Transcript ssDNA ligands can specifically block trafficking of

Essentials of Glycobiology
Lecture 23
May 20th. 2004
Ajit Varki
The “I-type” Lectins and the Siglecs
Current Classification of Lectins
Families with known protein sequence homologies
Calnexin group (e.g., Calnexin, Calcireticulin, Calmegin)
*”L-type” lectins (e.g., (ERGIC-53 and VIP-36 in ER-Golgi pathway, Plant Lectins
*"P-type" lectins (Mannose-6-Phosphate Receptors)
*"C-type" lectins (e.g., Selectins, Collectins etc.)
* Galectins (formerly "S-type" lectins)
*"I-type" lectins (includes Siglec family)
*”R-type" lectins (e.g., GalNAc-SO4 receptors, Plant Lectins)
“Eglectins” (Frog Egg lectins)
Eel Agglutinins (Fucolectins)
Hyaluronan-binding proteins
Ficolins
Pentraxins
Sequence homologies not known (Examples)
CD11b/CD18 (beta3-integrin, CR3)
TNF, Interleukins & Cytokines
Annexins
Amphoterin
Complement Factor H
Ameoba lectin Tachylectins
*Have defined Carbohydrate
Recognition Domains (CRDs)
“I-type" Lectins
Proteins other than antibodies that can mediate
carbohydrate (glycan) recognition
via immunoglobulin (Ig)-like domains
Siglecs
Sialic Acid Recognizing Ig-superfamily Lectins
The Immunoglobulin Superfamily
• Proteins with Immunoglobulin(Ig)-like domains - phylogenetically
similar to domains of vertebrate antibodies
• Occur in bacteria, but greatly expanded/diversified in animals.
• Ig-like domains traditionally classified into:
•
V-set, related to Variable region of Antibodies
• C1- and C2-set, related to Constant region of Antibodies.
• Many cell adhesion molecules belong to Ig superfamily
• Until 1990s, Ig superfamily thought to mediate Protein:Protein
interactions, but never Protein:Glycan interactions
CD22 - a B Cell specific cell surface molecule
SS
Ig Superfamily member
Found Primarily on
mature resting
sIgD Positive B Cells
1
1
2 C2
3 C2
4
Gene disruption gives
Altered B cell
responses to antigens
SS
SS
V
V
1
S
S
S
S
2 C2
S
S
S
S
C2
2
3 C2
S
S
S
S
C2
3
C2SS
5 C2
S
S
6 C2
S
S
7
Tyrosine
phosphorylation
Association with SHP-1
V
N
ss
S
S
S
S
S
S
S
S
Hinge
region Human
IgG
Fc
Heavy
region Chain
CD22-Fc : A TOOL FOR
STUDYING CD22 FUNCTION
C2SS
C
Binding to Cells abolished
by Sialidase Treatment!
SIALIC ACIDS AT THE OUTER END OF SUGAR CHAINS
ATTACHED TO VERTEBRATE GLYCOCONJUGATES
= Sialic acid
S
N-LINKED CHAIN
O
Ser/Thr
N
Asn
O-LINKED CHAIN
GLYCOSPHINGOLIPID
Secreted Protein
S
O
Ser/Thr
N
Asn
Membrane Protein
OUTSIDE
CELL
MEMBRANE
INSIDE
CD22 RECOGNIZES N-GLYCANS ON A SUBSET
OF SIALOGLYCOPROTEINS OF T- AND B-CELLS
s
s
CD22ß
CD22ß
SURFACE- OR METABOLICALLYLABELLED GLYCOPROTEINS
Sia
Sia
DETERGENT
LYSATE
SIALIDASE
MILD PERIODATE
TARGET CELL LINE
PROTEIN A
SEPHAROSE
s
s
CD22ß
CD22ß
Sia
Sia
PNGaseF Sia
Sia
N-GLYCANS
SIALIDASE
MILD PERIODATE
SDS-PAGE
s
s
CD22ß
CD22ß
X
STRUCTURAL
ANALYSIS
RECOGNITION OF SIALYLATED GLYCANS BY CD22
• Reproducible upon re-chromatography
• Destroyed by sialidase
• Requires a
linked sialic acids
• Affected by the number and location of a
-linked Sias
• Unaffected by sialic acids in other linkages e.g., a
3-linked
• Destroyed by truncation of sialic acid side chains
• Does not require the tri-mannosyl core of N-glycans
• Identical results with natural and semi-synthetic glycans
• Kd for monovalent association of Siaa
• Kd for divalent association of Sia
Galb -4Glc =
Gal
-4Glc = 1 m
m
Sialoadhesin (Sn)
• Originally discovered by Crocker and Gordon as a lectin-like
activity expressed on specific macrophage subpopulations and
capable of binding to red blood cells
• Binding abolished by treatment of red cells with sialidase.
