Multiple Genetic Differences in Sialic Acid Biology …cmm.ucsd.edu/varki/varkilab/Video...

Multiple Genetic Differences in SialicAcid Biology between Humans

and “Great Apes”

Ajit Varki

Distinguished Professor of Medicine and Cellular & Molecular MedicineCo-Director, Glycobiology Research and Training Center Coordinator, Project for Explaining the Origin of Humans

University of California, San DiegoLa Jolla, CA, USA

RECOMB“Research in Computational Molecular Biology”

LIPIDS

DNA RNA PROTEINS

ORGANISM

MATRIXCELL

TISSUES & ORGANSPHYSICAL PHYSICAL ENVIRONMENTENVIRONMENT

GLYCOPROTEINSPROTEOGLYCANS

GLYCOLIPIDS

ENZYMES

MICROBESMICROBESPARASITESPARASITES

DNA

GLYCANS

DNA

An “Holistic” View of Research in Molecular Biology

SIGNALLINGMOLECULES

REGULATORYFACTORS DIETDIET

CULTURALCULTURALENVIRONMENTENVIRONMENT

Electron micrograph of a human lymphocyte(Ruthenium Red staining)

Sugar chains (Oligosaccharides)

(Glycans)

Sialic Acid

Neutral Sugars

Protein

Scale Model of 1/100,000 of Human Red Blood Cell Surface

Modified by Pascal Gagneux from Viitala & Järnefelt

Cell Membrane(Lipids)

Varki A. Nature 446: 1023-1029, 2007

Sialic Acids on Cell Surface and Secreted MoleculesSialic Acids on Cell Surface and Secreted Molecules

SELF

Ligands forIntrinsic

Receptors

SiglecsFactor HSelectins

Uterine AgglutininLamininsL1CAM

INTRINSIC RECEPTORINTRINSIC RECEPTOR

SIALYLATED GLYCAN = M = Micro-organism/Toxin

SELF

M

EXTRINSIC RECEPTOREXTRINSIC RECEPTOR

InfluenzaMalariaCholera

HelicobacterMycoplasma

RotavirusPolyoma virusCoronavirus

PertussisTetanus etc.

Ligands forExtrinsic

Receptors

Biological Roles of Sialic Acids

Structural/Physical Roles

MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosomaStreptococcus

Etc.

Two Major Kinds of Sialic Acids in Mammalian CellsN-ACETYLNEURAMINIC ACID

(Neu5Ac)

R= LINKAGE TOUNDERLYINGSUGAR CHAIN

OOR

HO

HO OH

HO HN

O

O O-

88

77

11

22

33

55

6699

44

HO

N-GLYCOLYLNEURAMINIC ACID

(Neu5Gc)

OOR

HO

HO OH

HO HN

O

O O-

88

77

11

22

33

55

6699

44

Neu5Gc reported inhuman tumors andfetuses but not in

normal humantissues: an

“Oncofetal Antigen”?

CMAH

Human

*Precise

TimingUncertain

10

Mil

lio

ns

of

Ye

ars

Ag

o*

20

30

40

50

Chimpanzee RatRhesus Mouse

60

Dog Cow

Neu5Ac Neu5Gc

Human-SpecificLoss of Neu5Gc

Sialic AcidExpression

Human-SpecificEvolutionary Loss ofNeu5Gc Expression

10

5

Millio

ns

of

Ye

ars

Be

fore

Pre

se

nt*

0

*Precise Timing Uncertain

Gorilla gorilla

GorillaPan paniscus

BonoboPan troglodytes

ChimpanzeeHomo sapiens

HumanPongo pygmaeus

Orangutan

Muchmore, E.A., et. Al (1998) Amer.J.Phys.Anthro. 107:187

Chou,H.-H. et al. (1998) PNAS 95, 11751Hayakawa,T. et al. (2001) PNAS 98, 11399

SialicAcids in

BloodNeu5Ac Neu5Gc

CMAH gene Mutation

Causing lossOf Neu5Gc

““GreatGreat ApesApes””

““HominidsHominids””

