Macroarray analysis of coelomocyte gene expression in ...€¦ · basis of the striking increase in...

doi:10.1152/physiolgenomics.00052.2005 22:33-47, 2005. First published Apr 12, 2005;Physiol. Genomics

Courtney Smith Sham V. Nair, Heather Del Valle, Paul S. Gross, David P. Terwilliger and L.unexpected immune diversity in an invertebrate response to LPS in the sea urchin. Identification of Macroarray analysis of coelomocyte gene expression in

You might find this additional information useful...

for this article can be found at: Supplemental material http://physiolgenomics.physiology.org/cgi/content/full/00052.2005/DC1

128 articles, 51 of which you can access free at: This article cites http://physiolgenomics.physiology.org/cgi/content/full/22/1/33#BIBL

including high-resolution figures, can be found at: Updated information and services http://physiolgenomics.physiology.org/cgi/content/full/22/1/33

can be found at: Physiological Genomicsabout Additional material and information http://www.the-aps.org/publications/pg

This information is current as of July 1, 2005 .

http://www.the-aps.org/.the American Physiological Society. ISSN: 1094-8341, ESSN: 1531-2267. Visit our website at July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 bytechniques linking genes and pathways to physiology, from prokaryotes to eukaryotes. It is published quarterly in January, April,

publishes results of a wide variety of studies from human and from informative model systems withPhysiological Genomics

on July 1, 2005 physiolgenom

ics.physiology.orgD

ownloaded from

http://physiolgenomics.physiology.org/cgi/content/full/00052.2005/DC1

http://physiolgenomics.physiology.org/cgi/content/full/22/1/33#BIBL

http://physiolgenomics.physiology.org/cgi/content/full/22/1/33

http://www.the-aps.org/publications/pg

http://www.the-aps.org/

http://physiolgenomics.physiology.org

Macroarray analysis of coelomocyte gene expression in responseto LPS in the sea urchin. Identification of unexpectedimmune diversity in an invertebrate

Sham V. Nair,1 Heather Del Valle,1 Paul S. Gross,2 David P. Terwilliger,1 and L. Courtney Smith1

1Department of Biological Sciences, George Washington University, Washington, District of Columbia;and 2Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina

Submitted 1 March 2005; accepted in final form 6 April 2005

Nair, Sham V., Heather Del Valle, Paul S. Gross, David P.Terwilliger, and L. Courtney Smith. Macroarray analysis of coelo-mocyte gene expression in response to LPS in the sea urchin. Iden-tification of unexpected immune diversity in an invertebrate. PhysiolGenomics 22: 33–47, 2005. First published April 12, 2005;10.1152/physiolgenomics.00052.2005.—The purple sea urchin,Strongylocentrotus purpuratus, is a member of the phylum Echino-dermata, which is basal to the phylum Chordata within the deuteros-tome lineage of the animal kingdom. This relationship makes theanalysis of the sea urchin immune system relevant to understandingthe evolution of the deuterostome immune system leading to theVertebrata. Subtractive suppression hybridization was employed togenerate cDNA probes for screening high-density arrayed, conven-tional cDNA libraries to identify genes that were upregulated incoelomocytes responding to lipopolysaccharide. Results from 1,247expressed sequence tags (ESTs) were used to infer that coelomocytesupregulated genes involved in RNA splicing, protein processing andtargeting, secretion, endosomal activities, cell signaling, and alter-ations to the cytoskeletal architecture including interactions with theextracellular matrix. Of particular note was a set of transcripts repre-sented by 60% of the ESTs analyzed, which encoded a previouslyuncharacterized family of closely related proteins, provisionally des-ignated as 185/333. These transcripts exhibited a significant level ofvariation in their nucleotide sequence and evidence of putative alter-native splicing that could yield up to 15 translatable elements. On thebasis of the striking increase in gene expression in response tolipopolysaccharide and the unexpected level of diversity of the 185/333 messages, we propose that this set of transcripts encodes a familyof putative immune response proteins that may represent a majorcomponent of an immunological response to bacterial challenge.

innate; echinoderm; lipopolysaccharide; 185/333

THE IMMUNE SYSTEM of the purple sea urchin, Strongylocentrotuspurpuratus, lacks the hallmarks of adaptive immunity (103).Yet, as with other invertebrates studied thus far, there areseveral commonalities among the innate immune reactions ofall animals. Current paradigms suggest that invertebrates rec-ognize conserved molecular structures, or pathogen-associatedmolecular patterns (PAMPs), displayed by invading pathogens.Recognition is mediated by pattern recognition receptors(PRRs), which lack the discriminative capacity of the immu-noglobulin-based receptors of the vertebrate adaptive immunesystem (52, 68). This paradigm is now being challenged bynewly emerging data from a number of laboratories, including

ours, which may necessitate a reappraisal of immune diversityin invertebrates (15, 16, 24, 63 and reviewed in Ref. 28).

Studies assessing the immune response in sea urchins haveshown that the coelomic cavity is rapidly cleared of microbes,foreign cells, and other materials by the activities of phagocyticcoelomocytes (reviewed in Ref. 104). Although there areseveral morphologically distinct classes of coelomocytes, in-cluding phagocytes, red spherule cells, colorless spherule cells,and vibratile cells (27, 54, 103), the phagocytes appear to havea central role in host defense. Phagocytes have extensivecytoskeletons and are amoeboid, macrophage-like cells thatundergo significant changes in shape during chemotaxis,phagocytosis, encapsulation, clot formation, secretion, anddegranulation, all of which are associated with immune re-sponses (32, 34, 99). Analysis of expressed sequence tags(ESTs) prepared from immune-activated coelomocytes identi-fied a simple complement system operating in sea urchins(101). The sea urchin homolog of vertebrate complementcomponent C3 (SpC3) was the first member of the thioester-containing family of proteins identified in an invertebrate (3).It appears that SpC3 is functionally similar to its vertebratehomologs (97) in that it mediates opsonization of foreignmatter and pathogens, which leads to augmented uptake byphagocytic coelomocytes (20, 96). A homolog of factor B (Bf),called SpBf, has also been identified in sea urchins (98, 113)along with two cDNAs that encode the mosaic proteins Sp5and Sp5013 that share a number of domains [including a factorI membrane attack complex (FIMAC) domain] with comple-ment regulatory proteins and members of the terminal comple-ment pathway (70).

The use of large-scale screening technologies employingmicroarrays, cDNA libraries, and proteomics (118) has pro-vided insights into transcriptional programs that are initiated inanimal cells responding to pathogenic threat. These approacheshave significantly accelerated the rate of gene discovery ininvertebrates, including two genera of shrimp, Penaeus (108)and Litopenaeus (33); two species of oyster, Crassostrea gigas(35) and C. virginica (53); and the sea urchin, S. purpuratus(101). Such studies have been fruitful even for the morefamiliar and well-characterized invertebrates such as the fruitfly, Drosophila melanogaster (18, 48a), and two mosquitospecies, Anopheles gambiae (17) and Aedes aegypti (94).Comparative analysis of the emerging data indicates that thereare general classes of genes that may be the common denom-inators of invertebrate immune responses. The molecules en-coded by these genes include peptidoglycan recognition pro-teins, thioester-containing proteins, gram-negative bindingproteins, multidomain scavenger receptor family, ficolins, C-

Article published online before print. See web site for date of publication(http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: L. C. Smith, Dept. ofBiological Sciences, 340 Lisner Hall, George Washington Univ., 2023 G St.NW, Washington, DC 20052 (e-mail: [email protected]).

Physiol Genomics 22: 33–47, 2005.First published April 12, 2005; 10.1152/physiolgenomics.00052.2005.

1094-8341/05 $8.00 Copyright © 2005 the American Physiological Society 33


ics.physiology.orgD

ownloaded from


type lectins, and galectins. Other commonly expressed groupsof proteins comprise proteases (serine proteases, cysteine pro-teases, metalloproteases), protease inhibitors (kazal-type serineprotease inhibitor, serpin, �2-macroglobulin), antimicrobialpeptides, superoxide dismutase, and metal-binding proteins.Proteolytic cascades (such as the prophenoloxidase and theclotting/coagulation systems) and certain signal transductionsystems, especially those leading to the activation of rel/NF-�B(17, 35, 108, 33), are also common components of immuneactivation in invertebrates.

By employing EST studies or differential display, a numberof genes in sea urchin coelomocytes have been shown to beinduced by immune challenge, including Sp064, which en-codes SpC3 (19), the transcription factors SpNFkB andSpRUNT (78), as well as other uncharacterized transcripts(83). The expression of a C-type lectin, Sp056 (GenBankaccession no. AY663300), is only detectible after LPS chal-lenge and has been used as a reliable marker of coelomocyteactivation (70, 113). On the other hand, there are some im-mune-related genes expressed in coelomocytes that are notinduced by immune challenge, including Sp152 (SpBf; Ref.113), Sp5 and Sp5013 (70), and a large set of scavengerreceptors with cysteine-rich (SRCR) domains (76, 77). Fur-thermore, the transcription factor SpGATAc has been shown tobe downregulated in response to immune challenge (78).

At present, only a limited number of immune effectorcascades (e.g., complement components, a set of induciblelectins, and SRCR proteins) have been identified in sea urchins,but given the effectiveness of the sea urchin to repel infection,it is expected that the sea urchin immune system will havemany more as yet unidentified components and cascades. It ishypothesized that large-scale genomics-based screening ap-proaches should reveal additional genes with central impor-tance in sea urchin immunity. To test this hypothesis, normal-ized subtracted probes were used to identify expressed genesthat were induced by LPS through screening two arrayed,conventional, coelomocyte cDNA libraries, one of which wasconstructed from bacterially activated cells. Reported here arethe detailed results from an analysis of ESTs, which revealedthat a number of genes were induced, including many novelones. Matches to known sequences suggested that coelomo-cytes responded to gram-negative bacteria by increased expres-sion of genes encoding proteins involved in RNA splicing,protein processing and secretion, signaling pathways, modifi-cation of the cytoskeletal architecture, and altered interactionswith the extracellular matrix. Of particular note was the obser-vation that the vast majority of the ESTs represented a set ofsimilar transcripts that encoded a family of putative immuneresponse proteins. It is hypothesized that these proteins may bea major immune effector system in the sea urchin based on astriking increase in gene expression in response to LPS plussignificant sequence diversity due to variations in the primarynucleotide sequence and putative alternative splicing.

MATERIALS AND METHODS

Sea urchins. Animals were obtained and housed as previouslydescribed (34, 95). Immunoquiescent animals were generated bylong-term housing (�6–8 mo) in a closed aquarium without signifi-cant disturbance (19, 32, 95).

Animal treatments and RNA isolation. Coelomocytes were with-drawn from sea urchins as described (32, 70), and total RNA was

isolated using an RNeasy Mini kit according to the manufacturer’sprotocol (Qiagen, Valencia, CA). Sea urchins were injected once onday 1 and twice on day 2 (�9-h interval) with 0.5 �g LPS/mlcoelomic fluid, according to the methods of Smith et al. (99). Coelo-mocytes collected for RNA isolation and cDNA subtractions wereobtained before injection (immunoquiescent) and on day 3 (LPSactivated).

RT-PCR and construction of suppressive subtractive hybridizationcDNA. Reverse transcription reactions with random hexamer primerswere carried out on 0.5–3.0 �g of total RNA, 200 U of Superscript IIRT (Life Technologies, Carlsbad, CA), and 20 U RNAsin (Promega,Madison, WI). The resultant cDNA (1 �l) was amplified by PCRusing 1 U of Taq polymerase (Life Technologies) and 1 �M eachprimer (Supplemental Table S1; available at the PhysiologicalGenomics web site).1 Construction of suppressive subtractive hybrid-ization (SSH) cDNA was carried out using the PCR-select cDNAsubtraction kit (Clontech, Palo Alto, CA) according to the manufac-turer’s instructions. Forward cDNA subtraction employed coelomo-cyte RNA from an LPS-activated sea urchin (tester) subtracted fromRNA prepared from the same animal before challenge (driver), whilereverse cDNA subtraction used immunoquiescent RNA (tester) sub-tracted from LPS-activated RNA from the same sea urchin (driver).The quality of the SSH reaction was assessed using a virtual Northernblot. Samples of forward- and reverse-subtracted cDNAs, in additionto unsubtracted cDNA controls, were amplified by PCR using Clon-tech primers, electrophoresed, and blotted using standard methods(87). Filters were probed with inserts from specific clones (Supple-mental Table S1) that were amplified by PCR and labeled withalkaline phosphatase according to the manufacturer’s instructions(AlkPhos Direct; Amersham Pharmacia Biotech, Piscataway, NJ).

Riboprobe preparation. Forward- and reverse-subtracted cDNAsthat served as riboprobe templates were amplified by PCR using theP1 forward primer and the nested P2R reverse primer (PCR-selectcDNA subtraction kit, Clontech). The amplified cDNAs were labeledwith the use of T7 RNA polymerase (Promega) and [32P]rUTP (ICN,Costa Mesa, CA). The eluates from G50 Sephadex spin columns(Amersham Pharmacia Biotech) were quantified in an LS6500 scin-tillation counter (Beckman Instruments, Fullerton, CA). Probes werediluted to a final volume of 500 �l in hybridization solution [50%deionized formamide, 0.25 M phosphate buffer, 0.1% (wt/vol) BSA,1 mM EDTA, 7% (wt/vol) SDS] and heated to 65°C for 4 min beforeaddition to the filters.

Riboprobes were also generated by run-off polymerization usingthree 185/333 clones (Sp0323, Sp0324, Sp0325). Each clone waslinearized with XhoI, and [32P]rUTP (ICN) was incorporated into ariboprobe with T3, T7, or Sp6 RNA polymerase, depending on thetemplate (see Ref. 70). Unincorporated [32P]rUTP was removed withthe use of a G50 Sephadex spin column (Amersham PharmaciaBiotech). Probes were heated to 60°C for 4 min before hybridization.

Macroarray filter screens and clone selection. Two arrayed cDNAlibraries (5 filters per library of 92,160 clones) were used for analysis,one constructed from bacterially activated coelomocytes (14, 78, 82)and a second from nonactivated coelomocytes (3). For both libraries,coelomocytes were pooled from five sea urchins. Each library wasscreened with 32P-labeled riboprobes derived from the SSH cDNAsand specific cloned templates that were also used for virtual Northernblot analysis (Supplemental Table S1). Briefly, macroarray filterswere prehybridized for 1 h at 42°C followed by hybridization with theriboprobe at 42°C overnight in a rotating oven. Filters were washedtwice for 30 min at 65°C in each of the following wash buffers: 4�SSC with 1% SDS, 2� SSC with 1% SDS, and 1� SSC with 1%SDS. Wet filters were sealed within plastic bags and exposed to film

1 The Supplemental Material for this article (Supplemental Tables S1 andS2) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00052.2005/DC1.

34 IMMUNE DIVERSITY IN A SEA URCHIN

Physiol Genomics • VOL 22 • www.physiolgenomics.org


ics.physiology.orgD

ownloaded from


at �70°C using intensifying screens for varying periods. After auto-radiography, filters were stripped of the probes by denaturation in 0.4M NaOH for 30 min at 45°C followed by incubation in strippingbuffer (0.1� SSC, 0.1% SDS, 0.2 M Tris �HCl, pH 7.5) for 30 min at65°C. Final wash was in stripping buffer with 0.2 M EDTA for 10 minat room temperature. After stripping, filters were exposed to film toensure that there was no residual radioactivity associated with thefilters and stored at �20°C.

Clones identified during screening were manually picked, isolated,robotically arrayed into 96-well plates (as described in Ref. 33), andsequenced (Amplicon Express, Pullman, WA). Sequence similaritieswere identified in the nonredundant nucleic acid database and the ESTdatabase (GenBank) using basic local alignment search tool(BLAST)n and BLASTx algorithms accessed from the NationalCenter for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/Blast/). Only matches with e-values of �10�6

were considered significant. The ESTs generated in this study havebeen submitted to GenBank (accession nos. CK828301–CK829214and CV652690–CV652795).

Nonsynonymous-synonymous (dn/ds) ratio analysis. The 5-ends of293 of the best quality 185/333 ESTs were analyzed for nucleotidediversity. The region was 195 bases long and was manually aligned inBioEdit (38). Analysis of the alignment using WinClada (75) indi-cated that 250 of the 293 sequences were duplicates, which wereremoved to simplify further analysis. Two clones, Sp0267 andSp0291, that had stop codons in element 2 were also removed, leaving42 unique EST clones for phylogenetic analysis by maximum likeli-hood (PAML) (126). A neighbor-joining tree was generated in mo-lecular evolutionary genetics analysis (MEGA) v.3.0 (56) and used forthe PAML analysis.

RESULTS AND DISCUSSION

The availability of immunoquiescent sea urchins has been animportant tool for the investigation of specific responses toinjury and pathogenic challenge. This is a limiting factor whenattempting to assess immune responses in animals collectedwithin a few days or weeks from the wild. Contact withmicrobes and pathogens in a natural environment activates hostdefenses continuously in these animals and therefore precludestheir use as baseline or downregulated controls. Stress fromcollection and shipping exacerbates immune activation. Hence,in this study we have used only immunoquiescent animals togenerate a baseline of immunological activation. Immunoqui-escence has been defined as the condition in which the abun-dance of certain immune-specific transcripts becomes dimin-ished in sea urchin coelomocytes compared with animals livingin their natural environment (32). Immunoquiescence has beendemonstrated in S. purpuratus after animals have been housedin closed-circulation aquaria in the laboratory for 6–8 mo (19,20, 32, 34). Long-term housing does not induce immunosup-pression, only immunoquiescence, as immune reactivity can bereversed with injections of bacteria or LPS or from injury.Such challenges result in increases in the relative abundance ofthe immune-specific transcripts within individual animals (19,20). In a modified approach to that reported previously (101),immunoquiescent sea urchins were used to obtain RNA fromcoelomocytes pre- and post-LPS challenge for normalizedsubtractions. Subtracted cDNAs generated from coelomocytescollected from the same animal were used to produce ribo-probes for screening filters from arrayed coelomocyte cDNAlibraries. By employing the same animal for these purposes, weavoided inherent problems associated with the genetic diversitypresent among out-bred sea urchins (99, 100).