• Purified Sn from macrophages had Mr of 185kDa - adhered in sialic
acid dependent manner to various lymphohematopoietic cells.
• Found concentrated in vivo at sites of interaction between
macrophages and blood cells
• Purified Sn shown to directly recognize certain glycolipids and
glycoproteins in a sialic acid-dependent manner
Human Siglecs
Sialic acid-recognizing Ig-superfamily lectins)
Trp2
G
Neu5Ac
A
Trp106
Arg97
F
10x
1
2
4
3
Sialoadhesin
CD22
MAG
CD33
Macrophage
Subset
B cells
?Basophils
Glia
Myeloid
Precursors
Monocytes
Macrophages
V-set domain
C2-set domain
Biosynthetic pathways for sialylated chains recognized by mammalian lectins
GlcNAcb1Siaa2-6Galb1-4GlcNAcb1-
CD22
RECOGNIZED BY:
Sn MAG CD33 Selectins
+
+
Galb1-(3)4GlcNAcb1-
+
Siaa2-3Galb1-(3)4GlcNAcb1-
+
Siaa2-3Galb1-(3)4GlcNAcb1(4)3
1
Fuca
+
+
Siaa2-3Galb1-3GalNAcba1-
+
Galb1-3GalNAcba1-R
GalNAcba1-R
Siaa2-6GalNAcba1-
+
Siglec subfamily
+
Myelin-Associated Glycoprotein (MAG) / Siglec-4
•Most highly conserved Siglec, from fish to man. Rat
and human MAG sequences 96% identical, with only
two differences in first 183 amino acids
•Mediates cell-cell interactions between myelinating
glial cells and neurons in CNS and PNS
•Implicated in formation and maintenance of myelin
10x
1
2
Sialoadhesin
CD22
Macrophage
Subset
B cells
?Basophils
•V-set domain recognizes a2-3-linked Sias on Oglycans or glycolipids - with extended binding site
•MAG null mutants have defects in initiation of CNS
myelination, formation of intact myelin
sheaths and to a minor extent, integrity
of
3 myelin. In the PNS, only maintenance
4
of myelin is impaired.
CD33
MAG
•Glycosyltransferase KO eliminating
Glia
Myeloid
glycolipid ligands gives similar phenotype.
Precursors
Monocytes
Macrophages
Human
Siglecs
Human
Siglecs
Sialic acid-recognizing
lectins)
(Sialic acid-bindingIg-superfamily
Ig-superfamily lectins)
V-set domain
ITIM
ITAM
C2-set domain
Putative Tyr-based motif
10x
CD33(Siglec-3)-related Siglecs
1
2
4
3
Sialoadhesin
CD22
MAG
CD33
Macrophage
Subset
B cells
?Basophils
5
6
7
8
OBBP-1
AIRM-1
SAF-2
Neutrophils
Monocytes
Glia
Myeloid
Precursors
Monocytes
Macrophages
NK cells
Monocytes
T cell subset
Placenta
B-cells
Eosinophils
9
10
Monocytes
Granulocytes
T cell
Monocytes
subset
Eosinophils
B-cells
11
MacroPhage
Subset
Alignment of C-terminal Cytoplasmic Tails of CD33-related
Siglecs reveals Two Conserved Tyrosine-containing Motifs
Adapted from: Crocker P. and Varki, A.