Exon

92-bp

Exon 6 Exon

Great Ape Genome

Human genome

5 7

CMP-Neu5Ac Hydroxylase (CMAH) gene (18 exons)

Chou,H.-H. et al. (1998) PNAS 95, 11751

Hayakawa,T. et al. (2001) PNAS 98, 11399

CMP-Neu5Ac CMP-Neu5Gc

Humans ++++ -

“Great Apes” ++ ++

CMAHCMAH(Activity missing(Activity missing

In Humans)In Humans)

AluSq

AluY

4

2

Millio

ns o

f Y

ears

Befo

re P

resen

t*

*Precise Timing Uncertain

Bonobo

Chimpanzee

HomoSapiens

HomoNeanderthalensis

HomoErectus

HomoErgaster

Australopiths

6

Paranthopus

Timing ofNeu5Gc loss

during HumanEvolution

??

?

Mutation of Mutation of CMAHCMAHHsun-Hua Chou, Toshi Hayakawa - collaboration with

Meave Leakey, Svante Paabo, Yuki TakahataProc.Nat'l.Acad.Sci.U.S.A. 99:11736–11741, 2002

CoalescenceCoalescencetime oftime of

CMAH CMAH MutationMutation

Hayakawa et al. Genetics, 172:1139, 2006.

Brain sizeStone toolsHunting Meat Eating

Neu5Ac Neu5Gc

SELF


Receptors


Uterine AgglutininLamininsL1-CAM



SELF

M







Receptors


MolecularMimicry

E.ColiGonococcus


Etc.

Biological Roles of Sialic Acids : Siglecs

Human

Neu5Ac

Neu5Gc

Great Ape

Human Cells are Unusual Compared to Great ApesLoss of Neu5Gc and Excess of Neu5Ac

Glycophorin A

Binding TargetFor MalarialMerozoite

• Resistant to Neu5Gc-recognizing pathogens?

• Over-sensitive to Neu5Ac-recognizing pathogens?

Differential Expression of !2-6-linked Sialic Acids(Binding Sites for Human Influenza A) in Humans and Great Apes

Human Chimp

Mouse Gorilla

Gagneux et al. J.Biol.Chem. 278: 48245–48250, 2003

Monica Cheriyan, Pascal Gagneux & Nissi Varki

Dan Anderson, Harold MacClure, Yerkes Primate Center

Red arrows:Bronchialepithelium

Green arrows:Goblet Cells

(Mucins)

!2-6-linkedSialic Acidsstained by

SNA

SELF


Receptors





SELF

M







Receptors


MolecularMimicry

E.ColiGonococcus


Etc.

Biological Roles of Sialic Acids : Siglecs

From: Crocker P, Paulson J. & Varki, A. Nature Reviews Immunol. 7:255-266, 2007.

Biological Interactions Involving Siglecs

CD33/Siglec-3–related Siglecs (CD33rSiglecs)

SiglecsSiglecs (SiSialic Acid-binding IgIg-Superfamily LecLectinss)

in Hominids (Humans & “Great Apes”)1

4

Sialoadhesin

CD22

MAG

Y

V-set domain (Sia Recognition)

C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y

(Figure Modified from Takashi Angata)

5

6 7 8 9

OB-BP1AIRM1

MonocytesGranulocytes

PlacentaB cells

NK cellsMonocytes

EosinophilsMast Cells


Y Y Y Y Y

YY

Y Y Y Y

10

MonocytesEosinophils

B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y

ITIM (Inhibitory motif)

Putative Signaling motif

ITAM (Activating motif)Y

Y

R

14

Y

?

R

13

Y

? ?