Immunoquiescence and RT-PCR analysis of LPS activation.The immunoquiescent status of four randomly chosen seaurchins was assessed by RT-PCR for expression of severalcoelomocyte-specific genes. Two animals were found to beimmunoquiescent, as evidenced by the lack of initial expres-sion of the gene encoding a C-type lectin (Sp056; Fig. 1A) andits subsequent induction after challenge (Fig. 1B). This resultwas in agreement with previous work wherein Sp056 proved tobe a useful marker for assessing immune activation to LPSchallenge (70, 113). A second gene, whose pre- and postchal-lenge induction is more variable among individual animalsunder laboratory-rearing conditions, is Sp064, which encodesSpC3 (19). It was found to have low expression or wasundetectable before challenge (Fig. 1A, animals 2 and 3) butwas inducible in all animals after LPS injection, with oneindividual exhibiting a large change in expression (Fig. 1B,animal 2). The expression of three additional genes, SpNFkB(78), Sp152 (113) and cytoplasmic actin (CyI; Ref. 58), wasanalyzed by RT-PCR but showed no change in response to LPS(data not shown). Animal 2, which showed the largest detect-able increase in Sp064 expression, was chosen for furtheranalysis.

cDNA subtraction and virtual Northern blot analysis. For-ward- and reverse-subtracted cDNAs were generated by SSHfrom isolated RNA samples obtained from animal 2. Forwardsubtraction selectively enriches the pool of transcripts thatincrease upon LPS challenge and injury, whereas the reversesubtraction enriches for transcripts that diminish upon LPSchallenge and injury. The efficiency of both the forward andreverse subtractions was evaluated by comparing subtractedand unsubtracted cDNAs using virtual Northern blot analysis.Reverse subtraction effectively removed CyI and Sp056 tran-scripts from the immunoquiescent cDNA pool (Fig. 2, lanes 3and 11), whereas these cDNAs were enriched in the LPS-activated or forward-subtracted cDNA preparation (Fig. 2,lanes 4 and 12). This indicated that successful subtraction hadoccurred in both directions. Both Sp056 and CyI were identi-fied in the unsubtracted cDNA from both activated and immu-noquiescent coelomocytes, although greater hybridization wasobserved in the LPS-activated cDNA pools (Fig. 2, lanes 2 and10). The change in CyI expression may actually reflect a real

Fig. 1. Gene expression in coelomocytes from immunoquiescent sea urchinsbefore and after LPS activation. Coelomocytes were collected from fouranimals (1–4), and gene expression was analyzed by RT-PCR before (A) andafter (B) LPS challenge. Primers for Sp056 and Sp064 were used to amplifycDNAs (Supplementary Table S1). Animals 2 and 3 showed increased bandintensity after LPS challenge using primers for Sp064. None of the sea urchinsamples supported amplification with Sp056 (lectin) primers before challenge;however, all samples demonstrated expression of Sp056 after challenge. Foreach set of reactions, positive controls (C) employed a cloned cDNA as thetemplate. Negative controls (—) omitted cloned templates from the PCRreactions: 1, Sp056; 2, Sp064.

35IMMUNE DIVERSITY IN A SEA URCHIN



ics.physiology.orgD

ownloaded from


increase in actin gene expression in LPS-activated coelomo-cytes and would correspond with increased profilin expression(99, 100), which is involved in modifications to cytoskeletalshape (81).

Macroarray filter screens. Macroarray filters from two con-ventionally constructed and arrayed coelomocyte cDNA librar-ies were screened twice each with reverse-subtracted ribo-probes and then stripped and rescreened with forward-sub-tracted riboprobes. Spots that were positive either for thereverse-subtracted probe or for both probes were not consid-ered further. Filters were also screened for CyI (actin), Sp056(lectin), and Sp064 (SpC3), and positive clones were excludedfrom analysis (data not shown). From �6,000 clones that wererepeatedly identified from the bacterially activated library asgenes that were differentially expressed during LPS challengeand/or injury, 1,025 randomly chosen clones were sequencedinitially. Approximately 73% (746) showed significantmatches to hitherto uncharacterized cDNAs known as DD185(Ref. 78, GenBank accession no. AF228877) and EST333(Ref. 101, GenBank accession no. R62081), hereafter provi-sionally called 185/333. Because there was such a high pre-ponderance of 185/333 sequences among the clones analyzed,filters were rescreened using 185/333 riboprobes, and positiveswere excluded from the analysis of a subsequent 222 positiveclones. Of the 1,247 clones that were analyzed in total, 71sequences (5.7%) contained too many indeterminate base callsfor successful matching, while 328 sequences (26%) werecategorized as unknowns either because there was no signifi-cant similarity to any sequence (11%) or poor (e-value �10�6)similarity (15%) by BLAST searches of the nonredundantdatabase. Only 61 ESTs matched known sequences fromStrongylocentrotus species, and these represented 23 distinctgene products. Thus this project has deposited �1,100 ESTsinto the public domain. These represent novel sea urchintranscripts that are synthesized by immune-activated coelomo-cytes.

Sequences that could be assigned a putative identificationcould be further clustered by associated function (Fig. 3). Thevast majority of ESTs sequenced were 185/333 transcripts, buta number of immune-related genes were also identified. Inaddition, a significant number of genes involved in cytoskeletalmodulation (cytoskeletal architecture, motility, and cell adhe-sion), nuclear activities (RNA splicing and transcription), sig-

nal transduction, protease activity, and metabolism/energy gen-eration were identified (Table 1; for a complete list see Sup-plemental Table S2).

185/333 encodes a putative defensive protein. Previous stud-ies identified 185/333 as a transcript that was expressed in seaurchin coelomocytes in response to LPS (101) or to microbialchallenge and physical injury (83). Evaluation of 185/333transcript accumulation in coelomocytes by differential displayand Northern blots indicated that expression was maximal at6 h after bacterial challenge and at 12 h after injury. Inagreement with these results, filter screens from both arrayedlibraries indicated that the 185/333 transcripts constituted anestimate of 5,929 of the 91,920 clones (6.45%) in the bacteri-ally activated coelomocyte library, whereas there were only 79positive clones (0.086%) in the nonactivated coelomocytelibrary of the same size. On the basis of the extraordinarychange in expression level in response to LPS challenge,185/333 was investigated in detail.

Because 60% of the 1,247 ESTs in our database matched to185/333, the entire coding sequence of the 185/333 messagecould be reconstructed from these partial sequences. Optimalalignment of 185/333 ESTs could only be accomplished byinserting large gaps between matching stretches of nucleotidesthat delineated possible exons. This analysis indicated that thecomplete 185/333 translated sequence was composed of 15elements (Fig. 4). Because not all elements were present inevery 185/333 sequence, this suggested that many of thetranscripts might be alternatively spliced. Nucleotide variationsin the form of base substitutions were evident throughout thelength of the 185/333 messages. Figure 5 illustrates thesevariations and shows a partial sequence that includes the 3-endof the leader and the first element. An inspection of thenucleotide substitutions indicated that they were not randomlydistributed but occurred at specific positions along the se-quences. While many substitutions were conservative, a sub-stantial number of changes resulted in amino acid changeswhen translated. To determine the ratio of nonsynonymous tosynonymous nucleotide changes (dn/ds), 42 unique ESTs (of293) with high-quality 5-ends were analyzed with PAML(126). The analysis was restricted to 193 nt, which encoded theleader and element 1, because it maximized the number ofESTs that could be employed in the analysis and also avoidedlarge gaps in the alignment. Results indicated that the dn/ds

Fig. 2. Virtual Northern blot analysis of genes ex-pressed in coelomocytes. The subtraction process wasanalyzed using a virtual Northern blot (see MATERIALS

AND METHODS for details). Filters were probed for cy-toplasmic actin (CyI), Sp064, or Sp056 as indicated.Lanes 1, 5, and 9: unsubtracted cDNA from immuno-quiescent coelomocytes. Lanes 2, 6, and 10: unsub-tracted cDNA from LPS-activated coelomocytes. Lanes3, 7, and 11: reverse-subtracted cDNA. Lanes 4, 8, and12: forward-subtracted cDNA.




ics.physiology.orgD

ownloaded from


ratio was 1.12. Any score above 1.0 implies that there are morenonsynonymous nucleotide changes than synonymouschanges, and therefore, nucleotide diversity for the 185/333genes is being maintained within the population to putativelyincrease fitness. However, about one-half of the sequence thatwas employed in the analysis encoded the leader, whichappears to be less diverse than the rest of the sequence (see Fig.4A). Consequently, this suggests that element 1 was the majorcontributor to this result and that elevated diversity may bepresent in all downstream elements.

The NH2 termini of the translated sequences contained ahydrophobic leader terminating with a Ser at position 18. Theremaining protein could be divided into three regions: 1) anNH2-terminal glycine-rich region (Fig. 4, A and C; gly-rich,elements 1–5) and 2) a histidine-rich central region (his-rich,elements 6–14) and a COOH-terminal region (element 15),which exhibited stop codons at two different positions, depend-ing on the 185/333 sequence examined. Two of the 618185/333 sequences that were analyzed revealed stop codons inelement 2 (Fig. 4A; Sp0269 and Sp0289) that would encode anextremely short protein. Nucleotide insertions/deletions were

identified in the leader region resulting in two translatedvariants (MEVK vs. M-VK) as well as in elements 9 and 15(Fig. 4A). While no signature sequences for O-linked glyco-sylation were present, conserved NH2-linked glycosylationsites (up to 12) were noted in the his-rich region, as well as onein the gly-rich region in some sequences (Fig. 4, A and C).Because no cysteines were found in any of the 185/333 codingregions, the structure of the encoded proteins would not bestabilized by disulfide bonds. In addition, a cell adhesion motif(RGD) was identified in element 5.

Short, repeated stretches of amino acids were identifiedwithin both the gly-rich and the his-rich regions, usingMegalign (DNASTAR, Madison, WI), and by visual inspec-tion [Fig. 4, A (colored brackets) and C, and Table 2]. Theseincluded imperfect repeats, which showed variations in theiramino acid sequences (Table 2, types 1, 3, 5). Repeat type 1was only found in the gly-rich region and consisted of fourfull-length repeats and one short repeat, which was missingfive residues from the NH2-terminal end [Table 2 and Fig. 4,A (red brackets) and C]. Repeats 2–5 were only distributedthroughout the his-rich region, while the COOH-terminal

Fig. 3. A distribution of expressed sequence tags (ESTs). A:distribution of ESTs matching to 185/333 vs. all other ESTs. B:the numbers of ESTs assigned to the various functional cate-gories were calculated as a fraction of the 1,247 ESTs minusthe 185/333 matches. ESTs containing too many ambiguousbase calls for satisfactory homology searching were catego-rized as “bad sequences.” Those in the “unknown” categoryhad significant similarities to sequences in the nonredundantdatabases, but had poor functional characterization. “Other”refers to sequences that did not fit into any category indicatedin the pie chart.




ics.physiology.orgD

ownloaded from


region only had repeat type 5. It was interesting that thepositioning of the elements and the positioning of therepeats in the gly-rich region did not correspond. Althoughrepeats 2–5 were not found in tandem arrays like type 1, aseries of several different repeats were positioned in atandem arrangement of 5-2-3-4 in elements 14 and 15 (Fig.

4, A and C). Overall, the alignment indicated that thetranslated 185/333 sequences showed possible alternativesplicing, nucleotide polymorphisms, internal repeats, poten-tial NH2-linked glycosylation sites limited mostly to thehis-rich region, a variable positioning of the terminationcodon, and a lack of disulfide bonds.

Table 1. Partial list of ESTs

Sp No. Protein Match e-Value Species Match Accession No.

Defense-related proteins

0014 glaectin 9 3e-24 Rattus norvegicus CK8289890434 cathepsin L e-142 Strongylocentrotus purpuratus CK8283401107 thrombospondin 4e-53 Homo sapiens CV6527381108 cathepsin B 1e-44 Urechis caupo CK8283561186 phosphatidyleothanolamine-binding protein 4e-17 Arabidopsis thaliana CV6527201200 amssin 6e-18 Strongylocentrotus purpuratus CV652694

Nuclear activities and RNA splicing

0576 ET putative translation product 0.67 Mus musculus CV6527050874 nuclear RNA- and DNA-Binding Protein 1e-06 Homo sapiens CV6527170896 nuclear protein NHP2 1e-40 Homo sapiens CV6527161034 LPS-induced TNF alpha 3e-12 Mus musculus CV6527101093 DNA methyl transerfase-associated protein 3e-37 Mus musculus CV6527011122 heterogeneous nuclear ribonucleoprotein R 1e-31 Chironomus tentans CV6527061157 orphan steriod hormone receptor 2 e-129 Strongylocentrotus purpuratus CV6527181176 paraspeckle protein 4e-56 Homo sapiens CV6527191184 DNA-binding (hexamer-binding) protein 1e-25 Leishmania major CV6527001188 immediate early response 5 3e-06 Homo sapiens CV6527081214 splicing factor 30 8e-22 Homo sapiens CV652736

Protein synthesis, processing, and degradation

0057 Sec22 vesicle trafficking protein 4e-40 Rattus norvegicus CV6527320565 coated vesicle membrane protein (p24A) 2e-30 Mus musculus CV6526980694 vacuolar sorting protein 4b 3e-41 Mus musculus CV6527460744 Rab-7 e-113 Strongylocentrotus purpuratus CV6527280906 Sec61, ER transport protein 1e-49 Strongylocentrotus purpuratus CV6527331059 peptide chain release factor, subunit e-112 Drosophila melanogaster CV6527471074 translation elongation factor e-146 Strongylocentrotus purpuratus CV6527401094 vacuolar ATP synthase subunit G 6e-09 Manduca sexta CV6527451118 translocon associated protein gamma 9e-51 Xenopus laevis CV6527421136 ER calcistorin/Protein disulfide-isomerase e-165 Strongylocentrotus purpuratus CV6527251141 ribosomal protein 40S e-123 Tripneustes gratilla CK8290561197 mannose-6-phosphate receptor 4e-24 Mus musculus CV6527481209 transport protein 1e-24 Mus musculus CV6527341210 Rab5-interacting protein 8e-33 Mus musculus CV6527301228 presenilin 2e-24 Anopheles gambiae CV6527221244 translation initiation factor 4e-93 Homo sapiens CV6527411246 p24B precursor protein 4e-60 Mus musculus CV652712

Cytoskeleton and cell motility

0029 mena neural variant 1e-36 Mus musculus CV6527130042 thymosin beta e-112 Strongylocentrotus purpuratus CV6527390165 receptor, activated protein kinase C (RACK) 1e-67 Biomphalaria glabrata CK8290010188 integrin -C 3e-06 Strongylocentrotus purpuratus CV6527090350 protein tyrosine phsophatase receptor type F 9e-43 Homo sapiens CK8290001027 gelsolin 2e-68 Lumbricus terrestris CK8283211044 protein tyrosine kinase 9-like protein 3e-21 Mus musculus CV6527261089 tubulin 5e-28 Drosophila melanogaster CV6527441096 Rho 6e-87 Hemicentrotus pulcherrimus CV6527311152 cofilin 7e-09 Neurospora crassa CV6526991189 avena 2e-11 Gallus gallus CV6526961195 microtubule associated protein 3e-74 Strongylocentrotus purpuratus CV652714

Cell proliferation/apoptosis

0139 translationally controlled tumor protein e-159 Hemicentrotus pulcherrimus CK8290701086 allograft inflammatory factor 1 3e-29 Suberites domuncula CV6526931117 polo-like kinase e-163 Hemicentrotus pulcherrimus CV6527211158 Bax inhibitor-1 3e-31 Paralichthys olivaceus CV652723

EST, expressed sequence tag.




ics.physiology.orgD

ownloaded from


Unexpected results were revealed from a nucleotide align-ment of 290 of the 185/333 ESTs that had the least number ofambiguous bases and included the 5-UTRs plus 190–196nucleotides of open reading frame (ORF). This was essentiallythe same set that was analyzed for the dn/ds ratio above. On thebasis of the sequence of the 5-UTRs, the ESTs selected forthis analysis could be allocated into 25 groups, of which 19groups had between 2 and 101 members and 6 occurred assingletons (Table 3). Thirteen groups, each composed of five ormore ESTs with identical 5-UTRs, were analyzed using Win-Clada (75) to identify the linkage of an ORF sequence with aparticular 5-UTR. Results indicated that between one andeight ORFs were linked to a given 5-UTR. If each 5-UTRsequence was representative of expression from an individualgene, this result suggested that multiple genes with identical5-untranslated regions were present in the genome and prob-ably encoded different proteins. The total number of differentORF sequences identified using this approach of analyzing 13groups (with 5 or more sequences) was 40 (Table 3). On theother hand, when the total number of distinct ORFs wasanalyzed from all 290 ESTs without grouping the sequences by5-UTR identity, there were only 33 different ORFs. Thisindicated that identical ORFs were associated with different5-UTRs.

