh1_pg.scaffold_116000022 predicted peptides only PAN (wobbly), EGF (4X) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|202154|fgenesh1_pg.scaffold_32000166 predicted peptides only
Pan (wobbly), EGF (Wobbly), FBG (wobbly) PAN-FBG
Appendix B JCUSMART survey of the cnidarian adhesome
211
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
PAN-FBG Nvecjgi|Nemve1|205103|fgenesh1_pg.scaffold_56000069 predicted peptides only Pan (wobbly), EGF, FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|199873|fgenesh1_pg.scaffold_17000162 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|201513|fgenesh1_pg.scaffold_28000130 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|207725|fgenesh1_pg.scaffold_83000086 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|220897|fgenesh1_pg.scaffold_598000002 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|243269|estExt_fgenesh1_pg.C_830083 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|205658|fgenesh1_pg.scaffold_61000090 predicted peptides only PAN, EGF, FBG (wobbly) PAN-FBG
PAN-FBG Nvecjgi|Nemve1|207720|fgenesh1_pg.scaffold_83000081 predicted peptides only PAN, EGF, FBG (wobbly) PAN-FBG
PAPPA Nvec jgi|Nemve1|10113|gw.217.3.1gb|AAC50543.1| pregnancy-associated plasma protein-A preproform … 0
LamGL, Notch (2x wobbly), Peptidase_M43, CCP (5x), Notch PAPPA
Rhamnospondin Acropora Contig6659
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 6.00E-66 Rhanspondin
Rhamnospondin Acropora Contig3825
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-91 Rhanspondin
Rhamnospondin Acropora Contig13375
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-99 Rhanspondin
Rhamnospondin Acropora Contig3488
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-71 Rhanspondin
Rhamnospondin Acropora Contig7659
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 3.00E-59 Rhanspondin
Rhamnospondin Acropora Contig28422
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 3.00E-57 Rhanspondin
Rhamnospondin Acropora Amil_c933
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 5.00E-51 Rhanspondin
Rhamnospondin Acropora Contig10705
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 3.00E-59 Rhanspondin
Rhamnospondin Acropora Contig8704
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-115 Rhanspondin
Rhamnospondin Acropora Contig5323
gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 6.00E-89 Rhanspondin
Rhamnospondin Cltyia SA0AAB1YK18RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-108 TSP1 (6x)Rhamnospondin Cltyia SA0AAA8YF13RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 8.00E-98 TSP1 (5x), Gal_lectin Gal_lectinRhamnospondin Cltyia SA0AAB114YB09RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-86 TSP1 (5x)Rhamnospondin Cltyia SA0AAA22YC16RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 2.00E-63 TSP1 (4x)Rhamnospondin Cltyia IL0ABA24YA14RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 6.00E-71 TSP1 (4x)Rhamnospondin Nvec jgi|Nemve1|86049|e_gw.12.133.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 5.00E-92 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|34322|gw.12.157.