Modified from Takashi Angata et al. Glycobiology 2007 17:838-846

Molecular phylogenetic tree based on the N-terminal region of human Siglecs

CD33-related Siglecs

Sialoadhesin

CD22

Myelin-associatedGlycoprotein

CD33-related Siglec Gene Clusters from 5 Species

100 Kb

Human

Chimpanzee

Baboon

Mouse

Rat

KLK gene cluster Siglec gene cluster

2 4 5 6 7 8 9 1011

12 13 14

2 4 5

4 5

5

9 7 3P1 P2 P3 P4 P5 P6 P7 P8

ETFBNKG7

LIM2 10

13

XIIP9P10 P11 P12 P13

8 6 5 5* HAS1FPR1

FPRL1ZNF175

Px

6 7 8 91011

12 13 14 9 7 3P1 P2 P3 P8

ETFBNKG7

LIM2 10 12P9P10 P11 P12 P13

8 6 V 5* HAS1FPR1

FPRL1ZNF175

6 7 8 9 1011

12 13 14 9 3P7 P8

ETFBNKG7

LIM2 10P10 P12 P13

8 VI 5 5* HAS1FPR1

FPRL1ZNF17513

6 7 891011

12 13 14

6 7 891011

12 13 14

ETFBNKG7

LIM2

ETFBNKG7

LIM2

E 3 G FPA

PA

PB

E 3 G F

ZNF-like

Kallikrein-like Gene Siglec or Siglec-like Gene Siglec Pseudogene Other Genes

Rodent Siglec Cluster: fewer Genes and Pseudogenes - more conserved

Kallikrein-Like (KLK) Genes are Highly Conserved but Siglec Genes are not

Angata T, Margulies EH, Green ED and Varki A Proc.Nat'l.Acad.Sci.U.S.A.. 101:13251-13256, 2004

Impetus for Sequencing the Chimpanzee Genome

• “Explaining Humans”

• Explaining Biomedical Differences betweenHumans and Chimpanzees

• Improving Understanding of the Human Genome

• Improving Care & Conservation of Chimpanzees

Varki, A. A Chimpanzee Genome Project is a Biomedical Imperative Genome Research10:1065-1070, 2000

McConkey, E.H. and Varki A. A Primate Genome Project Deserves High Priority. Science289: 1295-96, 2000.

Olson, M. V., Eichler, E. E., Varki, A., Myers, R.M., Erwin, J. M., and McConkey, E. H. A WhitePaper Advocating Complete Sequencing of the Genome of the Common Chimpanzee(white paper submitted to NHGRI, February 2002).

Reich, D.E., Lander, E. S., Waterston, R., Pääbo, S., Ruvolo, M., and Varki, A. Sequencingthe Chimpanzee Genome (white paper submitted to NHGRI, February 2002).

Olson, M. and Varki A. Sequencing the Chimpanzee Genome: Insights into Human Evolutionand Disease Nature Reviews Genetics., 4: 20-28, 2003.

“Initial Sequence of theChimpanzee Genome and

Comparison with the HumanGenome”

The Chimpanzee Sequencing andAnalysis Consortium

Tarjei S. Mikkelsen1,2, LaDeana W. Hillier3, Evan E.

Eichler4, Michael C. Zody1, David B. Jaffe1, Shiaw-Pyng

Yang3, Wolfgang Enard5, Ines Hellman5, Kerstin

Lindblad-Toh1, Tasha K. Altheide6, Nicoletta

Archidiacono7, Peer Bork8,9, Jonathan Butler1, Jean L.

Chang1, Ze Cheng4, Asif T. Chinwalla3, Pieter deJong10,

Kimberley D. Delehaunty3, Catrina C. Fronick3, Lucinda

L. Fulton3, Yoav Gilad11, Gustavo Glusman12, Sante

Gnerre1, Tina A. Graves3, Toshiyuki Hayakawa6, Karen

E. Hayden13, Xiaoqiu Huang14, Hongkai Ji15, W. James

Kent16, Mary-Claire King4, Edward J. Kulbokas III1,

Ming K. Lee4, Ge Liu13, Carlos Lopez-Otin17, Kateryna D.

Makova18, Orna Man19, Elaine R. Mardis3, Evan

Mauceli1, Tracie L. Miner3, William E. Nash3, Joanne O.

Nelson3, Svante Pääbo5, Nick J. Patterson1, Craig S.

Pohl3, Katherine S. Pollard16, Kay Prüfer5, Xose S.