These data suggested that the 185/333 ESTs representedexpression from a large family of closely related genes thatmay perhaps share sequences and are induced by immunechallenge with LPS. Message heterogeneity appears to be aresult of putative alternative splicing within and possibly be-tween genes, as well as nucleotide polymorphisms withinelements. The messages encode a family of similar but distinctproteins that show an unexpected level of sequence diversity,perhaps under positive selection for diversity as a result ofpathogen pressure. On the basis of their expression patterns inresponse to LPS and the presence of a leader, these proteinsmay be secreted from the coelomocytes into the coelomic fluidand have an important immune function.

Defense-related proteins. A number of defense-relatedgenes, in addition to the 185/333 family, were identified in thisstudy. Amassin (Sp1200), a 75-kDa sea urchin protein thatcontains an olfactomedin domain, may be secreted by coelo-mocytes in response to injury or infection (46). The olfacto-medin domain may represent an intercellular adhesion domaininvolved in coelomocyte-mediated clotting through amassinpolymerization (reviewed in Ref. 46). Amassin may also bindto cell surface receptors, such as integrins on coelomocytes. Itis noteworthy that a match to integrin-C (Sp0188) was iden-tified that has been reported previously from sea urchin em-bryos (13, 71). Both the C- and L-integrins can be detectedserologically on the surface of coelomocytes (R. D. Burke,personal communication).

A galectin-like sequence (Sp0014) was identified thatmatched with both galectin-8 and galectin-9. Galectins aremembers of the S-type lectin subfamily that bind -galactosidestructures (36). Galectin-9 functions in the induction of apop-tosis (55), acts as an eosinophil chemoattractant (67), and ispossibly involved in inflammatory reactions as well as cell-celladhesion (4). The expression of a galactose-binding lectin incoelomocytes may be involved in responses to microbial chal-lenge, perhaps by mediating chemoattraction and cellular ac-cumulations at points of contact with microbes displaying

galactoside structures. The induction of the C-type lectinSp056 in coelomocytes (Fig. 1) is another example of agalactose-binding lectin that is induced by LPS (70, 113).

Two sequences (Sp0157, Sp1107) matched domains that arefound in a family of proteins that include thrombospondin(TSP), properdin, semaphorin, angiogenesis inhibitor, hemi-centrin, and fibulin-6 (1). Mosaic proteins with TSP domainsfunction in cell-cell and cell-matrix interactions, cytoskeletalmodifications, cell motility and aggregation, inflammatory re-sponses, and wound healing (1). Although these activities arelikely to occur in coelomocytes responding to LPS, the iden-tification of a possible properdin homolog that might functionin the ancestral sea urchin complement system is of greatinterest (102). In higher vertebrates, properdin functions tostabilize the formation of C3-convertase by the alternativepathway, and its expression in coelomocytes suggests that thesea urchin complement system may initiate the generation ofan echinoderm C3-convertase (for review, see Refs. 98, 102).Sequence analysis of both Sp0157 and Sp1107 indicated thatthese ESTs represented two tandem TSP1 domains, and it isnotable that properdin monomers also have TSP1 domainsarranged in a tandem fashion (92).

A single EST (Sp1186) matched to the serine proteaseinhibitor phosphatidylethanolamine-binding protein (PEBP),which is a phospholipid-binding protein with inhibitory activ-ity toward serine proteases (42). PEBP is highly conserved, andhomologs have been identified in a number of species from theanimal, plant, and archeal kingdoms. The interaction of PEBPswith Raf-1 kinase leads to the inhibition of the mitogen-activated protein (MAP) kinase signaling pathway, which sug-gests that these proteins may inhibit mitogenic signaling (42,129). A number of cysteine proteases, such as cathepsin L(Sp0434) and cathepsin B (Sp1108), were identified. Cathep-sins are known to function in lysosomes of mammalian mac-rophages and act to degrade basement membranes and theextracellular matrix of phagocytosed microbes (26, 80). Inaddition, cathepsins may be released from cells by secretorylysosomes to act on invading microbes in the extracellularmilieu (106). Matches to these types of enzymes, as well as toarylsulfatase (EST003, EST004, EST072, EST401; Ref. 101),suggested that coelomocytes may respond to LPS challenge bysynthesizing a significant number of proteases that are requiredfor optimal functioning of the endosomal system.

RNA splicing and protein synthesis, processing, and degra-dation. Many sequence matches identified in this study, in-ferred from large-scale increases in the expression of genes thatencode proteins involved in protein synthesis, processing andtargeting, occur in coelomocytes after immunological chal-lenge. There were several matches to proteins involved in theregulation of transcription, such as steroid hormone receptor(Sp1157), LPS-induced tumor necrosis factor-� (Sp1034), im-mediate early-response protein (Sp1188), and DNA methyltransferase-associated protein-1 (Sp1093). Other matches in-cluded proteins involved in RNA splicing, such as the ETputative translation product (Sp0576), nuclear RNA- andDNA-binding protein (Sp0874), heterogeneous nuclear ribo-nucleoprotein R (Sp1122), splicing factor 30 (Sp1214),paraspeckle protein (Sp1176), and a conserved nonhistonenucleic acid-binding protein (Sp0896). Increased expression ofproteins associated with RNA splicing in coelomocytes sug-gested an overall increase in the expression of genes that




ics.physiology.orgD

ownloaded from


Fig. 4.




ics.physiology.orgD

ownloaded from


require postsynthetic RNA processing. Alternative splicing hasbeen characterized in the expression of Sp152, which encodesthe complement homolog SpBf, where one or two of the fiveshort consensus repeats may be spliced out (113). Two tran-scripts encoding mosaic proteins with FIMAC domains mayalso undergo alternative splicing (70). It is possible that splic-ing may also be involved in 185/333 message production.

Matches to proteins involved in protein synthesis includedribosomal proteins (Sp1141, others), translation initiation fac-tor (Sp1244), translation elongation factor (Sp1074), and pep-tide chain release factor subunit 1 (Sp1059). In addition,matches were identified to Sec61 (Sp0906) and the �-subunit ofthe signal sequence receptor or the translocon-associated pro-tein (TRAP; Sp1118). TRAP-� functions in a complex thatbinds the signal sequence of proteins that are subsequentlyproduced by ribosomes associated with the endoplasmic retic-ulum (ER) and fed through the translocation apparatus into theER lumen (39). Sec61 is a component of the translocationchannel through which nascent peptides pass (117). Thesematches suggested that coelomocytes responding to immunechallenge may upregulate protein production, including trans-lation on both free ribosomes and those associated with the ER.

Two protein chaperones were identified: presenilin (Sp1228)and the ER-localized calcistorin/protein disulfide isomerase(ERcalcistorin/PDI; Sp1136). Presenilin is a member of afamily of aspartyl proteases that are multipass transmembraneproteins found in rough ER that are involved in processingmembrane proteins (e.g., signal peptide removal, amino acidproteolysis of loops extending out from the membrane, andcleaving transmembrane regions) (125, 123). ERcalcistorin/PDI is a putative calcium storage protein in sea urchin eggs thatis also found in the ER (65). Its capacity to bind Ca2�, withconsequent changes in the conformation of the protein, may beimportant for its role as a Ca2�-storage protein as well as achaperone for proper folding of newly synthesized proteins inthe ER (57). The identification of protein chaperones in coe-lomocytes was further evidence of the upregulation of thegeneral cellular machinery for producing and processing pro-teins in response to immune challenge.

There were a number of matches to proteins involved inprotein sorting and trafficking of transport vesicles, includingSec22 (Sp0057), p24A (Sp0565), p24B (Sp1246), transportprotein (Sp1209), vacuolar protein sorting-4b (VPS4b;Sp0694), and Rab-7 (Sp0744). The Rab family of proteins

Fig. 4—Continued. Full-length protein alignment deduced from 185/333 EST sequences. A: ESTs matching to 185/333 (618 sequences) were aligned manuallyusing BioEdit (38). Poor sequences and some duplicates were excluded, resulting in an alignment that was optimal for illustration purposes. No ESTs werefull-length, and all are denoted with the clone number at the 5-end. *Incomplete 3-ends. Color coding of the amino acids is based on the default designationin BioEdit. Gaps are indicated by dashed lines. Each element is numbered above the alignment. The glycine-rich region includes elements 1–5, and thehistidine-rich region includes elements 6–14. Internal and terminal stop codons are indicated with a lowercase “s.” Vertical lines below the alignment indicatepositions of variability. An RDG sequence in element 5 is indicated above the alignment. NH2-linked glycosylation sites are denoted within the alignment byunderlining the conserved sites. They are also marked below the alignment (�) to indicate that only some of the sequences have an Asn residue that could serveas an oligosaccharide acceptor; Œ indicates that all sequences in the alignment have a conserved NH2-linked glycosylation site. Amino acid repeats are identifiedbelow the alignment with colored brackets and are listed in Table 2. Incomplete repeats are labeled as “short” on the alignment. X, unknown amino acid. Forreference, all elements are shown at top with the associated numbers. L indicates the leader. B: the alignment shown in A is illustrated as squares or rectanglessized to the relative length of each element. Lines connecting the squares or rectangles indicate where gaps were introduced to optimize the alignment. As inA, no EST was long enough to include the entire open reading frame. *Missing sequence at one or both ends. C: details of the structures and modifications ofthe deduced proteins are illustrated. }, Positions of conserved sites for NH2-linked glycosylation. An RGD site is shown within element 5. The glycine-rich andhistidine-rich regions are indicated with arrows. Repeats are shown as symbols and are numbered as in A and Table 2.




ics.physiology.orgD

ownloaded from


consists of small GTPases that function as major regulators ofintracellular vesicular traffic (121, 130). Sec22, a v-N-ethyl-maleimide-sensitive factor attachment protein receptor(vSNARE) protein and a member of the p24 protein family,

regulates the direction and targeting of transport vesicles trav-eling between the ER and the Golgi apparatus (11, 22, 41, 51,62, 64, 90, 91). VPS4b and Rab-7, on the other hand, directvesicular traffic between the cell surface and the endosomalsystem (12, 10, 89, 79, 112). Several matches were identified toproteins involved in the endosomal system, including Rab5-interacting protein (Sp1210), which functions in the fusion andmaturation of endosomal elements (47, 86, 124); the mannose-

Fig. 5. Nucleotide alignment of 185/333 ESTs shows significant sequence variation. The highest quality and longest 185/333 ESTs (n 290) were used in analignment of sequences that included the 5-UTR and the open reading frame encoding the leader and first element of the protein. The region shown includesthe 3-end of the leader (arrow) and the entire first element (see Fig. 4 for element locations). Duplicate sequences were reduced to two each for illustrationpurposes to show variations in nucleotide sequence. Periods (.) indicate conserved nucleotides, and sequence changes (for ESTs Sp0047 to Sp0405, relative toSp 0189) are shown as nucleotides. For example, all ESTs contain an adenine at position 1, except for Sp0562, which has a thymidine at that position.

Table 2. 185/333 Repeats

Type 1 (red brackets)

MQMGGSRQDGGPMGGRRFDGPDSGAPQMDGRRQDGGPMGGRRFDGPGFGAPEMDGRRQNGGPMGGRFGAPPMGGPRQDGGPMGGRRFDGPGFGTPQMDGRRQNGGPMGGRRFDGPVFGG

Type 2 (blue brackets)

NHTEGHQGHNQTEGHQGHNHTEGHQGHDHREGHQDHNHTEGHQGH

Type 3 (green brackets)

NETGDHPHNETGDNETGDHPHNETGDHPH

Type 4 (purple brackets)

RHHSKTVDGDQDRHHSKTGDGDQD

Type 5 (orange brackets)

DRPMFGMRPFRFNPFGRKPFGDHPFGRRDRPMFETRPFRFNPFGRKPFGGRPFDRRTRPFRFNHFGR-PFGDHPFGRR

See brackets in Fig. 4A and numbered symbols in Fig. 4C.

Table 3. Comparison of 185/333 coding* sequences†

Group No.‡ No. of Clones Length, ntNo. of Different

Sequences§

2 38 193 84 101 190 86 9 196 18 5 196 29 23 196 611 9 196 413 12 193 114 16 196 115 5 196 216 6 196 117 11 196 218 25 196 319 7 196 113 groups 267 clones 40 different sequences

*The leader and the first element (see Fig. 4). †Data excluded from thisanalysis were 6 groups with 4 or fewer members and 6 unique sequences.‡Groups were defined based on clustering of the 5-UTR sequences usingPAUP (109). The 5-UTR was not used in the analysis of the coding region.§The no. of different sequences in each group was calculated in WinClada (75)after subtracting the number of sequences that were different based onambiguous bases.




ics.physiology.orgD

ownloaded from


6-phosphate receptor (Sp1197), which targets proteins for de-livery to the endosomal system (31); and a subunit of thevacuolar H�-ATPase (Sp1094), which acidifies the contents oflate endosomes and lysosomes (40, 60, 74, 107). Overall, thematches to proteins involved in regulating vesicle transport,trafficking, and targeting and in endosomal function suggestedthat coelomocytes responding to LPS increased the productionof proteins that were processed through the ER, the Golgiapparatus and the endosomal system, thereby requiring in-creased activities in vesicular traffic.

Matches that suggested significant protein production andtargeting to the endosomal system were noteworthy in light ofexpression of cathepsin B (Sp1108), cathepsin L (Sp0434 andothers; EST052; Ref. 101), cathepsin S (EST118; Ref. 101),and arylsulfatase (EST003, EST004, EST072, EST401; Ref.101), which are proteases that are typically directed toward andfunction in the endosomal system. Furthermore, the mostprevalent ESTs identified in this study, 185/333, all have leaderregions suggesting that they may also be processed through theER/Golgi system and targeted to either the endosomal systemor the plasma membrane. Increased secretory activity in re-sponse to LPS has been inferred from immunolocalizationstudies showing that two subsets of phagocytes synthesize andstore the complement homolog, SpC3, in small cytoplasmicvesicles (34). After LPS challenge, significant increases inSpC3 occur in the coelomic fluid within 15 min postinjection(19, 102), indicating significant secretory activity. Overall,these data indicate that secretion and endosomal activity aresignificantly upregulated in responses to LPS.

Cell motility and the cytoskeleton. The majority of coelo-mocytes are macrophage-like, amoeboid, phagocytic cells thatappear to be actively engaged in secretion and have extensiveand malleable cytoskeletons (43, 44). Many of the coelomocyteESTs identified in this study matched to structural componentsof the cytoskeleton or to proteins involved in modifying cy-toskeletal shape with consequent changes in cell behavior.These included actin (Sp1170 and others) and actin-bindingproteins such as profilin (99, 100), cofilin (Sp1152), thymo-sin- (Sp0042), and gelsolin (Sp1027). Protein tyrosine ki-nase-9 (Sp1044), which has a conserved ATP-binding regionand a cofilin-like domain, may function in depolymerizingactin microfilaments (9). Matches to the mouse mena protein(Sp0029) and the chicken avena protein (Sp1189) were alsoidentified. These proteins are both proline-rich cytoplasmicproteins, and mena is known to bind profilin, perhaps promot-ing actin polymerization (30). The presence of mena and avenahomologs in coelomocytes implies active changes in cytoskel-etal shape and cellular motility, which has been suggestedpreviously based on increased expression of sea urchin profilin(99, 100).

Matches to cell surface receptors included proteins thatmodulate the cytoskeleton and regulate links to the extracellu-lar matrix. For example, liprin-�2 (Sp0350) is believed to beinvolved in cell-cell and cell-matrix interactions, based on thesimilarity of its extracellular domain to adhesion proteins (93),while integrins (integrin-C, Sp0188; Refs. 13, 71) are local-ized to focal adhesions, where they are known to interact withthe extracellular matrix and are important in cell migration (49,50). Furthermore, integrins interact with -catenin and G-cadherin, which have been identified in sea urchin embryos(69). Together, expression of this set of genes, which encodes

cell surface and cytoskeletal proteins, suggested interactionsbetween the cell and the extracellular environment, perhapsinducing active modulation of the cell shape and behavior incoelomocytes.

Signal transduction, which links cell surface events to intra-cellular pathways, underpins the responsiveness of coelomo-cytes to immune challenge. Several components that functionin a range of signaling systems were identified in this study,including the receptor for activated protein kinase C (RACK1;Sp0165), which is a member of the G protein (-subunit)superfamily of proteins and functions at signaling nodes (128).RACK1-mediated signaling is known to affect cytoskeletalrearrangements that manifest in cell adhesion, formation oflamellipodia, and cell migration (23). In coelomocytes, theRACK1 homolog may be a key regulator of chemotacticresponses to LPS. Rho GTPase (Sp1096), a member of the Rasfamily of proteins, interacts with a multitude of proteins and isanother key signal transducer involved in modulating thecytoskeleton (21, 37, 66, 72, 110, 111) in addition to mediatingchanges in transcriptional regulation, protein translation, pro-liferation, motility, apoptosis, and membrane trafficking (21,116). The deactivation of Rho proteins is mediated by GTPase-activating proteins (GAPs) and GDP dissociation inhibitors(84), which has been identified previously (EST229; Ref. 101)The expression of Rho GTPase in activated coelomocytes maybe involved in coordinating a variety of signaling pathways andcellular responses to immune challenge.