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 2.00E-88 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|138675|e_gw.439.15.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 5.00E-87 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|2608|gw.2423.1.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 7.00E-85 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|2130|gw.3298.1.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 4.00E-80 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|82974|e_gw.6.92.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 8.00E-56 TSP1 (6x) Rhamnospondin
SPOCK Cltyia SA0AAB33YO03RM1sparc/osteonectin, cwcv and kazal-like domains pr... 4.00E-28 Kazal, efhand (wobbly), TY SPOCK
ref|XP_002155894.1| PREDICTED: similar to predicted protein [Hyd... 2.00E-78ref|XP_001641707.1| predicted protein [Nematostella vectensis] >... 3.00E-43
SPOCK Hydra CL2620Contig1gb|EDL32181.1| sparc/osteonectin, cwcv and kazal-like domains pr… 7.00E-23 Kazal, efhand (wobbly), TY SPOCK
SPOCK Nvecjgi|Nemve1|196524|fgenesh1_pg.scaffold_2000146
sparc/osteonectin, cwcv and kazal-like domains pr... 1.00E-28
FOLN (wobbly), Kazal, efhand (2x wobbly), TY SPOCK
Thrombospondin Cltyia IL0ABA26YN08RM1 HyTSR1 protein [Hydra vulgaris] 0 TSP1 (16x) HyTSRThrombospondin Cltyia IL0ABA4YL14RM1 HyTSR1 protein [Hydra vulgaris] 1.00E-144 TSP1 (8x) HyTSR
Appendix B JCUSMART survey of the cnidarian adhesome
212
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Thrombospondin Cltyia SA0AAA6YH14RM1 HyTSR1 protein [Hydra vulgaris] 4.00E-84 TSP1 (5x) HyTSRThrombospondin Cltyia IL0ABA18YA16RM1 HyTSR1 protein [Hydra vulgaris] 3.00E-83 TSP1 (5x) HyTSRThrombospondin Cltyia SA0AAB5YL07RM1 HyTSR1 protein [Hydra vulgaris] 8.00E-55 TSP1 (3x) HyTSRThrombospondin Cltyia SA0AAA8YF14CTG HyTSR1 protein [Hydra vulgaris] 6.00E-37 TSP1 (6x), MAM (wobbly) HyTSRThrombospondin Hydra gb|CX055898.1 HyTSR1 protein [Hydra vulgaris] 1.00E-124 TSP1 (4x) HyTSRThrombospondin Hydra gb|CF777333.1 HyTSR1 protein [Hydra vulgaris] 1.00E-113 TSP1 (3x) HyTSRThrombospondin Hydra gb|DR436385.1 HyTSR1 protein [Hydra vulgaris] 4.00E-74 TSP1 (2x) HyTSRThrombospondin Hydra CL954Contig1 HyTSR1 protein [Hydra vulgaris] 6.00E-45 TSP1 (3x) HyTSRThrombospondin Nvec jgi|Nemve1|116985|e_gw.142.42.1 HyTSR1 protein [Hydra vulgaris] 5.00E-78 TSP1 (6x) HyTSRThrombospondin Nvec jgi|Nemve1|35366|gw.12.184.1 HyTSR1 protein [Hydra vulgaris] 6.00E-66 TSP1 (5x) HyTSRThrombospondin Nvec jgi|Nemve1|33857|gw.12.136.1 HyTSR1 protein [Hydra vulgaris] 2.00E-48 TSP1 (6x) HyTSR
Appendix B JCUSMART survey of the cnidarian adhesome
213
Planar Cell Polarity Signalling
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion NotesPrickle Acropora Contig10161 Prickle like 2 1.00E-126 PET, 3xLIM PricklePrickle Acropora Contig12413 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Acropora Contig2872 Lim-9 1.00E-69 PET, 3xLIM PricklePrickle Acropora Contig14408 Lim-9 1.00E-92 PET, 6x LIMPrickle Acropora run001daytona_1711137 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Acropora run002_436495 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Clytia SA0AAB127YD03CTG Prickle 1.00E-86 3x LIM Prickle
