Essentials of Glycobiology Lecture 23 May 20th. 2004 Ajit Varki

Essentials of Glycobiology Lecture 23

May 20th. 2004

Ajit Varki

The “I-type” Lectins and the Siglecs

Current Classification of LectinsFamilies 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) Complement Factor HTNF, Interleukins & Cytokines Ameoba lectin TachylectinsAnnexins

Amphoterin*Have defined Carbohydrate Recognition Domains (CRDs)

“I-type" Lectins

Proteins other than antibodies that can mediate carbohydrate (glycan) recognition

via immunoglobulin (Ig)-like domains

SiglecsSialic 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

C

Tyrosine phosphorylation

Association with SHP-1

SS

SS

5

6

7 SS

C2

C2

SS

SS

SS

V

C2

3

4

C2

1

2

C2

S S

C2

Ig Superfamily member Found Primarily on

mature resting sIgD Positive B Cells

Gene disruption gives Altered B cell

responses to antigens

N

CD22-Fc : A TOOL FOR STUDYING CD22 FUNCTION

Binding to Cells abolishedby Sialidase Treatment!

SS

SS

SS

SS

SS

SS

SS

SS

Fc region

Hinge region

ssHuman

IgGHeavy Chain

V1

S S

V

S S

C2

3 C2

2 C2

3C2

2

1

SIALIC ACIDS AT THE OUTER END OF SUGAR CHAINS ATTACHED TO VERTEBRATE GLYCOCONJUGATES

OSer/Thr

NAsn

N-LINKED CHAINN-LINKED CHAIN

O-LINKED CHAINO-LINKED CHAIN

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

OUTSIDE

INSIDE

S

CELLMEMBRANE

Membrane ProteinMembrane Protein

OSer/Thr

NAsn

S

= Sialic acid

Secreted ProteinSecreted Protein

CD22 RECOGNIZES N-GLYCANS ON A SUBSET OF SIALOGLYCOPROTEINS OF T- AND B-CELLS

CD22ß

CD22ß

ss DETERGENT

LYSATESiaSia

TARGET CELL LINE

SURFACE- OR METABOLICALLY-LABELLED GLYCOPROTEINS

SIALIDASE MILD PERIODATE

SDS-PAGE STRUCTURAL ANALYSIS

SIALIDASE MILD PERIODATE

CD22ß

CD22ß

ss

CD22ß

CD22ß

ss

PROTEIN ASEPHAROSE

SiaSia

PNGaseF N-GLYCANSSiaSia

X

RECOGNITION OF SIALYLATED GLYCANS BY CD22

• Reproducible upon re-chromatography

• Destroyed by sialidase

• Requires linked sialic acids

• Affected by the number and location of -linked Sias

• Unaffected by sialic acids in other linkages e.g., 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 Sia Gal -4Glc =

• Kd for divalent association of Sia Gal-4Glc = 1

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

10x

V-set domain

C2-set domain

1Sialoadhesin

MacrophageSubset

2CD22

B cells?Basophils

4

MAG

Glia

3CD33

MyeloidPrecursorsMonocytes

Macrophages

Human SiglecsSialic acid-recognizing Ig-superfamily lectins)

A

F

G

Arg97

Trp2

Trp106

Neu5Ac

Biosynthetic pathways for sialylated chains recognized by mammalian lectins

Gal1-(3)4(3)4GlcNAc1-

GlcNAc1- RECOGNIZED BY: CD22 Sn MAG CD33 Selectins

+ + Sia2-33Gal1-(3)4(3)4GlcNAc1-

GalNAc1-R

Gal1-33GalNAc1-R

+ + +Sia2-33Gal1-33GalNAc1-

+

Sia2-66GalNAc1-

Sia2-33Gal1-(3)4(3)4GlcNAc1- (4)3(4)3 1Fuc

+

Siglec subfamily

Sia2-66Gal1-44GlcNAc1- +

+

10x

1Sialoadhesin

MacrophageSubset

2CD22

B cells?Basophils

4

MAG

Glia

3CD33

MyeloidPrecursorsMonocytes

Macrophages

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•V-set domain recognizes 2-3-linked Sias on O-glycans 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 myelin. In the PNS, only maintenance of myelin is impaired. •Glycosyltransferase KO eliminating glycolipid ligands gives similar phenotype.