Puente17, David Reich20,1, Mariano Rocchi7, Kate

Rosenbloom16, Maryellen Ruvolo21, Daniel J. Richter1,

Stephen F. Schaffner1, Arian F.A. Smit12, Scott M.

Smith3, Mikita Suyama8, James Taylor18, David

Torrents8, Eray Tuzun4, Ajit Varki6, Gloria Velasco17,

Mario Ventura7, John W. Wallis3, Michael C. Wendl3,

Richard K. Wilson3, Eric S. Lander1,22,23,24, & Robert H.

Waterston4.

SLC35A1

CMP-Sia

STs

CMP-Sia+

CMP

Sia

CMP-Neu5Gc

CMAH

CMP-Neu5AcCMAS

NPL

ManNAc

GNE

NEU3Sia

?LAMA1, 2

HF1

SELE

SELP

SELL

PlasmaMembrane

Cytosol

SIGLECs

L1C

AM

ExtracellularFluids/Matrix

Neu5GcCMAS

Nucleus

Golgi

NEU2

?

Adjacent Cell

ManNGc

NPL

ManNGc-6P

Neu5Gc-9P

GNE

NANS

?

NANS NANP

Neu5AcManNAc ManNAc-6P Neu5Ac-9PGNE

UDP-GlcNAc

GNE

GlcNAc-6P

GlcNAc-1P

GlcNH2-6P

CMP-KdnKdnMan Man-6P Kdn-9PNANS?

CMAS?

NANP?HK

GlcNAc

RENBP

NAGK

NAGK

Sia Sia

SiaSia

Sia

SiaSia

Sia

Sia

Sia

Sia

SiaAcO

Lysosome

Sia

NEU1/PPGBSLC17A5

Sia

Sia

NEU4

Genes Involved in Mammalian Sialic Acid Biology

Tasha Altheide & Toshi Hayakawa With Tarjei Mikkelson (Broad Institute)

SiaAcO

CMP-AcOSia

AcCoA

CoA

SIAESiaAcO

SiaAcO

SiaAcO

Altheide et al. J.Biol.Chem. 281: 25689-25702, 2006

How to Undertake A Systematic Approach to the

Comparative Genomics of Sialic Acid Biology?

• These genes fall into diverse and illogical groupswithin the typical Gene Ontology classification.

• They should be evaluated together from functionaland evolutionary perspectives in a “biochemicalsystems” approach, considering the steps of:

– Biosynthesis

– Activation

– Transport

– Modification

– Transfer

– Recycling

– Degradation

– Recognition.

Ka/Ks Ratios Show Different Evolutionary Rates in DifferentClasses of Genes Involved in Sialic Acid Biology

Tasha Altheide & Toshi Hayakawa


0

0.1

0.2

0.3

0.4

0.5

Activation

Transport& Transfer

Biosynthesis Recognition Recycling&

Degradation

(Av

era

ge

Ka

)/(A

ve

rag

e K

s)

Human/Chimp average Ka/Ks

Mouse/Rat average Ka/Ks

Within the Recognition Category, the SiglecsPrimarily Drive the Higher Ka/Ks Values.

# loci used

Average Ka

Average Ks

(Average Ka) (Average Ks)

Siglec lociPrimates 9 0.012 0.019 0.614Rodents 5 0.119 0.194 0.612

Non-Siglec lociPrimates 7 0.005 0.018 0.263Rodents 7 0.067 0.252 0.265



0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Human-ChimpanzeeHuman-Chimpanzee Human-BaboonHuman-Baboon Chimpanzee-BaboonChimpanzee-Baboon Mouse-RatMouse-Rat

Ka

/Ks

ra

tio

Only the Sialic Acid-Recognizing Amino-terminal V-setDomain of CD33-related Siglecs Shows Rapid Evolution

Humans Show the Most Rapid Evolution of V-set Domains



V-set domain

C2-set domain




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Human-Human-SpecificSpecific

Changes!Changes!




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-7 and -9 (and likely others)

• Chimpanzee and Gorilla Siglec-7 and -9 preferentially bind to Neu5Gc.