Prickle Nvecjgi|Nemve1|177334|estExt_GenewiseH_1.C_20501 Lim-9 1.00E-112 PET, 6x LIM
Prickle Nvec jgi|Nemve1|79617|e_gw.2.68.1 Prickle 2 1.00E-129 PET, 3xLIM PricklePrickle Hydra CL9755Contig1 prickle 1.00E-31 PET PrickleDishevelled Acropora Contig1475 Dsh (Lv) 1.00E-135 clonedDishevelled Nvec jgi|Nemve1|50431|gw.98.139.1 Dsh 1.00E-176 clonedDishevelled Clytia IL0ABA16YH15RM1 Dsh 1.00E-44
Dishevelled Hydra gb|DN603381.2 Dsh (Hydra) 1.00E-67cloned -XP_002162745
Van Gogh Hydra CL8947Contig1 Vangl 1.00E-67Van Gogh Acropora Contig17842 Vangl 1.00E-100
Van Gogh Nvecjgi|Nemve1|113928|e_gw.121.14.1 Vangl 1.00E-96
Van Gogh Clytia SA0AAA8YK14RM1 Vangl 1.00E-92Inversin Acropora Contig20726 Vangl 1.00E-56Inversin Acropora Contig28177 Vangl 1.00E-73Inversin Acropora Contig499 Vangl 1.00E-63
Inversin Nvecjgi|Nemve1|94580|e_gw.37.192.1 Vangl 1.00E-63
beta-catenin Acropora !"#$%"%&'()*+,-..beta-catenin Nvec Genbank: AAL49498beta-catenin Hydra Genbank: AAC47137beta-catenin Clytia SA0AAA13YE16CTG 1.00E-170LRP5/6 Acropora Contig26866 LRP6 1.00E-156LRP5/6 Acropora run001daytona_1726367 LRP6 1.00E-126LRP5/6 Acropora run002_425839 LRP6 0 *LRP5/6 Acropora Contig33249 LRP6 0LRP5/6 Acropora Contig14656 LRP6 1.00E-127LRP5/6 Acropora Contig34020 LRP6 1.00E-87LRP5/6 Nvec jgi|Nemve1|32389|gw.3.133.1 LRP6 1.00E-81Kynepk Acropora Contig1784 1.00E-67Kynepk Acropora Amil_c20217 1.00E-74Kynepk Nvec 1.00E-84
Kynepk Hyrda CL2103Contig1Genbank XP_002157574
Atrophin Acropora Contig34266 1.00E-125Atrophin Hydra CL5991Contig1 1.00E-129Atrophin Hydra CL6620Contig1 1.00E-114
Atrophin Nvecjgi|Nemve1|93577|e_gw.33.159.1 1.00E-46
Frizzled Acropora Amil_c46307 + Amil_c11915
Frizzled Acropora Contig20909 + EST_C007-H4 Fz4 Human 1.00E-156Frizzled domain
ref|XP_001622965.1| 3063bp
Frizzled Acroporarun001daytona_628203 + Contig 7411 Fz7 1.00E-172
Frizzled domain XP_001647540.1 0
Frizzled Acropora Contig30897 + EST_D019-E7 Fz2 Xenoppus 1.00E-32 FRI domains 3286bp
jgi|Nemve1|247677|estExt_fgenesh1_pg.C_3350009
Appendix B JCUSMART survey of the cnidarian adhesome
214
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Frizzled Acropora
EST_D016-E8 + Amil_rep_c193699 + Amil_c4306 + run001daytona_1734729 + Contig24514 893bp
Frizzled Acropora Contig6070 Fz8 Danio 1.00E-175Frizzled domain
ref|XP_001634995.1| 2958bp
Frizzled Acropora run001daytona_1732102 Fz2 Xenoppus 1.00E-32 FRI domainsFrizzled Acropora Contig8892 Fz7 Danio 1.00E-41 FRI domains ref|XP_001647540.1|Frizzled Acropora Contig4001 Frizzled-related 1.00E-38 FRI domains 976bpFrizzled Acropora Contig12602 Fz8 Mus 1.00E-21 FRI domains 954bp
Frizzled Acropora Contig11982 Fz10 Danio 1.00E-147Frizzled domain
ref|XP_001630630.1| 3063bp
Frizzled Acropora Contig8177 latrophillin3Frizzled domain
Frizzled Acropora Contig8091 smoothenedFrizzled domain
Frizzled Clytia SA0AAB17YK01RM1frizzled 2 [Hydra magnipapillata] 1.00E-122
Frizzled/7tm_2 Frizzled
Frizzled Clytia IL0ABA3YD02RM1frizzled-8 [Xenopus laevis] >gi|3869266|gb|A... 2.00E-22 Fz Frizzled - putative There are described Frizzled proteins in Hydra.