Cell proliferation. A finely tuned balance exists between cellproliferation and apoptosis in the vertebrate immune response,and this may hold true for sea urchins as well. In terms ofproliferation, Sp1117 matched to polo-like kinase, a serine/threonine protein kinase known to participate in a number ofactivities associated with mitosis, including mitotic spindleorganization and centrosome maturation. Furthermore, polo-like kinase is known to interact with -tubulin (Sp1089),microtubule-associated protein (Sp1195), and microtubule-sta-bilizing proteins including the translationally controlled tumorprotein (Sp0139; Refs. 61, 127). In addition, Sp1188 matchedto immediate early response protein-5, a nuclear protein thatregulates cellular responses to mitogenic signals (122), whileSp1086 matched to allograft inflammatory factor-1, a cytoplas-mic Ca2�-binding protein expressed in response to injury (7)whose overexpression has been linked to increased cell prolif-eration (5, 6). Sp1184 matched to a family of DNA-bindingproteins with known helicase activity that includes a DNA-binding protein characterized from Leishmania major (120), aDEAD/box transcription factor (85) identified in several spe-cies of tunicates (29), and the cellular nucleic acid-bindingprotein, a conserved protein found in a variety of vertebrates(119). The presence and increased expression of helicasescould be interpreted as an indicator of DNA replication leadingto mitosis. On the other hand, proliferative responses mayactually be due to decreased apoptosis. A match to Baxinhibitor-1 (Sp1158), which is conserved in animals, fungi andplants, suggested that the apoptotic pathway in coelomocytesmay be inhibited (48). Overall, the increased expression ofgenes encoding proteins involved in proliferative and apoptoticpathways is in general agreement with prior studies indicatingthat numbers of coelomocytes in the coelomic fluid increase inresponse to injections of LPS (19).




ics.physiology.orgD

ownloaded from


In summary, the change from an immunoquiescent to animmunologically activated state significantly alters the tran-scriptional program in coelomocytes and invokes a wide rangeof cellular activities. Sea urchins appear to respond to immu-nological challenges by active cellular remodeling of theircirculating coelomocytes, with commensurate transcriptionalupregulation of genes functioning in RNA processing, proteinsynthesis, protein processing, and trafficking. Among the genesidentified in this study that appear to be involved in trafficking,vesicle fusion, and protein sorting, many of the encodedproteins may be targeted to or function within the endosomesand lysosomes, indicating increased activity of these cellularcompartments.

The most striking finding among the genes inferred to beupregulated in response to LPS was the identification of a setof closely related transcripts, designated 185/333, which werethe most abundant single transcript species in immune-acti-vated coelomocytes. The 185/333 transcripts exhibited highlevels of sequence variability and putative alternative splicingof the mRNAs, resulting in encoded proteins with a wide rangeof size and sequence diversity. On the basis of the observeddiversity and the striking increase in expression, as illustratedby the vast difference in the number of 185/333 clones in thetwo cDNA libraries and from Northern blots (78), a putativefunction of the encoded proteins is likely to include someaspect of immunity. The implication from the dn/ds ratio forthe sequence at the 5-end of the messages suggests that thegenes may be under positive selection to diversify, and wespeculate that it may be pathogen pressure that underlies thisobservation. It is not currently known how many 185/333genes are present in the genome of an individual purple seaurchin. The data presented here give the impression that theremay be hundreds; however, this may have been a result of thelibrary structure, which used pooled coelomocytes from fivesea urchins (see MATERIALS AND METHODS). Preliminary andongoing efforts to quantitate the gene numbers have been basedon a variety of approaches. Southern blots indicate that thereare not hundreds of genes in a single genome; however, if the185/333 genes are linked, interpretation of high-molecular-weight bands is difficult (unpublished observation). Compari-sons among full-length cDNA sequences from four individualsea urchins responding to LPS have indicated that each animalgenerates between 11 and 30 different messages based on acombination of both element usage patterns and sequencediversity within elements (unpublished observation). Searchesof the trace archives of genome sequences maintained atCaltech (http://issola.caltech.edu/�t/su-traces/blast.cgi) anda direct analysis of the sea urchin genome (http://www.hgsc.bcm.tmc.edu/projects/seaurchin/) have indicated thatthere are 17 different sequences encoding part of the 5-UTR plus the leader (unpublished observation). Together,these analyses have indicated that there may be as few as 8and perhaps as many as 15 loci, and that the number of locimay vary among individual animals. Although our currentestimates of gene numbers may be low, as implied from“mixing” between the 5-UTR and the coding region se-quences (Table 3), our preliminary results indicate that thereare too few genes to explain the observed nucleotide vari-ability in the ESTs. This suggests that there may be mech-anisms for generating sequence diversity in the 185/333transcripts that have not been previously characterized. On

the basis of the similarities among the 185/333 cDNAsequences (and presumably among the genes), speculationson the mechanisms to generate diversity might include geneconversion, gene duplication, and RNA or DNA editing inaddition to possible alternative splicing.

The unexpected level of diversity among the 185/333 se-quences demonstrates a significantly greater level of complex-ity in the sea urchin immune system than had been imaginedpreviously. Yet, there are other examples of nucleotide poly-morphisms and significant sequence diversity in immune genesfrom other invertebrates. The sperm receptor located on eggsurfaces of the tunicate Halocynthia roretzi, which mediatesself sterility in these hermaphroditic animals, is highly variableand is speculated to function in allorecognition (88). Fibrino-gen-related peptides (FREPs), which show significant diver-sity, are expressed by freshwater snail hemocytes respondingspecifically to the presence of parasitizing trematode worms intheir hemocoel (45, 59, 131, 132). Current data suggest thatFREP sequence diversity may be a result of both alternativesplicing and gene conversion (63). In shrimp, the predominanttranscript (6.5–21%) in hemocyte cDNA libraries was from asingle family of antimicrobial peptides, the penaeidins, whichexhibit significant sequence diversity, giving rise to multipleclasses and isoforms within each class (25, 33). Differentclasses of penaeidins have differing effectiveness against spe-cific microbial and fungal pathogens (24). Hyperdiversity hasalso been identified in an immunoglobulin-type variable regionof an innate immune receptor in the cephalochordate Bran-chiostoma floridae, which may have chitin-binding activity(15, 16). The diversity shown in the innate immune responsesof the sea urchin, snail, shrimp, and Branchiostoma respondingto bacterial, parasitic, fungal, and viral challenges suggests thatthese animals, and perhaps most animals, may have hithertounrecognized mechanisms to diversify their responses to for-eignness. These mechanisms may either result in broad pro-tection against pathogens or in directed expression of specificpeptides to combat specific infecting microbes. The analysis ofthe sea urchin system promises to uncover mechanisms thatgenerate diversity in the immune response, the results of whichwill contribute to a paradigm shift in our understanding ofinvertebrate immunity, as suggested by Flajnik and DuPasquier (28). Identification of novel mechanisms for gen-erating immune diversity in invertebrates, which has impli-cations for innate immune capabilities in all animals, mayresult in a better understanding of innate immunity in highervertebrates.

ACKNOWLEDGMENTS

We acknowledge and thank Dr. Eleanor Shepard, who assisted with thesubtractions, and Cynthia Tallent, Sohum Mehta, and students in Cell Biologyat George Washington University, who assisted with BLAST searches. Dr.Lakshmi Vasudevan helped to modify the Microsoft Access database programto serve our purposes. Drs. Gary Litman, David Raftos, Virginia Brockton, andKatherine Buckley provided constructive improvements to the manuscript.

Present address of S. V. Nair: Dept. of Biological Sciences, MacquarieUniv., North Ryde, NSW, 2109, Australia.

GRANTS

This research was supported by funding from the National Science Foun-dation (MCB-0077970 to L. C. Smith and EPS-0083102 to P. S. Gross).




ics.physiology.orgD

ownloaded from


REFERENCES

1. Adams JC and Lawler J. The thrombospondins. Int J Biochem Cell Biol36: 961–968, 2004.

2. Aicher B, Lerch MM, Muller T, Schilling J, and Ullrich A. Cellularredistribution of protein tyrosine phosphatases LAR and PTPsigma byinducible proteolytic processing. J Cell Biol 138: 681–696, 1997.

3. Al-Sharif WZ, Sunyer JO, Lambris JD, and Smith LC. Sea urchincoelomocytes specifically express a homologue of the complement com-ponent C3. J Immunol 160: 2983–2997, 1998.

4. Asakura H, Kashio Y, Nakamura K, Seki M, Dai S, Shirato Y,Abedin MJ, Yoshida N, Nishi N, Imaizumi T, Saita N, Toyama Y,Takashima H, Nakamura T, Ohkawa M, and Hirashima M. Selectiveeosinophil adhesion to fibroblast via IFN-�-induced galectin-9. J Immu-nol 169: 5912–5918, 2002.

5. Autieri MV, Carbone C, and Mu A. Expression of allograft inflam-matory factor-1 is a marker of activated human vascular smooth musclecells and arterial injury. Arterioscler Thromb Vasc Biol 20: 1737–1744,2000.

6. Autieri MV and Carbone CM. Overexpression of allograft inflamma-tory factor-1 promotes proliferation of vascular smooth muscle cells bycell cycle deregulation. Arterioscler Thromb Vasc Biol 21: 1421–1426,2001.

7. Autieri MV, Kelemen SE, and Wendt KW. AIF-1 is an actin-poly-merizing and Rac1-activating protein that promotes vascular smoothmuscle cell migration. Circ Res 92: 1107–1114, 2003.

8. Banfield MJ, Barker JJ, Perry ACF, and Brady RL. Function fromstructure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction. Struc-ture 6: 1245–1254, 1998.

9. Beeler JF, Patel BK, Chedid M, and LaRochelle WJ. Cloning andcharacterization of the mouse homolog of the human A6 gene. Gene 193:31–37, 1997.

10. Beyer A, Scheuring S, Muller S, Mincheva A, Lichter P, and KohrerK. Comparative sequence and expression analyses of four mammalianVPS4 genes. Gene 305: 47–59, 2003.

11. Blum R, Feick P, Puype M, Vandekerckhove J, Klengel R, Nastainc-zyk W, and Schulz I. Tmp21 and p24A, two type I proteins enriched inpancreatic microsomal membranes, are members of a protein familyinvolved in vesicular trafficking. J Biol Chem 271: 17183–17189, 1996.

12. Buczynski G, Bush J, Zhang L, Rodriguez-Paris J, and Cardellit J.Evidence for a recycling role for Rab7 in regulating a late step inendocytosis and in retention of lysosomal enzymes in Dictyosteliumdiscoideum. Mol Biol Cell 8: 1343–1360, 1997.

13. Burke RD, Murray G, Rise M, and Wang D. Integrins on eggs: the Csubunit is essential for formation of the cortical actin cytoskeleton in seaurchin eggs. Dev Biol 265: 53–60, 2004.

14. Cameron RA, Mahairas G, Rast JP, Martinez P, Biondi TR, Swart-zell S, Wallace JC, Poustka AJ, Livingston BT, Wray GA, EttensohnCA, Lehrach H, Britten RJ, Davidson EH, and Hood L. A sea urchingenome project: Sequence scan, virtual map, and additional resources.Proc Natl Acad Sci USA 97: 9514–9518, 2000.

15. Cannon JP, Haire RN, and Litman GW. Identification of diversifiedgenes that contain immunoglobulin-like variable regions in a protochor-date. Nat Immun 3: 1200–1207, 2002.

16. Cannon JP, Haire RN, Schnitker N, Mueller MG, and Litman GW.Individual protochordates have unique immune-type receptor repertoires.Curr Biol 14: R465–R466, 2004.

17. Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, BlandinS, Blass C, Brey PT, Collins FH, Danielli A, Dimopoulos G, Hetru C,Hoa NT, Hoffmann JA, Kanzok SM, Letunic I, Levashina EA,Loukeris TG, Lycett G, Meister S, Michel K, Moita LF, Muller H,Osta MA, Paskewitz SM, Reichhart J, Rzhetsky A, Troxler L,Vernick KD, Vlachou D, Volz J, Mering C, Xu J, Zheng L, Bork P,and Kafatos FC. Immunity-related genes and gene families in Anoph-eles gambiae. Science 298: 159–165, 2002.

18. Clay FJ, McEwen SJ, Bertoncello I, Wilks AF, and Dunn AR.Identification and cloning of a protein kinase-encoding mouse gene, Plk,related to the polo gene of Drosophila. Proc Natl Acad Sci USA 90:4882–4886, 1993.

19. Clow LA, Gross PS, Shih SC, and Smith LC. Expression of SpC3, thesea urchin complement component, in response to lipopolysaccharide.Immunogenetics 51: 1021–1033, 2000.

20. Clow LA, Raftos DA, Gross PS, and Smith LC. The sea urchincomplement homologue, SpC3, functions as an opsonin. J Exp Biol 207:2147–2155, 2004.

21. Coleman ML, Marshall CJ, and Olson MF. Ras and Rho GTPases inG1-phase cell-cycle regulation. Nat Rev Mol Cell Biol 5: 355–366, 2004.

22. Cosson P and Letourneur F. Coatomer interaction with di-lysineendoplasmic reticulum retention motifs. Science 263: 1629–1631, 1994.

23. Cox E, Bennin D, Doan A, O’Toole T, and Huttenlocher A. RACK1regulates integrin-mediated adhesion, protrusion, and chemotactic cellmigration via its src binding site. Mol Biol Cell 14: 658–669, 2003.

24. Cuthbertson BJ, Bullesbach EE, Fievet J, Bachere E, and Gross PS.A new class (penaeidin class 4) of antimicrobial peptides from theAtlantic white shrimp (Litopenaeus setiferus) exhibits target specificityand an independent proline-rich-domain function. Biochem J 381: 79–86, 2004.

25. Cuthbertson BJ, Shepard EF, Chapman RW, and Gross PS. Diver-sity of the penaeidin antimicrobial peptides in two shrimp species.Immunogenetics 54: 442–445, 2002.

26. Delaisse JM, Ledent PA, and Vaes G. Collagenolytic cysteine protein-ases of bone tissue: cathepsin-B, (pro)cathepsin-L and cathepsin-L-like70-kDa proteinase. Biochem J 279d: 167, 1991.

27. Edds KT. Effects of cytochalasin and colcemid on cortical flow incoelomocytes. Cell Motil Cytoskeleton 26: 262–273, 1993.

28. Flajnik MF and Du Pasquier L. Evolution of innate and adaptiveimmunity: can we draw a line? Trends Immunol 25: 640–644, 2004.

29. Fujimura M and Takamura K. Characterization of an ascidian DEAD-box gene, Ci-DEAD1: specific expression in the germ cells and itsmRNA localization in the posterior-most blastomeres in early embryos.Dev Genes Evol 210: 64–72, 2000.

30. Gertler FB, Niebuhr K, Reinhard M, Wehland J, and Soriano P.Mena, a relative of VASP and Drosophila enabled, is implicated in thecontrol of microfilament dynamics. Cell 87: 227–239, 1996.

31. Ghosh P, Dahms NM, and Kornfeld S. Mannose 6-phosphate recep-tors: new twists in the tale. Nat Rev Mol Cell Biol 4: 202–212, 2003.

32. Gross PS, Al-Sharif WZ, Clow LA, and Smith LC. Echinodermimmunity and the evolution of the complement system. Dev CompImmunol 23: 429–442, 1999.

33. Gross PS, Bartlett TC, Browdy CL, Chapman RW, and Warr GW.Immune gene discovery by expressed sequence tag analysis of hemocytesand hepatopancreas in the Pacific white shrimp, Litopenaeus vannamei,and the Atlantic white shrimp, L. setiferus. Dev Comp Immunol 25:565–577, 2001.

34. Gross PS, Clow LA, and Smith LC. SpC3, the complement homologuefrom the purple sea urchin, Strongylocentrotus purpuratus, is expressedin two subpopulations of the phagocytic coelomocytes. Immunogenetics51: 1034–1044, 2000.

35. Gueguen Y, Cadoret JP, Flament D, Barreau-Roumiguiere C, Gi-rardot AL, Garnier J, Hoareau A, Bachere E, and Escoubas JM.Immune gene discovery by expressed sequence tags generated fromhemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene303: 139–145, 2003.

36. Hadari YR, Paz K, Dekel R, Mestrovic T, Accili D, and Zick Y.Galectin-8. J Biol Chem 270: 3447–3453, 1995.

37. Hall A. Rho GTPases and the actin cytoskeleton. Science 279: 509–514,1998.

38. Hall TA. BioEdit: a user-friendly biological sequence alignment editorand analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser41: 95–98, 1999.

39. Hartmann E, Gorlich D, Kostka S, Otto A, Kraft R, Knespel S,Burger E, Rapoport TA, and Prehn S. A tetrameric complex ofmembrane proteins in the endoplasmic reticulum. Eur J Biochem 214:375–381, 1993.

40. Harvey WR. Physiology of V-ATPases. J Exp Biol 172: 1–17, 1992.41. Hay JC, Hirling H, and Scheller RH. Mammalian vesicle trafficking

proteins of the endoplasmic reticulum and Golgi apparatus. J Biol Chem271: 5671–5679, 1996.

42. Hengst U, Albrecht H, Hess D, and Monard D. The phosphatidyleth-anolamine-binding protein is the prototype of a novel family of serineprotease inhibitors. J Biol Chem 276: 535–540, 2001.

43. Henson JH, Kolnik SE, Fried CA, Nazarian R, McGreevy J, Schul-berg KL, Detweiler M, and Trabosh VA. Actin-based centripetal flow:phosphatase inhibition by calyculin-A alters flow pattern, actin organi-zation, and actomyosin distribution. Cell Motil Cytoskeleton 56: 252–266, 2003.




ics.physiology.orgD

ownloaded from


44. Henson JH, Svitkina TM, Burns AR, Hughes HE, MacPartland KJ,Nazarian R, and Borisy GG. Two components of actin-based retrogradeflow in sea urchin coelomocytes. Mol Biol Cell 10: 4075–4090, 1999.