• Loss of Neu5Gc in Humans would have resulted in loss of Ligands.

• Human-specific changes have since occurred in the Sia-recognizing V-set

domain, allowing preferential recognition of Neu5Ac

• Are Humans fully adjusted yet?

Justin SonnenburgTasha Altheide

Proposed Evolutionary Scenario for MultipleProposed Evolutionary Scenario for MultipleHuman-Specific Changes in Sialic Acid BiologyHuman-Specific Changes in Sialic Acid Biology

Varki A. Nature 446: 1023, 2007

SELF


Receptors





SELF

M







Receptors


MolecularMimicry

E.ColiGonococcus


Etc.

Biological Roles of Sialic Acids : Molecular Mimicry by Pathogens




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-1 (Sialoadhesin)(Gene Expression Change)• Binds only Neu5Ac, not Neu5Gc.

• Increased Ligand Density in Human Cells. • Increased Frequency of Sialoadhesin-positive Macrophages

• Altered Expression Pattern of Sialoadhesin-positive Macrophages• Alterations in Human Macrophage Biology?

Els BrinkmanWith

Nissi VarkiPaul Crocker

Brinkman-Van der Linden, et al. J. Biol. Chem. 275:8633-40, 2000.


Varki A. Nature 446: 1023, 2007

9 7

P4

3 10 8 XII 6

P1 P2 P3 P5 P6 P7 P8 P10 P11P9

5

P12 P13 5*

11

P16CEN TEL

1 Mb

Siglec-11 P16

1234567891011

8.7 kb

87654321

Human-Specific Gene Conversion(s) betweenSiglec-11 and Siglec Pseudogene P16

Toshi HayakawaTakashi Angata

WithTarjei Mikkelson(Broad Institute)

Hayakawa et al., Science

309: 1693, 2005

HumanHuman Siglec Siglec-11 is the-11 is thefirst first ““Human-specificHuman-specific””

protein!protein!

>99.9%Identity

CD33-related Siglec Gene Cluster

Human-Specific Expression of Siglec-11 in Brain Microglia

8 human brains positive, 5 chimp and 2 orangutans negative

Nissi Varki


309: 1693, 2005




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-11Gene Conversion and Expression Change in Humans

• Human-specific gene conversion by adjacent pseudogene

• First “Human-specific” Protein

• Loss of Neu5Gc binding preference

• New expression in Brain Microglia

• Ligands present in Neurons/Glia


WithTarjei Mikkelson(Broad Institute)


309: 1693, 2005

Figure 1 . Brinkman van der Linden et al. Siglec-6 Expression in Human but not Great Ape Placentae

Els Brinkman Nissi VarkiKurt Benirschke

Fluorescence

Hu

man

Ch

imp

an

zee

Bo

no

bo

Go

rilla

Ora

ng

uta

n

% o

f M

ax

imu

m

Control

Anti-Siglec-6

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Siglec-6 is Expressed on Human and Great Ape B cellsNancy Hurtado-Ziola

Frozen Sections

Sig

lec

-6

1000X

BonoboHuman Chimpanzee

Human Chimpanzee Gorilla

Sig

lec

-6

1000X

Paraffin Sections

Brinkman-Van der Linden, et al. Glycobiology, 17: 922-931 2007




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-6 (OBBP-1)Gene Expression Change in Humans• Human-Specific Placental Expression.

• Sialylated ligands already present in Placenta.

• Enhanced Expression in Labor.

• Siglec-6 also binds Leptin, which also shows increased expression in Labor.

• Is Siglec-6 involved in regulating the tempo of Human Labor?

Els BrinkmanNancy Hurtado-Ziola

withKurt Benirschke

Nissi Varki

Brinkman-Van der Linden, et al.

