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
Ligands forIntrinsic
Receptors
SiglecsFactor HSelectins
Uterine AgglutininLamininsL1-CAM
INTRINSIC RECEPTORINTRINSIC RECEPTOR
SIALYLATED GLYCAN = M = Micro-organism/Toxin
SELF
M
EXTRINSIC RECEPTOREXTRINSIC RECEPTOR
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirus
PertussisTetanus etc.
Ligands forExtrinsic
Receptors
Structural/Physical Roles
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosomaStreptococcus
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
Ligands forIntrinsic
Receptors
SiglecsFactor HSelectins
Uterine AgglutininLamininsL1-CAM
INTRINSIC RECEPTORINTRINSIC RECEPTOR
SIALYLATED GLYCAN = M = Micro-organism/Toxin
SELF
M
EXTRINSIC RECEPTOREXTRINSIC RECEPTOR
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirus
PertussisTetanus etc.
Ligands forExtrinsic
Receptors
Structural/Physical Roles
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosomaStreptococcus
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
MonocytesGranulocytes
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
Altheide et al. J.Biol.Chem. 281: 25689-25702, 2006
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
Tasha Altheide & Toshi Hayakawa
Altheide et al. J.Biol.Chem. 281: 25689-25702, 2006
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
Tasha Altheide & Toshi Hayakawa
Altheide et al. J.Biol.Chem. 281: 25689-25702, 2006
V-set domain
C2-set domain
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
MonocytesGranulocytes
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
? ?
Human-Human-SpecificSpecific
Changes!Changes!
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
MonocytesGranulocytes
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
? ?
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
Ligands forIntrinsic
Receptors
SiglecsFactor HSelectins
Uterine AgglutininLamininsL1-CAM
INTRINSIC RECEPTORINTRINSIC RECEPTOR
SIALYLATED GLYCAN = M = Micro-organism/Toxin
SELF
M
EXTRINSIC RECEPTOREXTRINSIC RECEPTOR
InfluenzaMalariaCholera
HelicobacterMycoplasma
RotavirusPolyoma virusCoronavirus
PertussisTetanus etc.
Ligands forExtrinsic
Receptors
Structural/Physical Roles
MolecularMimicry
E.ColiGonococcus
MeningococcusCampylobacterTrypanosomaStreptococcus
Etc.
Biological Roles of Sialic Acids : Molecular Mimicry by Pathogens
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
MonocytesGranulocytes
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
? ?
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.
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
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
Hayakawa et al., Science
309: 1693, 2005
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
MonocytesGranulocytes
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
? ?
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
Toshi HayakawaTakashi Angata
WithTarjei Mikkelson(Broad Institute)
Hayakawa et al., Science
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
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
MonocytesGranulocytes
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
? ?
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
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
MonocytesGranulocytes
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
? ?
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
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
MonocytesGranulocytes
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
? ?
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
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
MonocytesGranulocytes
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
? ?
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
Toshi HayakawaTakashi Angata
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
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
MonocytesGranulocytes
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
? ?
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
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
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”
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
ST6GAL1ST6GAL1
SIGLEC7SIGLEC7 SIGLECSIGLEC99
DELETIONAMINO ACID CHANGEEXPRESSION CHANGE
GENE CONVERSION
SIGLEC6SIGLEC6SIGLEC11SIGLEC11SIGLEC5/14SIGLEC5/14SIGLEC13SIGLEC13
CMAHCMAH
SIGLEC12SIGLEC12
SIGLEC1SIGLEC1