45. Hertel LA, Adema CM, and Loker ES. Differential expression ofFREP genes in two strains of Biomphalaria glabrata after exposure to thedigenetic trematodes Schistosoma mansoni and Echinostoma paraensei.Dev Comp Immunol 29: 295–304, 2005.

46. Hillier BJ and Vacquier VD. Amassin, an olfactomedin protein, medi-ates the massive intercellular adhesion of sea urchin coelomocytes. J CellBiol 160: 597–604, 2003.

47. Hoffenberg S, Liu X, Nikolova L, Hall HS, Dai W, Baughn RE,Dickey BF, Barbieri MA, Aballay A, Stahl PD, and Knoll BJ. A novelmembrane-anchored Rab5 interacting protein required for homotypicendosome fusion. J Biol Chem 275: 24661–24669, 2000.

48. Huckelhoven R. BAX inhibitor-1, an ancient cell death suppressor inanimals and plants with prokaryotic relatives. Apoptosis 9: 299–307,2004.

48a.Hultmark D. Immune reactions in Drosophila and other insects: a modelfor innate immunity. Trends Genet 9: 178–183, 1993.

49. Huttenlocher A, Ginsberg MH, and Horwitz AF. Modulation of cellmigration by integrin-mediated cytoskeletal linkages and ligand-bindingaffinity. J Cell Biol 134: 1551–1562, 1996.

50. Hynes RO. Integrins: versatility, modulation, and signaling in celladhesion. Cell 69: 11–25, 1992.

51. Jackson MR, Nilsson T, and Peterson PA. Identification of a consensusmotif for retention of transmembrane proteins in the endoplasmic retic-ulum. EMBO J 9: 3153–3162, 1990.

52. Janeway CA and Medzhitov R. Innate immune recognition. Annu RevImmunol 20: 197–216, 2002.

53. Jenny MJ, Ringwood AH, Lacy ER, Lewitus AJ, Kempton JW,Gross PS, Warr GW, and Chapman RW. Potential indicators of stressresponse identified by expressed sequence tag analysis of hemocytes andembryos from the American oyster, Crassostrea virginica. Mar Biotech-nol (NY) 4: 81–93, 2002.

54. Johnson PT. The coelomic elements of sea urchins (Strongylocentrotus).I. The normal coelomocytes; their morphology and dynamics in hangingdrops. J Invertebr Pathol 13: 42–62, 1969.

55. Kashio Y, Nakamura K, Abedin MJ, Seki M, Nishi N, Yoshida N,Nakamura T, and Hirashima M. Galectin-9 induces apoptosis throughthe calcium-calpain-caspase-1 pathway. J Immunol 170: 3631–3636,2003.

56. Kumar S, Tamura K, and Nei M. MEGA3: Integrated software forMolecular Evolutionary Genetics Analysis and sequence alignment. BriefBioinform 5: 150–163, 2004.

57. Lebeche D and Kaminer B. Characterization of a calsequestrin-likeprotein from sea-urchin eggs. Biochem J 287: 741–747, 1992.

58. Lee JJ, Shott RJ, Rose SJ, Thomas TL, Britten RJ, and DavidsonEH. Sea urchin actin gene subtypes. Gene number, linkage and evolu-tion. J Mol Biol 172: 149–176, 1984.

59. Leonard PM, Adema CM, Zhang SM, and Loker ES. Structure of twoFREP genes that combine IgSF and fibrinogen domains, with commentson diversity of the FREP gene family in the snail Biomphalaria glabrata.Gene 269: 155–165, 2001.

60. Lepier A, Graf R, Azuma M, Merzendorfer H, Harvey WR, andWieczorek H. The peripheral vomplex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G. J Biol Chem 271: 8502–8508, 1996.

61. Li Q, Callaghan M, and Suprenant KA. The 77-kDa echinodermmicrotubule-associated protein (EMAP) shares epitopes with the mam-malian brain MAPs, MAP-2 and Tau. Biochem Biophys Res Commun250: 502–505, 1998.

62. Liu Y and Barlowe C. Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function.Mol Biol Cell 13: 3314–3324, 2002.

63. Loker ES, Adema CM, Zhang SM, and Kepler TB. Invertebrateimmune systems—not homogeneous, not simple, not well understood.Immunol Rev 198: 10–24, 2004.

64. Lowe M and Kreis TE. In vivo assembly of coatomer, the COP-I coatprecursor. J Biol Chem 270: 31364–31371, 1995.

65. Lucero HA, Lebeche D, and Kaminer B. ERcalcistorin/protein disul-fide isomerase (PDI). Sequence determination and expression of a cDNAclone encoding a calcium storage protein with PDI activity from endo-plasmic reticulum of the sea urchin egg. J Biol Chem 269: 23112–23119,1994.

66. Mackey DJG and Hall A. Rho GTPases. J Biol Chem 273: 20685–20688, 1998.

67. Matsumoto R, Matsumoto H, Seki M, Hata M, Asano Y, KanegasakiS, Stevens RL, and Hirashima M. Human ecalectin, a variant of humangalectin-9, is a novel eosinophil chemoattractant produced by T lympho-cytes. J Biol Chem 273: 16976–16984, 1998.

68. Medzhitov R and Janeway CA. Decoding the patterns of self andnonself by the innate immune system. Science 296: 298–300, 2002.

69. Miller JR and McClay DR. Changes in the pattern of adherens junction-associated -catenin accompany morphogenesis in the sea urchin em-bryo. Dev Biol 192: 310–322, 1997.

70. Multerer KA and Smith LC. Two cDNAs from the purple sea urchin,Strongylocentrotus purpuratus, encoding mosaic proteins with domainsfound in factor H, factor I, and complement components C6 and C7.Immunogenetics 56: 89–106, 2004.

71. Murray G, Reed C, Marsden M, Rise M, Wang D, and Burke RD.The � B C integrin is expressed on the surface of the sea urchin eggand removed at fertilization. Dev Biol 227: 633–647, 2000.

72. Narumiya S, Ishizaki T, and Watanabe N. Rho effectors and reorga-nization of actin cytoskeleton. FEBS Lett 410: 68–72, 1997.

73. Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P,Sleeman J, Lamond A, and Mann M. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceo-some complex. Nat Genet 20: 46–50, 1998.

74. Nishi T and Forgac M. The vacuolar (H�)-ATPases—nature’s mostversatile proton pumps. Nat Rev Mol Cell Biol 3: 94–103, 2002.

75. Nixon KC. WinClada Ver. 1.0000. KC Nixon: Ithaca, NY, 1999–2002.76. Pancer Z. Dynamic expression of multiple scavenger receptor cycsteine-

rich genes in coelomocytes of the purple sea urchin. Proc Natl Acad SciUSA 97: 13156–13161, 2000.

77. Pancer Z. Individual-specific repertoires of immune cells SRCR recep-tors in the purple sea urchin (S. purpuratus). Adv Exp Med Biol 484:31–40, 2001.

78. Pancer Z, Rast JP, and Davidson EH. Origins of immunity: transcrip-tion factors and homologues of effector genes of the vertebrate immunesystem expressed in sea urchin coelomocytes. Immunogenetics 49: 773–786, 1999.

79. Pfeffer S. Membrane domains in the secretory and endocytic pathways.Cell 112: 507–517, 2003.

80. Podgorski I and Sloane BF. Cathepsin B and its role(s) in cancerprogression. Biochem Soc Symp 70: 263–276, 2003.

81. Pollard TD and Borisy GG. Cellular motility driven by assembly anddisassembly of actin filaments. Cell 112: 453–465, 2003.

82. Rast JP, Amore G, Calestani C, Livi CB, Ransick A, and DavidsonEH. Recovery of developmentally defined gene sets from high-densitycDNA macroarrays. Dev Biol 228: 270–286, 2000.

83. Rast JP, Pancer Z, and Davidson EH. New approaches towards anunderstanding of deuterostome immunity. Curr Top Microbiol Immunol248: 3–16, 2000.

84. Rivero F, Illenberger D, Somesh BP, Dislich H, Adam N, and MeyerAK. Defects in cytokinesis, actin reorganization and the contractilevacuole in cells deficient in RhoGDI. EMBO J 21: 4539–4549, 2002.

85. Rocak S and Linder P. DEAD-Box proteins: the driving forces behindRNA metabolism. Nat Rev Mol Cell Biol 5: 232–241, 2004.

86. Saito K, Murai J, Kajiho H, Kontani K, Kurosu H, and Katada T. Anovel binding protein composed of homophilic tetramer exhibits uniqueproperties for the small GTPase Rab5. J Biol Chem 277: 3412–3418,2002.

87. Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, NY: Cold Spring HarborLaboratory Press, 1989.

88. Sawada H, Tanaka E, Ban S, Yamasaki C, Fujino J, Ooura K, AbeY, Matsumoto K, and Yokosawa H. Self/nonself recognition in ascid-ian fertilization: vitelline coat protein HrVC70 is a candidate allorecog-nition molecule. Proc Natl Acad Sci USA 101: 15615–15620, 2004.

89. Scheuring S, Rohricht RA, Schoning-Burkhardt B, Beyer A, MullerS, Abts HF, and Kohrer K. Mammalian cells express two VPS4proteins both of which are involved in intracellular protein trafficking. JMol Biol 312: 469–480, 2001.

90. Schimmoller F, Singer-Kruger B, Schroder S, Kruger U, Barlowe C,and Riezman H. The absence of Emp24p, a component of ER-derivedCOPII-coated vesicles, causes a defect in transport of selected proteins tothe Golgi. EMBO J 14: 1329–1339, 1995.




ics.physiology.orgD

ownloaded from


91. Schutze MP, Peterson PA, and Jackson MR. An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmicreticulum. EMBO J 13: 1696–1705, 1994.

92. Schwaeble WJ and Reid KBM. Does properdin crosslink the cellularand the humoral immune response? Immunol Today 20: 17–21, 1999.

93. Serra-Pages C, Kedersha NK, Fazikas L, Medley Q, Debant A, andStreuli M. The LAR transmembrane protein tyrosine phosphatase and acoiled-coil LAR-interacting protein co-localize at focal adhesions.EMBO J 14: 2827–2838, 1995.

94. Severson DW, Knudson DL, Soares MB, and Loftus BJ. Aedesaegypti genomics. Insect Biochem Mol Biol 34: 715–721, 2004.

95. Shah M, Brown KM, and Smith LC. The gene encoding the sea urchincomplement protein, SpC3, is expressed in embryos and can be upregu-lated by bacteria. Dev Comp Immunol 27: 529–538, 2003.

96. Smith LC. The complement system in sea urchins. In: PhylogeneticPerspectives on the Vertebrate Immune Systems. Advances in Experi-mental Medicine and Biology, edited by Beck M, Sugumaran M, andCooper EL. New York: Kluwer Academic/Plenum, 2001, vol. 484, p.363–372.

97. Smith LC. Thioester function is conserved in SpC3, the sea urchinhomologue of the complement component C3. Dev Comp Immunol 26:603–614, 2002.

98. Smith LC, Azumi K, and Nonaka M. Complement systems in inver-tebrates. The ancient alternative and lectin pathways. Immunopharma-cology 42: 107–120, 1999.

99. Smith LC, Britten RJ, and Davidson EH. SpCoel1: a sea urchinprofilin gene expressed specifically in coelomocytes in response toinjury. Mol Biol Cell 3: 403–414, 1992.

100. Smith LC, Britten RJ, and Davidson EH. Lipopolysaccharide activatesthe sea urchin immune system. Dev Comp Immunol 19: 217–224, 1995.

101. Smith LC, Chang L, Britten R, and Davidson EH. Sea urchin genesexpressed in activated coelomocytes are identified by expressed sequencetags. Complement homologues and other putative immune responsegenes suggest immune system homology within the deuterostomes.J Immunol 156: 593–602, 1996.

102. Smith LC, Clow LA, and Terwilliger DP. The ancestral complementsystem in sea urchins. Immunol Rev 180: 16–34, 2001.

103. Smith LC and Davidson EH. The echinoid immune system and thephylogenetic occurrence of immune mechanisms in deuterostomes. Im-munol Today 13: 356–362, 1992.

104. Smith LC and Davidson EH. The echinoderm immune system: char-acters shared with the vertebrate immune system, and characters arisinglater in deuterostome phylogeny. In: Primordial Immunity: Foundationsfor the Vertebrate Immune System, edited by Beck G, Cooper EL,Habicht GS, and Marchalonls JJ. New York: New York Academy ofSciences, 1994, p. 213–226.

105. Smith LC, Shih CS, and Dachenhausen SG. Coelomocytes expressSpBf, a homologue of factor B, the second component in the sea urchincomplement system. J Immunol 161: 6784–6793, 1998.

106. Stinchcombe J, Bossi G, and Griffiths GM. Linking albinism andimmunity: the secrets of secretory lysosomes. Science 305: 55–59, 2004.

107. Sun-Wada GH, Wada Y, and Futai M. Lysosome and lysosome-related organelles responsible for specialized functions in higher organ-isms, with special emphasis on vacuolar-type proton ATPase. Cell StructFunct 28: 455–463, 2003.

108. Supungul P, Klinbunga S, Pichyankura R, Jitrapakdee S, Hirono I,Aoki T, and Tassanakajon A. Identification of immune-related genes inhemocytes of black tiger shrimp (Panaeus monodon). Mar Biotechnol(NY) 4: 487–494, 2002.

109. Swofford DL. PAUP*. Phylogenetic Analysis Using Parsimony (*andOther Methods), Version 4. Sunderland, MA: Sinauer Associates, 2002.

110. Symons M and Settleman J. Rho family GTPases: more than simpleswitches. Trends Cell Biol 10: 415–419, 2000.

111. Takai Y, Sasaki T, and Matozaki T. Small GTP-binding proteins.Physiol Rev 81: 153–208, 2001.

112. Tanaka H, Fujita H, Katoh H, Mori K, and Negishi M. Vps4-A(vacuolar protein sorting 4-A) is a binding partner for a novel Rho familyGTPase, Rnd2. Biochem J 365: 349–353, 2002.

113. Terwilliger DP, Clow LA, Gross PS, and Smith LC. Constitutiveexpression and alternative splicing of the exons encoding SCRs in Sp152,the sea urchin homologue of complement factor B. Implications on theevolution of the Bf/C2 gene family. Immunogenetics 56: 531–543, 2004.

114. Utans U, Arceci RJ, Yamashita Y, and Russell ME. Cloning andcharacterization of allograft inflammatory factor-1: a novel macrophagefactor identified in rat cardiac allografts with chronic rejection. J ClinInvest 95: 2954–2962, 1995.

115. Utans U, Quist WC, McManus BM, Wilson JE, Arceci RJ, WallaceAF, and Russell ME. Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted humanhearts. Transplantation 61: 1387–1392, 1996.

116. Van Aelst L and D’Souza-Schorey C. Rho GTPases and signallingnetworks. Genes Dev 11: 2295–2322, 1997.

117. Van den Berg B, Clemons WM, Collinson I, Modis Y, Hartmann E,Harrison SC, and Rapoport TA. X-ray structure of a protein-conduct-ing channel. Nature 427: 36–44, 2004.

118. Vierstraete E, Verleyen P, Baggerman G, D’Hertog W, Van denBergh G, Arckens L, De Loof A, and Schoofs L. A proteomic approachfor the analysis of instantly released wound and immune proteins inDrosophila melanogaster hemolymph. Proc Natl Acad Sci USA 101:470–475, 2004.

119. Warden CH, Krisans SK, Purcell-Huynh D, Leete LM, Daluiski A,Diep A, Taylor BA, and Lusis AJ. Mouse cellular nucleic acid bindingproteins: a highly conserved family identified by genetic mapping andsequencing. Genomics 24: 14–19, 1994.

120. Webb J and McMaster W. Molecular cloning and expression of aLeishmania major gene encoding a single-stranded DNA-binding proteincontaining nine “CCHC” zinc finger motifs. J Biol Chem 268: 13994–14002, 1993.

121. Wickner W and Haas A. Yeast homotypic vacuole fusion: a window onorganelle trafficking mechanisms. Annu Rev Biochem 69: 247–275, 2000.

122. Williams M, Lyu MS, Yang YL, Lin EP, Dunbrack R, Birren B,Cunningham J, and Hunter K. Ier5, a novel member of the slow-kinetics immediate-early genes. Genomics 55: 327–334, 1999.

123. Wolfe MS and Kopan R. Intramembrane proteolysis: theme and vari-ations. Science 305: 1119–1123, 2004.

124. Woodman PG. Biogenesis of the sorting endosome: the role of Rab5.Traffic 1: 695–701, 2000.

125. Xia W and Wolfe MS. Intramembrane proteolysis by presenilin andpresenilin-like proteases. J Cell Sci 116: 2839–2844, 2003.

126. Yang Z. PAML: a program package for phylogenetic analysis bymaximum likelihood. CABIOS 13: 555–556, 1997.

127. Yarm FR. Plk phosphorylation regulates the microtubule-stabilizingprotein TCTP. Mol Cell Biol 22: 6209–6221, 2002.

128. Yarwood SJ, Steele MR, Scotland G, Houslay MD, and Bolger GB.The RACK1 signaling scaffold protein selectively interacts with thecAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 274:14909–14917, 1999.

129. Yeung K, Seitz T, Li S, Janosch P, Mcferran B, Kaiser C, Fee F,Katsanakis KD, Rose D, Mischak H, Sedivy JM, and Kolch W.Suppression of Raf-1 kinase activity and MAP kinase signalling byRKIP. Nature 401: 173–177, 1999.