Glycobiology, 17: 922-931 2007

Human 1 MLLLLLLL--PPLLCGRVGAKEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQNGWTASDPVHGYWFRAGD

Bonobo 1 MLLLLLLLLLPPLLCGRVGAKEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQNGWTDSDPVHGYWFRAGD

Chimp 1 MLLLLLLLLLPPLLCGRVGAKEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQNGWTDSDPVHGYWFRAGD

Gorilla 1 MLLVLLLL--PPLLCGRVGAKEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQNGWTDSDPVHGYWFRAGD

Orang 1 MLLLLLLL--PPLLCGRVGAKEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQDDWTDSDPVHGYWFRAGD

Human 69 HVSRNIPVATNNPARAVQEETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYVFCVERGNMKWNYKYDQLS

Bonobo 71 HVSRNVPVATNNPARAVQEETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYVFRVERGNMKWNYKYDQLS

Chimp 71 HVSRNVPVATNNPARAVQEETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYVFRVERGNMKWNYKYDQLS

Gorilla 69 HVSRNVPVATNNPARAVQEETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYVFRVERGNMKWNYKYDQLS

Orang 69 HVSRNILVATNNPARAVREETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYFFRVERGNIKWSYKHDQLS

• Human Siglec-12 does not recognize sialic acids, due to apoint-mutation changing the “essential arginine”

• The Chimpanzee ortholog (Siglec-12) and “Chimpanized”

(arginine-restored) human one prefer Neu5Gc over Neu5Ac

Angata, T., Varki N.M. and Varki, A J.Biol.Chem. 276:40282-40287, 2001.

The Siglec-12 Arginine MutationIs Human-Specific

TakashiAngata




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-12Human-specific Mutation of Functionally-Important Residue

• Human-specific Mutation of “Essential Arginine” Residue

Required for Optimal Sia recognition.

• Chimpanzee Siglec-12 Preferentially Recognizes Neu5Gc

• Pseudogenization ongoing in Human Populations

Tasha AltheideTakashi AngataNivedita MitraXiaoxia Wang




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-13(Human-specific Gene deletion)

• Human-specific Alu-mediated Deletion of entire Siglec-13 gene.

• Expression patterns in Chimpanzees Under Study

• Biological Roles Unknown

Nivedita MitraNancy Hurtado-Ziola

Takashi AngataNissi Varki

CD22/Siglec-2

Siglec-5 (lo)Siglec-6

Siglec-9 (lo)

Siglec-10

Siglec-7Siglec-9

Siglec-10

Siglec-8

Siglec-10 (lo)

Stem

cell

B cell NK cell Monocyte EosinophilNeutrophil Basophil

Macrophage

CD33Siglec-7

CD33

Siglec-5Siglec-7Siglec-9

Siglec-10 (lo)

Siglec-5

Siglec-9CD22?

Siglec-8 (lo)

Sn/Siglec-1

CD33/Siglec-3Siglec-5Siglec-11

CD33/Siglec-3

CD33CD33

Dendritic

cell

CD8 T cell

subset

Siglec-7(lo)

Siglec-9(lo)

Lymphoidprogenitor

Myeloidprogenitor

Human T cells are Unusual Among Mature Leukocytes in Not Expressing Siglecs

CD4 T cell

None

10

Siglec

1

2

3

3 5 6 78 910

Ch

imp

an

zee

4

5

7

8

9

10

11

12

13

6

14

15

16

1

2

3

Bo

no

bo

12

Go

r.

4

5

1

2

3 5 6 78 9

H1

1

2

3

H2

4

Siglec

1

2H3

1

2H4

1

2H5

3

1

2H63

4H7

1

2H8

% Positive

<2 2-4 4-8 8-25 25-50 >50

Chimp

100

101

102

103

104

FL2-H: CD3

0

20

40

60

80

100

% o

f M

ax

Fluorescence Intensity

100

101

102

103

104

FL2-H

0

20

40

60

80

100

% o

f M

ax

Sig-3

Sig-5

Sig-7

Sig-9

Control

Human

Fluorescence Intensity

High Expression of CD33-related Siglecs on Great Ape

lymphocytes, but not Human Lymphocytes

Dzung NguyenNancy Hurtado-Ziola

withPascal Gagneux

Nguyen. et al. Proc.Nat'l.Acad.Sci.U.S.A. 103: 7765, 2006




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-5Human-specific Expression Change

• Human-specific Suppression of Expression on Lymphocytes

• Possible Explanation for Enhanced Proliferative Response of Human T cells.