10x

V-set domain

C2-set domain

1Sialoadhesin

MacrophageSubset

2CD22

B cells?Basophils

4

MAG

Glia

3CD33

MyeloidPrecursorsMonocytes

Macrophages

Human Siglecs (Sialic acid-binding Ig-superfamily lectins)

5

NeutrophilsMonocytes

6

OBBP-1

PlacentaB-cells

11

Macro-PhageSubset

10

MonocytesEosinophils

B-cells

9

MonocytesGranulocytes

T cellsubset

8SAF-2

Eosinophils

7AIRM-1

NK cellsMonocytes

T cell subset

CD33(Siglec-3)-related Siglecs

ITIMITAM Putative Tyr-based motif

Human SiglecsSialic acid-recognizing Ig-superfamily lectins)

Alignment of C-terminal Cytoplasmic Tails of CD33-related Siglecs reveals Two Conserved Tyrosine-containing Motifs

Proximal motifClassical ITIM

Distal motif

Immunoreceptor Tyrosine-based Inhibition Motif(Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val)

Phosphorylation by src-family kinasesRecruitment of tyrosine phosphatases

SHP-1 and SHP-2, sometimes also inositol phosphatase SHIP

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

Adapted from: Crocker P. and Varki, A. Trends in Immunology 22: 337, 2001

Distribution of Human Siglecs in the Hematopoietic System(based on incomplete data available to date)

Stemcell

Siglec-7Siglec-9

Siglec-9Siglec-10

NK cellCD16+CD56+

NK cellCD16++CD56–

Siglec-2 (CD22)Siglec-5Siglec-6Siglec-9Siglec-10

B-cell

CD8+ T-cellsubset

Siglec-7Siglec-9

Lymphoidprogenitor

Dendriticcell

Macrophage

CD33Siglec-7

Siglec-1(Sialoadhesin)

CD33Siglec-5, Siglec-11

Siglec-3 (CD33)Myeloidprogenitor

Neutro-phil

Siglec-5Siglec-9 Siglec-8

Siglec-10 (low)

Eosin-ophil

Baso-phil

CD22?Siglec-8

(low)

Monocyte

CD33Siglec-5Siglec-7Siglec-9

Siglec-10 (low)

Adapted from: Crocker P. and Varki, A. Trends in Immunology 22: 337, 2001

1122

3344

55

6677

8899 --

CLEAVAGE SITE FOR SIALIDASE (NEURAMINIDASE)

LINKAGE TO UNDERLYING SUGAR CHAIN (RELATIVE OR

ABSOLUTE PREFERENCE)

Structural Features of Sialic Acids Involved in Recognition by Siglecs

NEGATIVELY CHARGED CARBOXYLATE (REQUIRED)

Carbon Oxygen Nitrogen Hydrogen

5--N-ACYL-GROUP (AFFECTS BINDING OF

SOME)

SIDE CHAIN (REQUIRED FOR BINDING OF MOST)

A Generic Siglec

TM

Ig C2-set domain(s): 1 to 17

NH2

COOH

Ig V-set domain (Sialic Acid Binding site)

ITIM Motif (Tyrosine kinase Target site) Tyrosine Phosphatase Binding site

(S/I/L/VxYxxL/V)

Tyrosine kinase target?SAP Binding Site?(TxYxxI/V)

What exactly is theMechanistic Connection?

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

Mildperiodateoxidation

P-B

Sialidase

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)

How Interactions of Siglecs with other cells might be affected by cis-interactions with sialylated glycoconjugates

Cell-cell interactions(e.g., sialoadhesin)

CD33-related Siglecs?

ConstitutivelyUnmasked

Siglecexpressing

cell

Ligandexpressing

cell

Masked

Cell signalling(e.g., CD22)

CD33-related Siglecs?

Siglecexpressing

cell

Ligandexpressing

cell

?

i ii

Unmasked onactivation orexposure to

sialidase (e.g., viral infection)

Adhesion/Signalling?(e.g., CD22)

CD33-related Siglecs?

Siglec expressing

cell

Ligandexpressing

cell

?

iii

Reduced Adhesion/Signalling?

CD33-related Siglecs?