Trends in Immunology 22: 337, 2001
Siglec-9
Siglec-8
Siglec-7
Siglec-6
Siglec-5
Siglec-3(CD33)
Siglec-10
ASARSSVGEGELQYASLSFQMVKPWDSR-GQEATDTEYSEIKIHR-----------------AVAPSSGEEGELHYATLSFHKVKPQDPQ-GQEATDSEYSEIKIHKRETAETQACLRNHNPSSK
LAAHSSGEEREIQYAPLSFHKGEPQDLS-GQEATNNEYSEIKIPK-----------------EAGPISEDEQELHYAVLHFHKVQPQEP----KVTDTEYSEIKIHKDRLWE------------GDAPPLEEQKELHYASLSFSEMKSREPKDQEAPSTTEYSEIKTSK-----------------GAAPTVEMDEELHYASLNFHGMNPSKD------TSTEYSEVRTQ------------------QAPESQESQEELHYATLNFPGVRPRPEARMPKGTQADYAEVKFQ------------------Proximal motif
Classical ITIM
Immunoreceptor Tyrosine-based Inhibition Motif
(Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val)
Phosphorylation by src-family kinases
Recruitment of tyrosine phosphatases
SHP-1 and SHP-2,
sometimes also inositol phosphatase SHIP
Distal motif
Similar to (Thr-Ile-Tyr-X-X-(Val/Ile) in
SLAM (signalling lymphocyte activation
molecule) and SLAM-related proteins
that bind SAP (SLAM-associated
protein), a T-cell-specific molecule that
inhibits SHP-2 recruitment to SLAM
Distribution of Human Siglecs in the Hematopoietic System
(based on incomplete data available to date)
Stem
cell
Adapted from: Crocker P. and Varki, A.
Trends in Immunology 22: 337, 2001
Myeloid
Siglec-3 (CD33)
progenitor
Lymphoid
progenitor
NK cell
CD16++
CD56–
CD8+
T-cell
subset
B-cell
Siglec-7
Siglec-9
Siglec-9
Siglec-10
Siglec-2 (CD22)
Siglec-5
Siglec-6
Siglec-9
Siglec-10
NK cell
CD16+
CD56+
Siglec-7
Siglec-9
Dendritic
cell
CD33
Siglec-7
Monocyte
Neutrophil
CD33
Siglec-5
Siglec-7
Siglec-9
Siglec-10
(low)
Siglec-5
Siglec-9
Eosinophil
Basophil
CD22?
Siglec-8
Siglec-10 Siglec-8
(low)
(low)
Macrophage
Siglec-1
(Sialoadhesin)
CD33
Siglec-5, Siglec-11
Structural Features of Sialic Acids Involved
in Recognition by Siglecs
SIDE CHAIN (REQUIRED
FOR BINDING OF MOST)
9
7
1
8
6
2
5
3
4
Oxygen
CLEAVAGE SITE FOR
SIALIDASE
(NEURAMINIDASE)
LINKAGE TO UNDERLYING
SUGAR CHAIN (RELATIVE OR
ABSOLUTE PREFERENCE)
5--N-ACYL-GROUP
(AFFECTS BINDING OF SOME)
Carbon
-
NEGATIVELY CHARGED
CARBOXYLATE (REQUIRED)
Nitrogen
Hydrogen
A Generic Siglec
NH2
Ig V-set domain (Sialic Acid Binding site)
Ig C2-set domain(s): 1 to 17
TM
What exactly is the
Mechanistic Connection?
(S/I/L/VxYxxL/V)
ITIM Motif (Tyrosine kinase Target site)
Tyrosine Phosphatase Binding site
Tyrosine kinase target?SAP Binding Site?
(TxYxxI/V)
COOH
Most Siglecs on Cell Surfaces are “masked” but can be
uncovered by removing or altering cell surface sialic acids
P-B: (Sialyl-LacNAc)n-PAA-Biotin
P-B
Sialidase
Mild
periodate
oxidation
P-B
“Unmasking” can also occur spontaneously during
cellular activation,via as yet unknown mechanisms
Three Possible Models for Functions of CD22-Sialic Acid
Interactions (Collins et al Glycobiology. 2002 12:563-71)
Adapted from:
Crocker P. and
Varki, A.