Frizzled Clytia SA0AAB36YE16RM17-transmembrane receptor frizzled-1 [Xenopus... 8.00E-20
Fz, Gal_lectin(wobbly) Frizzled - putative
Frizzled Clytia IL0ABA4YO10RM1frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 2.00E-19
Fz, Gal_lectin(wobbly) Frizzled - putative
Frizzled Hydra CL4401Contig1frizzled 2 [Hydra magnipapillata] 1.00E-101 Fz Frizzled
Frizzled Hydra CL6396Contig1frizzled receptor [Hydra vulgaris] 1.00E-127 Fz Frizzled
next best hit frizzled [Aedes aegypti] >gi|108875685|gb|EA... 6.00E-07
Frizzled Hydra CL10411Contig1 frizzled receptor [Hydra vulgaris] 7.00E-60 No domains Frizzled
Frizzled Hydra gb|CX835366.2PREDICTED: Frizzled4/9/10 [Hydra magnipapill... 3.00E-59 Fz (wobbly) Frizzled
Frizzled Hydra CL2039Contig1 Fzd7 protein [Mus musculus] 6.00E-25 Fz Frizzled - putative
Frizzled Hydra CL5686Contig1frizzled homolog 8 [Rattus norvegicus] >gi|1... 5.00E-16
Fz, gal_lectin (wobbly) Frizzled - putative
Frizzled Hydra gb|CN560484.1 frizzled 2 [Hydra magnipapillata] 1.00E-33 No domains Frizzled - putative
Frizzled Nvecjgi|Nemve1|171640|estExt_gwp.C_1830043
7-transmembrane receptor frizzled-1 [Xenopus... 1.00E-180 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|168924|estExt_gwp.C_1170009
frizzled homolog 10 [Xenopus (Silurana) trop... 1.00E-155 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|139208|e_gw.466.3.1
frizzled homolog 4 [Gallus gallus] >gi|17433062... 1.00E-137 Fz, Frizzled Frizzled
Frizzled Nvecjgi|Nemve1|183962|estExt_GenewiseH_1.C_530042
frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 0 Fz, Frizzled Frizzled
PREDICTED: similar to More Of MS family memb... 1.00E-107
Axin Acropora Contig4409 Axin2 1.00E-24Axin Clytia SA0AAB78YI15RM1 Axin1 1.00E-08Axin Hydra CL7700Contig1 Axin 1.00E-09
Axin Nvecjgi|Nemve1|248853|estExt_fgenesh1_pg.C_40840001 Axin1 1.00E-03
Axin Nvecjgi|Nemve1|182113|estExt_GenewiseH_1.C_340142 Axin1 1.00E-28
Appendix B JCUSMART survey of the cnidarian adhesome
215
Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes
Flamingo Nvec jgi|Nemve1|84228|e_gw.9.5.1 CELSR 0
CA (8x), EGF (2-3), LamG, EGF, LamG, EGF (3x), HormR_1, GPS, 7tm_2 No 1 Flamingo start and stop codons present
Flamingo Acropora Contig8737 + Contig3665APC Acropora Contig20548
APC Nvecjgi|Nemve1|137471|e_gw.401.17.1
APC Hydra CL4732Contig1Wnt16 Clytia SA0AAB3YI03CTG Hydra Wnt16 1.00E-86Wnt16 Hydra BAH23775.1Wnt16 Nvec ABF48091.1Wnt16 AcroporaGSK3beta Acropora Contig33988 GSK3b 0GSK3beta Clytia SA0AAB127YF01CTG GSK3b 0GSK3beta Hydra gb|CV985547.1 GSK3bGSK3beta Nvec 190252 GSK3bGroucho Acropora Contig21079
Groucho Nematostellajgi|Nemve1|184640|estExt_GenewiseH_1.C_600171
Groucho Clytia SA0AAB51YI19RM1Groucho Hydra CL3019Contig1
cloned from cDNA -Ukolova, unpublished