130. Zerial M and McBride H. Rab proteins as membrane organizers. NatRev Mol Cell Biol 2: 107–117, 2001.

131. Zhang S-M, Adema CM, Kepler TB, and Loker ES. Diversification ofIg superfamily genes in an invertebrate. Science 305: 251–254, 2004.

132. Zhang SM, Leonard PM, Adema CM, and Loker ES. Parasite-responsive IgSF members in the snail Biomphalaria glabrata: character-ization of novel genes with tandemly arranged IgSF domains and afibrinogen domain. Immunogenetics 53: 684–694, 2001.




ics.physiology.orgD

ownloaded from


Table S1. Primers used for coelomocyte cDNA analysis.

Primers Clones used as probe templates and positive controls for PCR

Gene, Accession No. and Reference

Sp056F; 5’ GCA CAG CCA GCA ACC AGC ACT ACA ATSp056R; 5’ ACG CCG ATG GGT TCT ACA GTG AAG GT

pSPORT-K15/182 Sp056, AY336600Multerer & Smith 2004, Terwilliger et al. 2004

Sp064F; 5’ ACT TAC AGG GCT CAA ACA GGT GGT GAA CGSp064R; 5’ TCC TTC CCG GTC AAA TCT TGT ATA TGG CC

pExCell064-063 Sp064, AF025526Al-Sharif et al. 1998

CyF; 5’ ACG ACG ATG TTG CCG CTC TTG TCA TCyR; 5’ GCT GTC CTT CTG TCC CAT ACC GAC CA

pExCell278 CyI (Cytoplasmic Actin), R62049Smith et al. 1996

Table S2. Summary of coelomocyte ESTs

Sp# Protein Match Species Match e value*#Accession

Number

0010 Actin S. purpuratus 4e-96 ck8283020011 185/333 S. purpuratus 1e-99* ck8283720012 Actin S. franciscanus 7e-86 ck8283030013 185/333 S. purpuratus 3e-62 ck8283730014 Lectin R. norvegicus 3e-24 ck8289890015 No significant match ck8290790016 185/333 S. purpuratus 1e-5 ck8283740017 185/333 S. purpuratus 4e-10 ck8283750018 ATP synthase P. lividus 8e-74 ck8283270019 Cytochrome C oxidase subunit 1 P. lividus 4e-79 ck8283580020 Ribosomal Protein 40S S3A H. sapiens 2e-70 ck8290020021 Actin H. tuberculata 1e-16 ck8283040022 185/333 S. purpuratus 2e-22 ck8283760023 Ribosomal protein 40S S7 R. norvegicus 4e-65 ck8290030024 185/333 S. purpuratus # ck8283770025 Actin A. phyllitidis 1e-31 ck8283050027 Ribosomal protein 60S L19 D. melanogaster 4e-57 ck8290040028 Actin S. franciscanus 1e-88 ck8283060029 Mena, neural variant M. musculus 1e-36 cv6527130030 No significant match ck8283780031 185/333 S. purpuratus 3e-22 ck8283790032 185/333 S. purpuratus 1e-17 ck8283800033 185/333 S. purpuratus ck8283810034 ATP Synthase B. taurus 1e-52 ck8283280037 185/333 S. purpuratus 1e-35 ck8283820038 Unknown M. musculus 2e-8 cv6527620039 Ribosomal protein 60S L21 D. melanogaster 5e-20 ck8290050040 ATP synthase D. melanogaster 5e-33 ck8283290041 Ribosomal RNA 12S H. pulcherrimus 0* ck8290060042 Thymosin beta S. purpuratus 1e-112* cv6527390043 Ribosomal protein 40S S2 U. caupo 2e-89 ck8290070044 Hdd 11 H. cunea 6e-13 cv6527490045 Translation elongation factor alpha S. purpuratus e-121 ck8290710047 185/333 S. purpuratus 1e-22 ck8283830048 185/333 S. purpuratus 5e-39 ck8283840049 Ribosomal RNA 16S S. purpuratus 0 ck8290080050 Cathepsin M. glutinosa 2e-44 ck8283570051 185/333 S. purpuratus 6e-18* ck8283850052 No significant match ck8290800053 No significant match ck8290810054 No significant match ck8290820056 No significant match ck8290830057 Vesicle trafficking protein R. norvegicus 4e-40 cv6527320058 Unknown H. sapiens 2e-37* ck8290840059 Ribosomal protein 60S L3 B. taurus 9e-52 ck8290090060 Ribosomal protein 60S L11 M. musculus 2e-21 ck8290100061 Ribosomal protein 60S L11 M. musculus 8e-12 ck8290110062 ATP synthase P. lividus 8e-82 CK8283300063 DD104 S. purpuratus 2e-97 ck8283650064 Ribosomal protein 60S L3 H. sapiens 1e-100 ck8290120065 185/333 S. purpuratus 9e-62 ck8283860066 Ribosomal protein 60S L17 D. melanogaster 1e-47 ck8290130067 NADH dehydrogenase subunit 4 S. purpuratus 2e-86 cv6527690068 185/333 S. purpuratus 4e-07 ck8283870069 Annexin C. elegans 1e-44 ck8283260071 Ribosomal Protein 60S L23A H. sapiens 4e-44 ck829014

0073 Ribosomal protein 60S L11 H. sapiens 2e-84 ck8290150074 Ribosomal protein L10A R. norvegicus 2e-84 ck8290160075 Ribosomal protein 60S L3 D. melanogaster 2e-84 ck8290170076 Translational elongation factor alpha M. musculus 9e-61 ck8290720077 Ribosomal protein 60S L26 H. sapiens 4e-49 ck8290180078 185/333 S. purpuratus ck8283880080 185/333 S. purpuratus ck8283890081 185/333 S. purpuratus ck8283900082 Ribosomal protein 60S L36 H. sapiens 3e-19 ck8290190083 185/333 G. cydonium 8e-09* ck8283910085 Ribosomal protein 40S S8 P. xylostella 2e-48 ck8290200088 Translational elongation factor gamma H. sapiens 1e-62 ck8290780089 Ribosomal protein 60S L26 H. sapiens 3e-49 ck8290210091 Ribosomal protein 40S S6 M. sexta 7e-04 ck8290220092 NADH dehydrogenase subunit 2 S. purpuratus 2e-70* ck8289930094 Ribosomal protein 60S L7a I. punctatus 2e-60 ck8290230095 No significant match ck8290850096 No significant match ck8290860097 Ribosomal protein 40S S13 C. griseus 6e-72 ck8290240099 Actin H. tuberculata 9e-71 ck8283070101 Cytochrome C oxidase subunit 1 P. lividus 1e-51 ck8283590102 No significant match ck8290870103 185/333 S. purpuratus ck8283920104 Ribosomal protein 60S L10 I. punctatus 1e-73 ck8290250105 185/333 S. purpuratus ck8283930106 Ribosomal protein 60S L26 O. sativa 3e-14 ck8290260107 185/333 S. purpuratus 6e-47 ck8290880108 185/333 S. purpuratus ck8283940110 185/333 S. purpuratus 1e-05 ck8283950111 185/333 S. purpuratus ck8283960112 185/333 S. purpuratus ck8283970114 185/333 S. purpuratus ck8283980115 No significant match ck8290890116 185/333 S. purpuratus ck8283990117 No significant match ck8290900118 Cathepsin O. mykiss 5e-38 ck8283330119 No significant match ck8290910120 185/333 S. purpuratus ck8284000122 185/333 S. purpuratus ck8284010124 185/333 S. purpuratus ck8284020125 185/333 S. purpuratus ck8284030126 185/333 S. purpuratus ck8284040127 Fatty acid desaturase H. sapiens 6e-24 ck8289880128 185/333 S. purpuratus ck8284050129 ATPase S. pallidus 8e-27 cv6526910130 Cathepsin O. mykiss 2e-27 ck8283340131 185/333 S. purpuratus ck8284060132 185/333 S. purpuratus ck8284070134 No significant match S. purpuratus 2e-10 ck8290920135 Ribosomal protein L6 M. musculus 2e-39 ck829027]0136 No significant match ck8290930137 185/333 S. purpuratus ck8284080138 No significant match ck8290940139 Translationally-controlled tumor protein D. rerio 4e-13 ck8290700140 No significant match ck8290950141 Ribosomal protein 60S L10, QM homologue B. mandarina 4e-58 cv6527750142 No significant match ck8290960143 Actin S. purpuratus 2e-72 ck8283080144 Unknown ck829097

0145 185/333 S. purpuratus ck8284090147 No significant match ck8290980149 No significant match ck8290990150 185/333 S. purpuratus ck8284100151 No significant match ck8291000152 No significant match ck8291010153 No significant match ck8291020154 No significant match ck8291030155 185/333 S. purpuratus ck8284110156 185/333 S. purpuratus ck8284120157 Thrombospondin C. elegans 4e-14 cv6527810158 No significant match ck8291040159 Translation elongation factor 1 alpha S. purpuratus 1e-81 ck8290730160 185/333 S. purpuratus ck8284130161 Ribosomal protein 60S L27 H. sapiens 2e-48 ck8290280162 No significant match ck8291050163 No significant match ck8291060164 185/333 S. purpuratus ck8284140165 Receptor for Activated Protein Kinase C (RACK) B. glabrata 1e-67 ck8290010166 Ribosomal protein 60S L7A T. rubripes 1e-03 ck8290290167 185/333 S. purpuratus ck8284150169 No significant match ck8291070170 Beta-thioketolase B. taurus 9e-41 cv6527430172 Actin S. purpuratus 8e-82 ck8283090173 185/333 S. purpuratus ck8284160174 185/333 S. purpuratus ck8284170175 185/333 S. purpuratus ck8284180177 No significant match ck8291080178 185/333 S. purpuratus ck8284190179 185/333 S. purpuratus ck8284200180 No significant match ck8284210182 Ribosomal protein 40S S19 M. musculus 1e-44 ck8290300183 185/333 S. purpuratus ck8284220184 Actin H. tuberculata 4e-53 ck8283100185 185/333 S. purpuratus ck8284230186 185/333 S. purpuratus 1e-10 ck8284240187 No significant match ck8291090188 Beta integrin L. variegatus 3e-06 cv6527090189 185/333 S. purpuratus 7e-10 ck8284250190 185/333 S. purpuratus 2e-03 ck8284260191 185/333 S. purpuratus 8e-06 ck8284270192 Translational elongation factor alpha S. purpuratus 5e-58 ck8290740193 185/333 S. purpuratus 1e-28* ck8284280194 No significant match ck8291100195 185/333 S. purpuratus 2e-04 ck8284290196 RNA Helicase H. sapiens 2e-27 cv6527290197 185/333 S. purpuratus 7e-10 ck8284300198 185/333 S. purpuratus 5e-12 ck8284310199 185/333 S. purpuratus 1e-135* ck8284320200 185/333 S. purpuratus 8e-97* ck8284330201 Ribosomal protein 60S L14 G. gallus 9e-34 ck8290310202 185/333 S. purpuratus 4e-66 ck8284340203 185/333 S. purpuratus 5e-06 ck8284350204 No signficant match ck8284360205 Guanine nucleotide binding protein, beta subunit D. melanogaster 2e-11 ck8289900206 185/333 S. purpuratus 2e-75* ck8284370207 185/333 S. purpuratus 8e-06 ck8284380208 185/333 S. purpuratus 1e-14 ck8284390209 185/333 S. purpuratus 2e-35 ck828440

0210 185/333 S. purpuratus 5e-04 ck8284410211 185/333 S. purpuratus 6e-14 ck8284420212 No significant match ck8291110213 185/333 S. purpuratus 6e-22 ck8284430214 185/333 S. purpuratus 6e-79* ck8284440215 185/333 S. purpuratus 1e-07 ck8284450216 185/333 S. purpuratus ck8284460217 185/333 S. purpuratus ck8284470219 185/333 S. purpuratus 2e-27 ck8284480221 Actin H. tuberculata 1e-68 ck8283110222 185/333 S. purpuratus ck8284490224 185/333 S. purpuratus ck8284500226 185/333 S. purpuratus ck8284510227 185/333 S. purpuratus ck8284520228 185/333 S. purpuratus ck8284530229 185/333 S. purpuratus ck8284540230 185/333 S. purpuratus ck8284550231 ATP/ADP Translocase R. sylvatica 1e-46 cv6527500232 185/333 S. purpuratus ck8284560233 185/333 S. purpuratus ck8284570234 185/333 S. purpuratus ck8284580235 185/333 S. purpuratus ck8284590236 185/333 S. purpuratus ck8284600237 185/333 S. purpuratus ck8284610238 185/333 S. purpuratus ck8284620239 185/333 S. purpuratus ck8284630241 185/333 S. purpuratus ck8284640242 Ribosomal protein 60S L29 P. ginseng 4e-18 ck8290320243 185/333 S. purpuratus ck8284650244 185/333 S. purpuratus ck8284660245 185/333 S. purpuratus ck8284670246 185/333 S. purpuratus ck8284680248 185/333 S. purpuratus ck8284690249 No significant match ck8291120251 185/333 S. purpuratus ck8284700252 185/333 S. purpuratus ck8284710253 185/333 S. purpuratus ck8284720254 185/333 S. purpuratus ck8284730255 Cathepsin G. gallus 1e-52 ck8283350256 185/333 S. purpuratus ck8284740257 185/333 S. purpuratus ck8284750259 185/333 S. purpuratus ck8284760260 185/333 S. purpuratus ck8284770261 185/333 S. purpuratus ck8284780262 185/333 S. purpuratus ck8284790263 185/333 S. purpuratus ck8284800264 185/333 S. purpuratus ck8284810265 185/333 S. purpuratus ck8284820266 Unknown M. musculus 3e-04 ck8291130267 185/333 S. purpuratus ck8284830268 185/333 S. purpuratus ck8284840269 Unknown M. musculus 3e-04 ck8291140270 185/333 S. purpuratus ck8284850271 185/333 S. purpuratus ck8284860272 185/333 S. purpuratus ck8284870273 185/333 S. purpuratus ck8284880274 185/333 S. purpuratus ck8284890275 185/333 S. purpuratus ck8284900277 185/333 S. purpuratus ck828491

0278 185/333 S. purpuratus ck8284920279 185/333 S. purpuratus ck8284930280 185/333 S. purpuratus ck8284940283 185/333 S. purpuratus ck8284950284 185/333 S. purpuratus ck8284960285 185/333 S. purpuratus ck8284970286 185/333 S. purpuratus ck8284980287 185/333 S. purpuratus ck8284990288 185/333 S. purpuratus ck8285000289 185/333 S. purpuratus ck8285010290 Ribosomal RNA, 18S H. sapiens 1e-07 ck8290330291 185/333 S. purpuratus ck8285020292 185/333 S. purpuratus ck8285030293 185/333 S. purpuratus ck8285040294 185/333 S. purpuratus ck8285050295 185/333 S. purpuratus ck8285060296 185/333 S. purpuratus ck8285070297 185/333 S. purpuratus ck8285080299 185/333 S. purpuratus ck8285090300 185/333 S. purpuratus ck8285100301 185/333 S. purpuratus 1e-167* cv6527870302 185/333 S. purpuratus ck8285110303 185/333 S. purpuratus ck8285120304 185/333 S. purpuratus ck8285130305 185/333 S. purpuratus ck8285140306 185/333 S. purpuratus ck8285150307 185/333 S. purpuratus ck8285160308 185/333 S. purpuratus ck8285170309 185/333 S. purpuratus ck8285180310 185/333 S. purpuratus ck8285190311 185/333 S. purpuratus ck8285200312 185/333 S. purpuratus ck8285210313 185/333 S. purpuratus ck8285220314 Cathepsin O. mykiss 5e-20 ck8283360315 185/333 S. purpuratus ck8285230316 Actin S. purpuratus 1e-77 ck8283120317 185/333 S. purpuratus ck8285240318 Actin S. purpuratus 2e-77 ck8283130319 185/333 S. purpuratus ck8285250320 185/333 S. purpuratus ck8285260321 185/333 S. purpuratus ck8285270322 185/333 S. purpuratus ck8285280323 185/333 S. purpuratus ck8285290324 185/333 S. purpuratus ck8285300325 Cathepsin O. mykiss 5e-24 ck8283370326 DD104 S. purpuratus 2e-53 ck8283660327 185/333 S. purpuratus ck8285310328 185/333 S. purpuratus ck8285320329 185/333 S. purpuratus ck8285330330 185/333 S. purpuratus ck8285340331 DD104 S. purpuratus 2e-54 ck8283670332 185/333 S. purpuratus 2e-06 ck8285350333 185/333 S. purpuratus 2e-10 ck8285360334 Ribosomal protein 60S L19 D. melanogaster 7e-17 ck8290340336 5-aminolevulinate synthase S. droebachiensis 5e-72 ck8283010337 185/333 S. purpuratus ck8285370339 185/333 S. purpuratus ck8285380340 185/333 S. purpuratus ck8285390341 185/333 S. purpuratus ck828540