Unmasked onactivation orexposure to

Sialidase (e.g., viral infection)

Siglec expressing

cell

Virallyinfected

cell(eg influenza)

iv

?Sialic-acid-containing glycan

Siglecs

Desialylated glycan

Adapted from: Crocker P. and

Varki, A. Trends in

Immunology 22: 337, 2001

SELF

Ligands forIntrinsic

Receptors

SiglecsFactor HSelectins

Uterine AgglutininLaminins

INTRINSIC RECEPTORINTRINSIC RECEPTOR

SIALYLATED GLYCAN = M = Micro-organism/Toxin

SELF

M

EXTRINSIC RECEPTOREXTRINSIC RECEPTOR

InfluenzaMalariaCholera

HelicobacterMycoplasma

RotavirusPolyoma virusCoronavirus

Pertussis Tetanus etc.

Ligands for ExtrinsicReceptors

Biological Roles of Sialic AcidsStructural/Physical Roles

MolecularMimicry

E.ColiGonococcus

MeningococcusCampylobacterTrypanosomaStreptococcus

Etc.

?

Possible interactions of Siglecs with Sialylated pathogens

Sialoadhesinexpressing

macrophage

Increased pathogen uptake?Decreased pathogen survival?

Siglecexpressing

cell

Siglec expressing

cell

unmasking

Decreased immune cell activation?Increased pathogen survival?

Sialylated pathogen

Adapted from: Crocker P. and Varki, A. Immunology 103: 137-145, 2001

Sialic-acid-containing glycan

Siglecs

Desialylated glycan

“Great Apes”

Evolutionary Relationships

amongst Humans and the

Great Apes

10

5

Mill

ions

of Y

ears

Bef

ore

Pres

ent* 0

*Precise Timing Uncertain

Gorilla gorillaGorilla

Pan paniscusBonobo

Pan troglodytes Chimpanzee

MEAN Amino AcidDifference

~0.5%<1.0%

Homo sapiens Human

Pongo pygmaeusOrangutan

Neu5Ac Neu5Gc

Genetic Mutation

Causing lossOf Neu5Gc

Alignment of 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 P138 6 5 5* HAS1

FPR1FPRL1ZNF175

Px

6 7 8 91011

12 13 14 9 7 3P1 P2 P3 P8

ETFBNKG7

LIM2 10 12P9P10 P11 P12 P138 6 V 5* HAS1

FPR1FPRL1ZNF175

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 Clusters have fewer Genes and Pseudogenes and are generally more conserved

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

Human-Specific Features in the Siglec Gene Cluster

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 P138 6 5 5* HAS1

FPR1FPRL1ZNF175

Px

6 7 8 91011

12 13 14 9 7 3P1 P2 P3 P8

ETFBNKG7

LIM2 10 12P9P10 P11 P12 P138 6 V 5* HAS1

FPR1FPRL1ZNF175

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

Deletion of Siglec-13

Largest Number of Pseudogenes“Essential Arginine” Mutation in Siglec-XII

Probable Orthologous Correspondences of CD33-related Siglecs

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

Human Chimpanzee Baboon Mouse RatCD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3 CD33/Siglec-3

Siglec-5 *Siglec-V Siglec-5Siglec-6 Siglec-6 *Siglec-VI Siglec-F Siglec-F

Siglec-7 Siglec-7 NF NF NFSiglec-8 Siglec-8 Siglec-8 NF NFSiglec-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 NFNF ? ? #Siglec H *#Siglec H

CD83 P0

1

Siglec-3-related subfamily

Siglecs Other I-type lectins

L1

NCAM

ICAM-1Hemolin

CD2

2

Sn

CD22

MAG

CD33

S2V 4

3

5

11

9 8 7 6

10L1

Ig V-setIg C2-setFNIIITransmembrane

ITAMITIMputative tyrosine-based motif

?

AIRM1OB-BP1

Tissue distribution and glycan recognition of Non-Siglec I-type lectins

Name Tissue distribution Glycan recognizedCD83 Dendritic cells Sia on monocytes and

HPB-ALL cells (T cell line)L1 Neurons, Glial cells, CD4+ T cells,

Monocytes, B cells*Alpha2-3 Sia on CD24

NCAM(CD56)

Neurons, Muscles, NK cells High mannose N-linked on L1(cis)N-linked (phosphacan)

P0 Schwann cells Hybrid/complex N-linked*HNK-1 on P0 (homophilic)

ICAM-1(CD54)

Endothelial cells, Lymphocytes,Monocytes

*Hyaluronan, CD43

CD2 T cells, NK cells N-glycans with fucose (mayberelated to Lewis X)

Hemolin Hemolymph, Hemocytes ofinsects

GlcNAc and/or Sia containingglycans?

*Evidence for Glycan-binding Definitive

CRD not defined in any of these