Trends in
Immunology
22: 337, 2001
How I nteractions of Siglecs with other cells might be affected
by cis-interactions with sialylated glycoconjugates
ii
i
Ligand
expressing
cell
iii
Ligand
expressing
cell
iv
Ligand
expressing
cell
Virally
infected
cell
(eg influenza)
Siglecs
?
Sialic-acidcontaining
glycan
Desialylated
glycan
Siglec
expressing
cell
Constitutively
Unmasked
Siglec
expressing
cell
Masked
?
?
Siglec
expressing
cell
Siglec
expressing
cell
Unmasked on
activation or
exposure to
sialidase
(e.g., viral infection)
Unmasked on
activation or
exposure to
Sialidase
(e.g., viral infection)
Cell-cell interactions
Cell signalling
Adhesion/Signalling?
Reduced
(e.g., sialoadhesin)
(e.g., CD22)
(e.g., CD22)
Adhesion/Signalling?
CD33-related Siglecs?
CD33-related Siglecs?
CD33-related Siglecs?
CD33-related Siglecs?
Biological Roles of Sialic Acids
Structural/Physical Roles
Siglecs
Factor H
Selectins
Uterine Agglutinin
Laminins
Ligands for
Intrinsic
Receptors
Influenza
Malaria
Cholera
Helicobacter
Mycoplasma
Rotavirus
Polyoma virus
Coronavirus
Pertussis
Tetanus etc.
Ligands for
Extrinsic
Receptors
EXTRINSIC RECEPTOR
SELF
SELF
M
INTRINSIC RECEPTOR
SIALYLATED GLYCAN =
?
M = Micro-organism/Toxin
Molecular
Mimicry
E.Coli
Gonococcus
Meningococcus
Campylobacter
Trypanosoma
Streptococcus
Etc.
Possible interactions of Siglecs with Sialylated pathogens
Adapted from:
Crocker P. and Varki, A.
Immunology 103: 137-145, 2001
Sialylated pathogen
Siglecs
Sialic-acidcontaining
glycan
unmasking
Sialoadhesin
expressing
macrophage
Desialylated
glycan
Increased pathogen uptake?
Decreased pathogen survival?
Siglec
expressing
cell
Siglec
expressing
cell
Decreased immune cell activation?
Increased pathogen survival?
Millions of Years Before Present*
MEAN
Amino Acid
Difference
0
5
<1.0%
Neu5Ac
Homo sapiens
Human
~0.5%
“Great Apes”
Neu5Gc
Gorilla gorilla
Pan paniscus Pan troglodytes
Bonobo
Chimpanzee
Gorilla
Pongo pygmaeus
Orangutan
Genetic
Mutation
Causing loss
Of Neu5Gc
10
*Precise Timing
Uncertain
Evolutionary
Relationships
amongst Humans
and the
Great Apes
Alignment of Siglec Gene Clusters from 5 Species
KLK gene c luster
2
4
5
6 7
11
8 9 10 12
Siglec gene cluster
P1
13 14
P2
9
P3 P4 P5 P6
7
P7 P8
P9 P10
NKG7
ETFB LIM2 10
3
P11
8
XII
P12 P13
6
ZNF175
5
FPR1
FPRL1
5*
HAS1
V 5*
HAS1
Human
2
4
P1
5 6 7
11
8 910 12
13 14
9
6 7
11
8 9 10 12
13 14
9
P2
7
P3
P8
P9 P10
NKG7
ETFB LIM2 10
3
13
P11
8
12
P12 P13
6
ZNF175
FPR1
FPRL1
Chimpanzee
P7 P8
NKG7
ETFB
LIM2
3
P10
10
13
8
Px
P12 P13
VI
ZNF175 5
5*
HAS1
FPR1
FPRL1
Baboon
4
5
11
6 7 8910 12
13
14
E
14
E
NKG7
PA
ETFB LIM2 G
3
F
PB
ZNF-like
Mouse
11
5 6 7 8910 12
13
3
NKG7
PA
ETFB LIM2 G
F
Rat
100 Kb
Kallikrein-like Gene
Siglec or Siglec-like Gene
Siglec Pseudogene
Other Genes
Kallikrein-Like (KLK) Genes are Highly Conserved but Siglec Genes are not