0342 185/333 S. purpuratus ck8285410343 185/333 S. purpuratus ck8285420344 Cathepsin O. mykiss 9e-25 ck8283380345 Actin S.purpuratus 1e-52 ck8283140346 185/333 S. purpuratus ck8285430347 Ribosomal protein 60S P1 G. gallus 1e-18 ck8290350348 185/333 S. purpuratus ck8285440349 185/333 S. purpuratus ck8285450350 Protein tyrosine phosphatase receptor type F H. sapiens 9e-43 ck8290000351 185/333 S. purpuratus ck8285460352 185/333 S. purpuratus ck8285470353 No significant match ck8291150354 185/333 S. purpuratus ck8285480355 185/333 S. purpuratus ck8285490356 185/333 S. purpuratus ck8285500357 185/333 S. purpuratus ck8285510358 185/333 S. purpuratus ck8285520359 185/333 S. purpuratus ck8285530360 185/333 S. purpuratus ck8285540361 185/333 S. purpuratus ck8285550362 185/333 S. purpuratus ck8285560363 185/333 S. purpuratus ck8285570364 Actin S. purpuratus 7e-77 ck8283130365 185/333 S. purpuratus ck8285580366 185/333 S. purpuratus ck8285590367 185/333 S. purpuratus ck8285600368 185/333 S. purpuratus ck8285610369 185/333 S. purpuratus ck8285620370 185/333 S. purpuratus ck8285630371 185/333 S. purpuratus ck8285640372 185/333 S. purpuratus ck8285650373 185/333 S. purpuratus ck8285660374 185/333 S. purpuratus ck8285670375 185/333 S. purpuratus ck8285680376 185/333 S. purpuratus ck8285690377 185/333 S. purpuratus ck8285700378 DD104 S. purpuratus 3e-74 ck8283680379 185/333 S. purpuratus ck8285710380 185/333 S. purpuratus ck8285720381 185/333 S. purpuratus ck8285730382 185/333 S. purpuratus 2e-20 ck8285740383 185/333 S. purpuratus ck8285750384 185/333 S. purpuratus ck8285760385 185/333 S. purpuratus ck8285770386 185/333 S. purpuratus ck8285780387 Ribosomal protein 40S S8 H. sapiens 2e-59 ck8290360388 185/333 S. purpuratus ck8285790389 185/333 S. purpuratus ck8285800390 185/333 S. purpuratus ck8285810391 185/333 S. purpuratus ck8285820392 185/333 S. purpuratus ck8285830393 185/333 S. purpuratus ck8285840394 185/333 S. purpuratus ck8285850395 185/333 S. purpuratus ck8285860396 185/333 S. purpuratus ck8285870397 185/333 S. purpuratus ck8285880398 185/333 S. purpuratus ck8285890399 No significant match ck8291160400 185/333 S. purpuratus ck828590

0401 185/333 S. purpuratus ck8285910402 185/333 S. purpuratus ck8285920403 185/333 S. purpuratus ck8285930404 185/333 S. purpuratus ck8285940405 185/333 S. purpuratus ck8285950406 185/333 S. purpuratus ck8285960407 185/333 S. purpuratus ck8285970408 185/333 S. purpuratus ck8285980409 185/333 S. purpuratus ck8285990410 EST040 S. purpuratus 1e-115 cv6527030411 185/333 S. purpuratus ck8286000412 185/333 S. purpuratus ck8286010413 185/333 S. purpuratus ck8286020414 185/333 S. purpuratus ck8286030415 Cathepsin O. mykiss 6e-39 ck8283390416 185/333 S. purpuratus ck8286040417 185/333 S. purpuratus ck8286050418 185/333 S. purpuratus ck8286060419 185/333 S. purpuratus ck8286070420 185/333 S. purpuratus ck8286080421 185/333 S. purpuratus ck8286090422 185/333 S. purpuratus ck8286100423 185/333 S. purpuratus ck8286110424 Actin S.purpuratus 2e-70 ck8283160425 185/333 S. purpuratus ck8286120426 Actin S. purpuratus 1e-79 ck8283170427 185/333 S. purpuratus ck8286130429 185/333 S. purpuratus ck8286140430 185/333 S. purpuratus ck8286150431 185/333 S. purpuratus ck8286160432 185/333 S. purpuratus ck8286170433 185/333 S. purpuratus ck8286180434 Cathepsin S. purpuratus 1e-142* ck8283400435 Actin S. purpuratus 3e-11* ck8283180436 185/333 S. purpuratus ck8286190437 185/333 S. purpuratus ck8286200438 185/333 S. purpuratus ck8286210439 Cathepsin E. japonicus 7e-12 ck8283410441 185/333 S. purpuratus ck8286220442 185/333 S. purpuratus ck8286230443 185/333 S. purpuratus ck8286240444 185/333 S. purpuratus ck8286250445 185/333 S. purpuratus ck8286260446 185/333 S. purpuratus ck8286270447 185/333 S. purpuratus ck8286280474 185/333 S. purpuratus ck8286290475 185/333 S. purpuratus ck8286300476 185/333 S. purpuratus ck8286310477 185/333 S. purpuratus ck8286320478 185/333 S. purpuratus ck8286330479 185/333 S. purpuratus ck8286340480 185/333 S. purpuratus ck8286350481 Cathepsin M. glutinosa 4e-25 ck8283420482 185/333 S. purpuratus ck8286360483 185/333 S. purpuratus ck8286370484 185/333 S. purpuratus ck8286380485 185/333 S. purpuratus ck8286390486 185/333 S. purpuratus ck8286400487 185/333 S. purpuratus ck828641

0488 Cathepsin O. mykiss 2e-24 ck8283430489 185/333 S. purpuratus ck8286420490 185/333 S. purpuratus ck8286430491 185/333 S. purpuratus ck8286440492 185/333 S. purpuratus ck8286450493 Translation elongation factor 1 alpha X. olivieri 8e-07 ck8290750494 185/333 S. purpuratus ck8286460495 185/333 S. purpuratus ck8286470496 185/333 S. purpuratus ck8286480497 Translation elongation factor 1 alpha S. cerevisiae 7e-61 ck8290760498 185/333 S. purpuratus ck8286490499 185/333 S. purpuratus ck8286500500 185/333 S. purpuratus ck8286510501 185/333 S. purpuratus 5e-15 ck8286520502 185/333 S. purpuratus ck8286530503 185/333 S. purpuratus ck8286540504 185/333 S. purpuratus ck8286550505 185/333 S. purpuratus ck8286560506 185/333 S. purpuratus ck8286570507 185/333 S. purpuratus ck8286580508 185/333 S. purpuratus ck8286590509 185/333 S. purpuratus ck8286600510 185/333 S. purpuratus ck8286610511 185/333 S. purpuratus ck8286620512 185/333 S. purpuratus ck8286630513 185/333 S. purpuratus ck8286640514 185/333 S. purpuratus ck8286650516 185/333 S. purpuratus ck8286660517 185/333 S. purpuratus ck8286670518 185/333 S. purpuratus ck8286680519 185/333 S. purpuratus ck8286690520 185/333 S. purpuratus ck8286700521 185/333 S. purpuratus ck8286710522 185/333 S. purpuratus ck8286720523 185/333 S. purpuratus ck8286730524 185/333 S. purpuratus ck8286740525 185/333 S. purpuratus ck8286750526 Cathepsin O. mykiss 3e-17 ck8283440527 185/333 S. purpuratus ck8286760528 185/333 S. purpuratus ck8286770529 185/333 S. purpuratus ck8286780530 185/333 S. purpuratus ck8286790531 185/333 S. purpuratus ck8286800532 185/333 S. purpuratus ck8286810533 185/333 S. purpuratus ck8286820534 185/333 S. purpuratus ck8286830535 185/333 S. purpuratus ck8286840536 185/333 S. purpuratus ck8286850537 185/333 S. purpuratus ck8286860538 185/333 S. purpuratus ck8286870539 185/333 S. purpuratus ck8286880540 185/333 S. purpuratus ck8286890541 185/333 S. purpuratus ck8286900542 185/333 S. purpuratus ck8286910543 185/333 S. purpuratus ck8286920544 185/333 S. purpuratus ck8286930545 185/333 S. purpuratus ck8286940547 185/333 S. purpuratus ck8286950548 185/333 S. purpuratus ck828696

0549 185/333 S. purpuratus ck8286970550 185/333 S. purpuratus ck8286980551 185/333 S. purpuratus ck8286990552 Cathepsin O. mykiss 1e-14 ck8283450553 185/333 S. purpuratus cv6527560554 185/333 S. purpuratus ck8287000555 185/333 S. purpuratus ck8287010556 185/333 S. purpuratus ck8287020557 185/333 S. purpuratus ck8287030558 185/333 S. purpuratus ck8287040559 185/333 S. purpuratus ck8287050560 185/333 S. purpuratus ck8287060561 185/333 S. purpuratus ck8287070562 185/333 S. purpuratus ck8287080563 185/333 S. purpuratus ck8287090564 185/333 S. purpuratus ck8287100565 Vesicle membrane protein, p24b M. musculus 2e-30 cv6526980566 185/333 S. purpuratus ck8287110567 185/333 S. purpuratus ck8287120568 185/333 S. purpuratus ck8287130569 Cathepsin O. mykiss 4e-51 ck8283460570 185/333 S. purpuratus ck8287140571 185/333 S. purpuratus ck8287150572 Cathepsin B. microplus 6e-21 ck8283470573 185/333 S. purpuratus ck8287160574 185/333 S. purpuratus ck8287170575 185/333 S. purpuratus ck8287180576 ET putative translation product H. sapiens 2e-23 cv6527050577 185/333 S. purpuratus cv6527880578 185/333 S. purpuratus ck8287190579 185/333 S. purpuratus ck8287200580 185/333 S. purpuratus ck8287210581 No significant match ck8291170582 185/333 S. purpuratus ck8287220583 185/333 S. purpuratus ck8287230584 Aconitase D. pulex 6e-04 ck8291180585 185/333 S. purpuratus ck8287240586 185/333 S. purpuratus ck8287250587 185/333 S. purpuratus 6e-17 ck8291190588 185/333 S. purpuratus ck8287260589 185/333 S. purpuratus ck8287270590 185/333 S. purpuratus ck8287280591 185/333 S. purpuratus ck8287290592 185/333 S. purpuratus ck8287300593 185/333 S. purpuratus ck8287310594 185/333 S. purpuratus ck8287320595 185/333 S. purpuratus ck8287330596 185/333 S. purpuratus ck8287340597 185/333 S. purpuratus ck8287350598 185/333 S. purpuratus ck8287360600 Actin S. purpuratus 1e-75 ck8283190601 185/333 S. purpuratus ck8287370602 185/333 S. purpuratus ck8287380603 185/333 S. purpuratus ck8287390604 185/333 S. purpuratus ck8287400605 185/333 S. purpuratus 2e-91* ck8287410606 185/333 S. purpuratus ck8287420607 185/333 S. purpuratus ck8287430608 185/333 S. purpuratus ck828744

0609 185/333 S. purpuratus ck8287450610 Cathepsin B. microplus 3e-34 ck8283480612 185/333 S. purpuratus ck8287460613 Actin S. franciscanus 1e-76 cv6527530614 185/333 S. purpuratus ck8287470615 185/333 S. purpuratus ck8287480616 185/333 S. purpuratus ck8287490617 185/333 S. purpuratus ck8287500618 185/333 S. purpuratus ck8287510619 185/333 S. purpuratus 1e-07 ck8287520620 185/333 S. purpuratus ck8287530621 185/333 S. purpuratus ck8287540622 185/333 S. purpuratus ck8287550623 185/333 S. purpuratus ck8287560624 185/333 S. purpuratus ck8287570625 185/333 S. purpuratus ck8287580626 185/333 S. purpuratus ck8287590627 185/333 S. purpuratus ck8287600628 Ribosomal protein 40S S8 A. mellifera 5e-22 ck8290370629 185/333 S. purpuratus ck8287610630 185/333 S. purpuratus ck8287620631 185/333 S. purpuratus ck8287630632 185/333 S. purpuratus ck8287640633 185/333 S. purpuratus ck8287650634 Cathepsin M. glutinosa 5e-19 ck8283490635 185/333 S. purpuratus ck8287660636 185/333 S. purpuratus 7e-07 ck8287670637 185/333 S. purpuratus ck8287680638 185/333 S. purpuratus ck8287690639 185/333 S. purpuratus ck8287700640 185/333 S. purpuratus ck8287710641 185/333 S. purpuratus ck8287720642 Complement component Factor (B SpBf) S. purpuratus 0* ck8289870643 185/333 S. purpuratus ck8287730644 185/333 S. purpuratus ck8287740645 185/333 S. purpuratus ck8287750646 185/333 S. purpuratus ck8287760647 185/333 S. purpuratus ck8287770648 185/333 S. purpuratus ck8287780649 185/333 S. purpuratus ck8287790650 185/333 S. purpuratus ck8287800651 185/333 S. purpuratus ck8287810652 185/333 S. purpuratus ck8287820653 185/333 S. purpuratus ck8287830654 185/333 S. purpuratus ck8287840655 185/333 S. purpuratus ck8287850656 185/333 S. purpuratus ck8287860657 185/333 S. purpuratus ck8287870658 185/333 S. purpuratus ck8287880659 185/333 S. purpuratus ck8287890660 185/333 S. purpuratus ck8287900661 185/333 S. purpuratus ck8287910662 185/333 S. purpuratus ck8287920663 185/333 S. purpuratus ck8287930664 185/333 S. purpuratus ck8287950665 185/333 S. purpuratus ck8287940666 185/333 S. purpuratus ck8287960667 185/333 S. purpuratus ck8287970668 185/333 S. purpuratus ck828798

0669 185/333 S. purpuratus ck8287990670 DD104 S. purpuratus 1e-52 ck8283690671 185/333 S. purpuratus ck8288000672 185/333 S. purpuratus ck8288010673 185/333 S. purpuratus ck8288020674 185/333 S. purpuratus ck8288030675 185/333 S. purpuratus ck8288040676 185/333 S. purpuratus ck8288050677 185/333 S. purpuratus ck8288060678 185/333 S. purpuratus ck8288070679 185/333 S. purpuratus ck8288080680 185/333 S. purpuratus ck8288090681 185/333 S. purpuratus ck8288100682 185/333 S. purpuratus ck8288110683 185/333 S. purpuratus ck8288120684 No significant match ck8291200685 185/333 S. purpuratus ck8288130686 185/333 S. purpuratus ck8288140687 185/333 S. purpuratus ck8288150688 No significant match ck8291210689 185/333 S. purpuratus ck8288160690 Cathepsin M. glutinosa 5e-19 ck8283500691 185/333 S. purpuratus ck8288170692 185/333 S. purpuratus ck8288180693 185/333 S. purpuratus ck8288190694 Vacuolar sorting protein M. musculus 3e-41 cv6527460695 185/333 S. purpuratus ck8288200696 185/333 S. purpuratus 7e-07 ck8288210697 185/333 S. purpuratus ck8288220698 185/333 S. purpuratus ck8288230699 185/333 S. purpuratus ck8288240700 185/333 S. purpuratus ck8288250701 185/333 S. purpuratus ck8288260702 185/333 S. purpuratus ck8288270703 185/333 S. purpuratus ck8288280704 185/333 S. purpuratus ck8288290705 Cathepsin (EST052) O. mykiss 4e-24 ck8283510706 185/333 S. purpuratus ck8288300708 185/333 S. purpuratus ck8288310709 185/333 S. purpuratus ck8288320710 185/333 S. purpuratus ck8288330712 185/333 S. purpuratus ck8288340713 Unknown S. purpuratus 2e-81* ck8291220714 185/333 S. purpuratus ck8288350715 185/333 S. purpuratus ck8288360717 185/333 S. purpuratus ck8288380718 185/333 S. purpuratus ck8288390719 185/333 S. purpuratus ck8288400720 185/333 S. purpuratus ck8288410721 185/333 S. purpuratus ck8288420722 185/333 S. purpuratus ck8288430723 185/333 S. purpuratus ck8288440724 185/333 S. purpuratus ck8288450726 185/333 S. purpuratus ck8288460727 185/333 S. purpuratus ck8288470728 185/333 S. purpuratus ck8288480729 EST198 S. purpuratus 3e-33* cv6527040730 185/333 S. purpuratus ck8288490731 Sec61 translocon-associated protein gamma X. laevis 2e-54 cv652785

0732 185/333 S. purpuratus ck8288500733 185/333 S. purpuratus ck8288510734 185/333 S. purpuratus ck8288520735 185/333 S. purpuratus ck8288530736 185/333 S. purpuratus ck8288540737 185/333 S. purpuratus ck8288550738 185/333 S. purpuratus ck8288560739 185/333 S. purpuratus ck8288570740 185/333 S. purpuratus ck8288580741 185/333 S. purpuratus ck8288590742 185/333 S. purpuratus ck8288600743 185/333 S. purpuratus ck8288610744 Rab-7 H. sapiens 8e-52 cv6527280745 185/333 S. purpuratus ck8288620746 DD104 S. purpuratus 2e-56 ck8283700747 185/333 S. purpuratus ck8288630748 DD104 S. purpuratus 2e-37 ck8283710749 185/333 S. purpuratus ck8288600750 185/333 S. purpuratus ck8288650751 185/333 S. purpuratus ck8288660753 185/333 S. purpuratus ck8288670754 185/333 S. purpuratus ck8288680755 185/333 S. purpuratus ck8288690756 185/333 S. purpuratus ck8288700757 185/333 S. purpuratus ck8288710758 185/333 S. purpuratus ck8288720759 185/333 S. purpuratus ck8288730760 185/333 S. purpuratus ck8288740761 185/333 S. purpuratus ck8288750762 185/333 S. purpuratus ck8288760763 185/333 S. purpuratus ck8288770858 185/333 S. purpuratus ck8288780860 185/333 S. purpuratus ck8288790861 185/333 S. purpuratus 1e-06 cv6527890862 185/333 S. purpuratus ck8288800863 185/333 S. purpuratus ck8288810864 185/333 S. purpuratus ck8288820865 185/333 S. purpuratus ck8288830866 185/333 S. purpuratus ck8288840867 185/333 S. purpuratus ck8288850868 185/333 S. purpuratus ck8288860869 185/333 S. purpuratus ck8288870870 185/333 S. purpuratus ck8288880871 185/333 S. purpuratus ck8288890872 185/333 S. purpuratus ck8288900873 185/333 S. purpuratus ck8288910874 Nuclear RNA- and DNA-binding protein H. sapiens 1e-06 cv6527170875 185/333 S. purpuratus ck8288920876 185/333 S. purpuratus ck8288930877 185/333 S. purpuratus ck8288940878 185/333 S. purpuratus ck8288950879 Cathepsin S. purpuratus 0* ck8283520880 185/333 S. purpuratus ck8288960881 185/333 S. purpuratus ck8288970882 185/333 S. purpuratus ck8288980883 185/333 S. purpuratus ck8288990884 185/333 S. purpuratus ck8289000885 185/333 S. purpuratus ck8289010886 185/333 S. purpuratus ck828902