• A Role in Human Propensity for T cell-mediated disorders?

Dzung NguyenNancy Hurtado-Ziola

withPascal Gagneux

SIGLEC5* “Pseudogene” is Actually

a Functional Siglec gene: SIGLEC14

Takashi Angata

Alignment of Siglec-5 and Siglec-14

YY

R

514

>99% identity

ITIM

Arg residue in TM domain- recruits DAP-12

Takashi Angata

0.01

Gorilla 14

Gorilla 5Human 14

Human 5

Chimp 14

Chimp 5

Orang 5

Orang 14

Baboon 14

Baboon 5

Repeated Intra-species Gene Conversions between 5 and 14

Molecular phylogenetic tree of SIGLEC5 and SIGLEC14

Actual

Independent gene conversions of

SIGLEC5/14 in each primate lineage

Schematic

• Expected

• Observed

5 14

H HC G O B C G O B

H CG O BH CG O B


Angata, T., et al.FASEB J,

20:1964-73 2006

“Essential Arg” Mutation in Great Ape 5 & 14

Gorilla 14His/Arg

Gorilla 5His

Human 14

Human 5

Chimp 14His

Chimp 5His

Orang 5Tyr

Orang 14Tyr

Baboon 14

Baboon 5

Siglecs with “Essential Arg” mutation

Chimpanzee Siglec-5

WTH119R “reverse” mutant

Angata, T., et al.FASEB J,

20:1964-73 2006




4

Sialoadhesin

CD22

MAG

Y


C2-set domain

Macrophages

B cells Glia

Y

Y

2

Y

Y

3CD33

MonocytesMyeloid

precursors

Y

Y


5

6 7 8 9

OB-BP1AIRM1


PlacentaB cells

NK cellsMonocytes



Y Y Y Y Y

YY

Y Y Y Y

10


B cells

Y

Y

11

Macrophages

Lumenal epithelium

Y Y

Y Y

12

K

15

Y

Y




Y

R

14

Y

?

R

13

Y

? ?

Siglec-14• Ongoing Gene Conversions with Siglec-5

Involving Exons encoding Amino-terminal regions

•Apparent Human-specific Restoration (?) of “Essential Arginine Residue”

required for optimal Sia recognition.

Takashi AngataToshi Hayakawa

Of the <60 Genes Involved in Sialic Biology, 11 so farshow Human-Specific Differences from the “Great Apes”

Was there a “Sialoquake” During Human Evolution?

Varki A. Nature 446: 1023-1029 , 2007


Varki A. Nature 446: 1023, 2007

Hypothesis

Major diseases of a given species are

likely to be related to (mal)adaptations

during the recent evolutionary past of

that species

Corollary

Comparisons of Disease Incidence and

Susceptibility between Humans with our closest

evolutionary relatives should be useful

Apparent Differences between Humans and “Great Apes” in Incidence/Severity ofMedical Conditions - Excluding those Fully Explained by Anatomical Differences

Varki, A. & Altheide, T.K.:Genome Research.15:1746-1758, 2005

**PossiblePossibleExplanations fromExplanations fromUniquely HumanUniquely HumanChanges in Changes in SialicSialic

Acid BiologyAcid Biologyand/or Neu5Gcand/or Neu5GcIncorporationIncorporation

**********

****

**

**

McConkey E & Varki, A. Science 309:1499-1501 (2005)

Varki, A et al. Science 282, 239-240 (1998)

The Need for A “Great Ape Phenome Project”


Varki A. Nature 446: 1023, 2007

ST6GAL1ST6GAL1

SIGLEC7SIGLEC7 SIGLECSIGLEC99

DELETIONAMINO ACID CHANGEEXPRESSION CHANGE

GENE CONVERSION

SIGLEC6SIGLEC6SIGLEC11SIGLEC11SIGLEC5/14SIGLEC5/14SIGLEC13SIGLEC13

CMAHCMAH

SIGLEC12SIGLEC12

SIGLEC1SIGLEC1

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