Rodent Siglec Clusters have fewer Genes and Pseudogenes and
are generally more conserved
Human-Specific Features in the Siglec Gene Cluster
KLK gene c luster
2
4
5
6 7
Siglec gene cluster
P1
11
8 9 10 12
13 14
P2
9
P3 P4 P5 P6
7
P7 P8
P9 P10
NKG7
ETFB LIM2 10
3
P11
8
XII
P12 P13
6
ZNF175
5
FPR1
FPRL1
5*
HAS1
V 5*
HAS1
Human
2
4
P1
5 6 7
11
8 910 12
13 14
9
6 7
11
8 9 10 12
13 14
9
P2
7
P3
P8
P9 P10
NKG7
ETFB LIM2 10
3
13
P11
8
P12 P13
12
6
ZNF175
FPR1
FPRL1
Chimpanzee
P7 P8
NKG7
ETFB
LIM2
3
P10
10
13
Px
8
P12 P13
VI
ZNF175 5
5*
HAS1
FPR1
FPRL1
Baboon
4
5
11
6 7 8910 12
13
14
E
14
E
NKG7
PA
ETFB LIM2 G
3
F
PB
ZNF-like
Mouse
11
5 6 7 8910 12
13
3
NKG7
PA
ETFB LIM2 G
F
Rat
100 Kb
Kallikrein-like Gene
Siglec or Siglec-like Gene
Siglec Pseudogene
Other Genes
Deletion of Siglec-13
Largest Number of Pseudogenes
“Essential Arginine” Mutation in Siglec-XII
Probable Orthologous Correspondences of CD33-related Siglecs
Human
Chimpanzee
Baboon
Mouse
Rat
CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3
Siglec-5
*Siglec-V
Siglec-5
Siglec-F
Siglec-F
Siglec-6
Siglec-6
*Siglec-VI
Siglec-7
Siglec-7
NF
NF
NF
Siglec-8
Siglec-8
Siglec-8
NF
NF
Siglec-9
Siglec-9
Siglec-9
Siglec-E
Siglec-E
Siglec-10
Siglec-10
Siglec-10
Siglec-G
Siglec-G
#Siglec-11
#Siglec-11
?
NF
NF
*Siglec-XII
Siglec-12
NF
NF
NF
NF
Siglec-13
Siglec-13
NF
NF
NF
?
?
#Siglec H
*#Siglec H
Based on published and on-line data as of Dec 2003
Criteria for ortholog assignment include sequence similarity of amino-terminal V-set
domains, map location, gene structure, and phylogenetic relationships.
NF: Corresponding V-Set domain not found in available data on this species.
* Siglec-like molecules missing Arg residue required for optimal Sia binding.
# Located outside CD33-related Siglecs gene cluster.
? Published genomic information not sufficient to definitively determine status
Siglecs
1 Sn
Other I-type lectins
Ig V-set
ITAM
ITIM
putative tyrosine-based motif
Ig C2-set
FNIII
Transmembrane
CD22
L1
NCAM
Siglec-3-related subfamily
2
ICAM-1
MAG
4
AIRM1
5 OB-BP1
CD33
6
7
8
3
10
9
Hemolin
11 S2V
L1
CD2
CD83
P0
?
Tissue distribution and glycan recognition of
Non-Siglec I-type lectins
Name
CD83
L1
NCAM
(CD56)
P0
ICAM-1
(CD54)
CD2
Hemolin
Tissue distribution
Dendritic cells
Glycan recognized
Sia on monocytes and
HPB-ALL cells (T cell lin e)
Neurons, Glial cells, CD4+ T cells, *Alpha2-3 Sia on CD24
Monocytes, B cells
Neurons, Muscles, NK cells
High mannose N-linked on L1(cis)
N-linked (phosphacan)
Schwann cells
Hybrid/complex N-linked
*HNK-1 on P0 (homophilic)
Endothelial cells, Lymphocytes, *Hyaluronan, CD43
Monocytes
T cells, NK cells
N-glycans with fucose (maybe
related to Lewis X)
Hemolymph, Hemocytes of
GlcNAc and/or Sia containing
insects
glycans?
*Evidence for Glycan-binding Definitive
CRD not defined in any of these