0887 185/333 S. purpuratus ck8289030888 185/333 S. purpuratus ck8289040889 185/333 S. purpuratus ck8289050890 EST040 S. purpuratus 1e-114* cv6527550891 185/333 S. purpuratus ck8289060892 185/333 S. purpuratus ck8289070893 185/333 S. purpuratus ck8289080894 No significant match ck8291230895 Cytochrome C oxidase subunit 4 U. caupo 2e-31 ck8283600896 Nuclear protein NHP2 H. sapiens 1e-40 cv6527160897 185/333 S. purpuratus ck8289090898 185/333 S. purpuratus ck8289100899 185/333 S. purpuratus ck8289110900 No significant match ck8291240902 185/333 S. purpuratus ck8289120903 185/333 S. purpuratus ck8289130904 185/333 S. purpuratus ck8289140905 185/333 S. purpuratus 4e-04 ck8289150906 Sec61 ER transport protein H. sapiens 2e-35 cv6527330907 185/333 S. purpuratus ck8289160908 185/333 S. purpuratus ck8289170909 185/333 S. purpuratus ck8289180910 185/333 S. purpuratus ck8289190911 185/333 S. purpuratus ck8289200912 185/333 S. purpuratus ck8289210913 185/333 S. purpuratus ck8289220914 185/333 S. purpuratus ck8289230915 No significant match ck8291250916 185/333 S. purpuratus ck8289240917 185/333 S. purpuratus ck8289250918 185/333 S. purpuratus ck8289260919 No significant match ck8291260920 185/333 S. purpuratus ck8289270921 185/333 S. purpuratus ck8289280922 185/333 S. purpuratus ck8289290923 185/333 S. purpuratus ck8289300924 185/333 S. purpuratus ck8289310925 185/333 S. purpuratus ck8289320926 185/333 S. purpuratus ck8289330927 185/333 S. purpuratus ck8289340928 185/333 S. purpuratus ck8289350929 185/333 S. purpuratus ck8289360930 185/333 S. purpuratus ck8289370931 185/333 S. purpuratus ck8289380932 185/333 S. purpuratus ck8289390933 185/333 S. purpuratus 2e-22 ck8289400934 185/333 S. purpuratus 3e-09 ck8289410935 185/333 S. purpuratus ck8289420936 185/333 S. purpuratus ck8289430937 185/333 S. purpuratus ck8289440938 185/333 S. purpuratus ck8289450939 No significant match ck8291270940 185/333 S. purpuratus ck8289460941 185/333 S. purpuratus ck8289470943 185/333 S. purpuratus ck8289480944 185/333 S. purpuratus ck8289490945 185/333 S. purpuratus ck8289500946 185/333 S. purpuratus ck8289510947 185/333 S. purpuratus ck828952

0948 185/333 S. purpuratus ck8289530949 No significant match ck8291280950 185/333 S. purpuratus ck8289540951 185/333 S. purpuratus ck8289550952 185/333 S. purpuratus ck8289560953 185/333 S. purpuratus ck8289570954 Ribosomal RNA 26S A. sinapina 1e-120* ck8290380955 185/333 S. purpuratus ck8289580956 Ribosomal protein 40S S11 C. carpio 1e-03 ck8290390957 No significant match ck8291290958 185/333 S. purpuratus ck8289590959 185/333 S. purpuratus ck8289600960 Cathepsin S. zeamais 5e-32 ck8283530961 185/333 S. purpuratus ck8289610962 185/333 S. purpuratus ck8289620963 Actin H. erythrogramma 5e-40 ck8283200964 185/333 S. purpuratus ck8289630965 185/333 S. purpuratus ck8289640966 185/333 S. purpuratus ck8289650967 185/333 S. purpuratus ck8289660968 Ribosomal RNA 26S A. gallica 1e-111* ck8290400969 185/333 S. purpuratus ck8289670970 185/333 S. purpuratus ck8289680971 Ribosomal protein 60S L23 H. sapiens 5e-48 ck8290410972 185/333 S. purpuratus ck8289690973 No significant match ck8291300974 No significant match ck8291310975 185/333 S. purpuratus ck8289700976 No significant match ck8291320977 185/333 S. purpuratus e-132 ck8289710978 185/333 S. purpuratus ck8289720979 185/333 S. purpuratus ck8289730980 185/333 S. purpuratus ck8289740981 No significant match ck8291330982 Cathepsin O. mykiss 2e-14 ck8283540983 185/333 S. purpuratus ck8289750984 No significant match ck8291340985 185/333 S. purpuratus e-163 cv6527650986 185/333 S. purpuratus ck8289760987 185/333 S. purpuratus ck8289770988 185/333 S. purpuratus ck8289780990 185/333 S. purpuratus ck8289790991 185/333 S. purpuratus ck8289800992 185/333 S. purpuratus ck8289810993 185/333 S. purpuratus e-167 ck8289820994 185/333 S. purpuratus ck8289830995 185/333 S. purpuratus ck8289840996 185/333 S. purpuratus ck8289850997 185/333 S. purpuratus ck8289861006 No significant match ck8291351027 Gelsolin L. terrestris 2e-68 ck8283211028 No significant match ck8291361029 No significant match ck8291371030 No significant match ck8291381031 185/333 R. norvegicus 2e-04 cv6527901032 IgE-dependent histamine release factor D. variabilis 3e-04 ck8291391033 Ribosomal protein 60S L7A T. rubripes 1e-98 ck8290421034 Small integral membrane protein M. musculus 3e-12 cv6527101035 No significant match ck829140

1036 No significant match ck8291411037 No significant match ck8291421038 NADH dehydrogenase subunit 1 P. lividus 4e-39 ck8289911039 No significant match ck8291431040 No significant match ck8291441041 DD104 S. purpuratus 2e-77 cv6527911042 Ribosomal protein 40S S14 P. carnea 8e-52 ck8290431043 NADH dehydrogenase subunit 2 S. purpuratus 4e-83 ck8289941044 Protein typrotein tyrosine kinase 9-like protein M. musculus 3e-21 cv6527261045 No significant match ck8291451046 Elongation factor-1 alpha A. crassispina 1e-136 ck8290771047 No significant match ck8291461048 No significant match ck8291471049 No significant match ck8291481050 No significant match ck8291491051 DD104 S. purpuratus 3e-10 cv6527921052 DD104 S. purpuratus 1e-131 ck8291501053 No significant match ck8291511054 No significant match ck8291521057 Ribosomal protein 60S L14 M. musculus 6e-38 ck8290441058 Protein disulfide S. purpuratus 1e-133 cv6527701059 Peptide chain release factor, subunit 1 D. melanogaster 1e-112 cv6527471060 Proteasome C. auratus 1e-108 cv6527241061 No significant match ck8291531062 Sec61 alpha transport protein A. aegypti 1e-162 cv6527761063 ATP/ADP translocase D. melanogaster 2e-92 cv6527511064 No significant match ck8291541065 185/333 H. sapiens 3e-13 cv6527681066 No significant match ck8291551067 No significant match ck8291561068 Proteasome clawed frog 1e-100 cv6527731069 Actin S. purpuratus 1e-118 ck8283221070 Cytochrome C oxidase subunit 1 P. lividus 1e-125 ck8283611071 Ribosomal protein 40S S14a D. melanogaster 4e-54 ck8290451072 185/333 Zea mays 9e-09 cv6527571073 Ribosomal protein 60S L15 M. musculus 1e-75 ck8290461074 Elongation factor S. purpuratus 1e-146 cv6527401075 No significant match cv6527581076 No significant match ck8291571077 Sec61 beta transport protein H. sapiens 7e-14 cv6527721079 Ribosomal protein 60S L24 H. sapiens 1e-37 ck8290471080 Ribosomal protein 60S L10a R. norvegicus 6e-82 ck8290481081 No significant match ck8291581082 Cytochrome C oxidase subunit 1 S. purpuratus 2e-77 ck8283621083 Nuclear antigen EBNA1 herpesvirus 4 5e-06 ck8289951084 Ribosomal protein 60S L3 R. rattus 1e-147 ck8290491085 No significant match ck8291591086 Allograft inflammatory factor 1 S. domuncula 3e-29 cv6526931087 No significant match ck8291601088 Complement component Factor B (SpBf) S. purpuratus 1e-119 cv6527781089 Tubulin, alpha D. melanogaster 5e-28 cv6527441090 Translationally-controlled tumor protein D. rerio 1e-16 cv6527801091 No significant match ck8291611092 Ribosomal protein 60S L35 H. sapiens 2e-27 ck8290501093 DNA methyl transferase-associated protein M. musculus 3e-37 cv6527011094 Vacuolar ATP synthase subunit M. sexta 6e-09 cv6527451095 No significant match ck8291621096 Rho H. pulcherrimus 6e-87 cv6527311097 No significant match ck829163

1098 No significant match ck8291641099 NADH dehydrogenase subunit 1 P. lividus 6e-47 ck8289921100 ATP synthase C. carpio 2e-28 ck8283311101 No significant match ck8291651102 Cathepsin O. mykiss 3e-98 ck8283551103 No significant match ck8291661104 No significant match ck8291671105 No significant match ck8291681106 No significant match ck8291691107 Thrombospondin H. sapiens 4e-53 cv6527381108 Cathepsin U. caupo 1e-44 ck8283561109 No significant match ck8291701110 Ribosomal protein 60S L21 A. irradians 1e-56 ck8290511111 No significant match L. major 1e-05 ck8291711112 No significant match ck8291721113 No significant match ck8291731114 Annexin S. domesticus 2e-68 cv6526951116 Ribosomal protein 60S L36 M. musculus 1e-19 ck8290521117 Polo-like kinase H. pulcherrimus 1e-163 cv6527211118 Translocon associated protein gamma X. laevis 9e-51 cv6527421119 Ribosomal protein 40S S8 S. frugiperda 7e-72 ck8290531121 Ribosomal protein 40S S16 H. sapiens 1e-56 ck8290541122 Heterogenous nuclear ribonuclear protein C. tentans 1e-31 cv6527061124 No significant match cv6527741125 Cytochrome C oxidase subunit 1 P. lividus 1e-125 ck8283631126 No significant match ck8291741127 No significant match ck8291751128 185/333 S. purpuratus cv6527931129 No significant match ck8291761130 No significant match ck8291771131 DD104 S. purpuratus 1e-131 ck8291781132 No significant match ck8291791133 Complement component Factor B (SpBf) S. purpuratus 1e-122 cv6527351135 Unknown V. carteri 3e-26 cv6527941136 Protein disulfide-isomerase S. purpuratus 1e-165 cv6527251137 Ribosomal protein 60S L24 H. sapiens 2e-33 ck8290551138 No significant match ck8291801139 Aldehyde dehydrogenase H. sapiens 1e-103 cv6526921140 Complement component Factor B (SpBf) S. purpuratus 1e-101 cv6527791141 Ribosomal protein 40S SA T. gratilla 1e-123 ck8290561143 Fatty acid desaturase H. sapiens 8e-23 cv6527591145 Cytochrome C oxidase subunit 3 A. lixula 1e-78 cv6527541146 Unknown, mitochondrial C. aruatus 4e-10 cv6527071147 Na+-dependent inorganic PO4 cotransporter A. thaliana 2e-39 cv6527271148 185/333 S. purpuratus 3e-47 ck8291811149 No significant match ck8291821150 No significant match ck8291831151 No significant match ck8291841152 Cofilin N. crassa 7e-09 cv6526991153 Citrate synthase A. gambiae 4e-85 cv6526971154 No significant match ck8291851155 Actin T. belangeri 9e-14 ck8283231157 Orphan steroid hormone receptor 2 S. purpuratus 1e-129 cv6527181158 Bax inhibitor-1 P. olivaceus 3e-31 cv6527231159 No significant match ck8291861160 No significant match ck8291871161 No significant match ck8291881162 No significant match ck8291891163 Nuclear antigen EBNA1 herpesvirus 4 7e-17 cv652763

1164 No significant match ck8291901165 No significant match ck8291911166 Translocon-associated protein A. gambiae 1e-17 cv6527821168 Translation elongation factor 1 gamma H. sapiens 3e-43 cv6527831169 Ribosomal protein 60S L3 S. frugiperda 1e-145 ck8290571170 Actin S. purpuratus 1e-172 ck8283241171 Midasin H. sapiens 2e-48 cv6527151172 Ribosomal protein 60S L24 H. sapiens 6e-39 ck8290581173 Ribosomal protein 60S L24 H. sapiens 6e-39 ck8290591174 Cytochrome C oxidase subunit 1 P. lividus 2e-67 ck8283641175 No significant match ck8291921176 Paraspeckle protein H. sapiens 4e-56 cv6527191177 Sec61 alpha transport protein A. aegypti 1e-126 cv6527771179 No significant match ck8291931180 Ribosomal protein 60S L6 I. punctatus 6e-62 ck8290601181 Ribosomal protein 60S L44 C. farreri e-41 ck8290611182 Nuclear antigen EBNA1 herpesvirus 4 1e-05 ck8289961183 Ribosomal protein 60S L19 D. melanogaster e-58 ck8290621184 HEXBP DNA-binding protein L. major 1e-25 cv6527001185 No significant match ck8291941186 Phosphatidyleothanolamine-binding protein A. thaliana 4e-17 cv6527201187 Ribosomal protein 40S S8 S. frugiperda 8e-76 ck8290631188 Immediate early response 5 H. sapiens 3e-06 cv6527081189 Avena G. gallus 2e-11 cv6526961190 No significant match ck8291951191 1-acylglycerol-3-phosphate O-acyltransferase 2 M. musculus 8e-26 cv6526911192 Protein synthesis initiation factor 4A M. musculus 1e-34 cv6527711193 No significant match ck8291961194 No significant match ck8291971195 Microtubule associated protein S. purpuratus 3e-74 cv6527141196 No significant match ck8291981197 Mannose-6-phosphate receptor M. musculus 4e-24 cv6527121198 Hypothetical protein, mitochondrial C. auratus 3e-09 cv6527641199 No significant match ck8291991200 Noelin, Amassin H. sapiens cv6526941201 No significant match ck8292001202 No significant match herpesvirus 4 5e-12 ck8289971203 No significant match ck8292011204 Glycine-rich cell wall structural protein 1 Petunia sp. 1e-22 cv6527611205 NADH/NADPH thyroid oxidase S. scrofa 2e-92 cv6527021206 Nuclear antigen EBNA1 herpesvirus 4 6e-12 ck8289981207 Ribosomal protein 60S L21 A. irradians 5e-52 ck8290641208 No significant match ck8292021209 Solute carrier transport protein M. musculus 1e-24 cv6527341210 Rab5-interacting protein M. musculus 8e-33 cv6527301211 Translation elongation factor A. crassispina 1e-153 cv6527841212 No significant match ck8292031213 Unknown H. sapiens 1e-109 cv6527371214 Splicing factor H. sapiens 8e-22 cv6527361215 No significant match ck8292041216 No significant match ck8292051217 No significant match ck8292061220 Ribosomal protein 60S L15 M. musculus 2e-75 ck8290651221 Ribosomal protein 40S S5 L. punctatus 2e-96 ck8290661224 No significant match ck8292071225 No significant match ck8292081226 Malate dehydrogenase S. idiastes 1e-116 cv6527111227 Ribosomal protein 60S L24 H. sapiens 2e-37 ck8290671228 Presenilin A. gambiae 2e-24 cv652722

1229 No significant match ck8292091230 Ribosomal protein 60S L10a R. norvegicus 5e-80 ck8290681231 Actin S. franciscanus 1e-122 ck8283251233 No significant match ck8292101234 Nuclear antigen EBNA1 herpesvirus 4 4e-13 ck8289991235 185/333 S. purpuratus 1e-75 cv6527951236 Ribosomal protein 60S L22 T. gratilla 4e-35 ck8290691237 ATPase beta chain Artemia sp. 2e-29 cv6527521238 No significant match ck8292111239 No significant match ck8292121240 185/333 S. purpuratus 4e-43 ck8292131241 185/333 T. aestivum 1e-07 cv6527601242 Tubulin, alpha-3 chain A. gambiae 1e-166 cv6527861243 No significant match ck8292141244 Translation initiation factor H. sapiens 4e-93 cv6527411246 p24B precursor protein M. musculus 4e-60 cv6527481247 ATP synthase S. purpuratus 9e-06 ck828332

________________* e values marked by * were obtained by BLASTn searches. All the others were obtained by BLASTx.#Missing e values for 185/333 matches indicates that the clones were identified by library screens rather than sequence

analysis.

Macroarray analysis of coelomocyte gene expression in ...€¦ · basis of the striking increase in...

Documents

Transcript of Macroarray analysis of coelomocyte gene expression in ...€¦ · basis of the striking increase in...