Essentials of Glycobiology Lecture 23 May 20th 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) Complement Factor H TNF, Interleukins & Cytokines Ameoba lectin Tachylectins Annexins 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 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 C Tyrosine phosphorylation Association with SHP-1S S S C2 S S S V 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 abolished by Sialidase Treatment! S S S S S S S S Fc region Hinge region ss Human IgG Heavy Chain V1 S V C

SIALIC ACIDS AT THE OUTER END OF SUGAR CHAINS ATTACHED TO VERTEBRATE GLYCOCONJUGATES O Ser/Thr N Asn N-LINKED CHAIN O-LINKED CHAIN GLYCOSPHINGOLIPID OUTSIDE INSIDE S CELL MEMBRANE Membrane Protein O Ser/Thr N Asn S = Sialic acid Secreted Protein

CD22 RECOGNIZES N-GLYCANS ON A SUBSET OF SIALOGLYCOPROTEINS OF T- AND B-CELLS DETERGENT LYSATE Sia TARGET CELL LINE SURFACE- OR METABOLICALLY- LABELLED GLYCOPROTEINS SIALIDASE MILD PERIODATE SDS-PAGE STRUCTURAL ANALYSIS SIALIDASE MILD PERIODATE PROTEIN A SEPHAROSE Sia PNGaseF N-GLYCANS Sia 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 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils 4 MAG Glia 3 CD33 Myeloid Precursors Monocytes Macrophages Human Siglecs Sialic acid-recognizing Ig-superfamily lectins) A F G Arg 97 Trp 2 Trp 106 Neu5Ac

Biosynthetic pathways for sialylated chains recognized by mammalian lectins (3)4 Gal  1-(3)4GlcNAc  1- GlcNAc  1- RECOGNIZED BY: CD22 Sn MAG CD33 Selectins + + 3(3)4 Sia  2-3Gal  1-(3)4GlcNAc  1- GalNAc  1-R 3 Gal  1-3GalNAc  1-R Sia  2-3Gal  1-3GalNAc  Sia  2-6GalNAc  1- 3(3)4 Sia  2-3Gal  1-(3)4GlcNAc  1- (4)3 1 Fuc  + Siglec subfamily 64 Sia  2-6Gal  1-4GlcNAc 

10x 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils 4 MAG Glia 3 CD33 Myeloid Precursors Monocytes 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 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils 4 MAG Glia 3 CD33 Myeloid Precursors Monocytes Macrophages Human Siglecs (Sialic acid-binding Ig-superfamily lectins) 5 Neutrophils Monocytes 6 OBBP-1 Placenta B-cells 11 Macro- Phage Subset 10 Monocytes Eosinophils B-cells 9 Monocytes Granulocytes T cell subset 8 SAF-2 Eosinophils 7 AIRM-1 NK cells Monocytes T cell subset CD33(Siglec-3)-related Siglecs ITIM ITAM Putative Tyr-based motif Human Siglecs Sialic acid-recognizing Ig-superfamily lectins)

Alignment of C-terminal Cytoplasmic Tails of CD33-related Siglecs reveals Two Conserved Tyrosine-containing Motifs Proximal motif Classical ITIM Distal motif 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 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) Stem cell Siglec-7 Siglec-9 Siglec-10 NK cell CD16+ CD56+ NK cell CD16++ CD56– Siglec-2 (CD22) Siglec-5 Siglec-6 Siglec-9 Siglec-10 B-cell CD8+ T-cell subset Siglec-7 Siglec-9 Lymphoid progenitor Dendritic cell Macrophage CD33 Siglec-7 Siglec-1 (Sialoadhesin) CD33 Siglec-5, Siglec-11 Siglec-3 (CD33) Myeloid progenitor Neutro- phil Siglec-5 Siglec-9 Siglec-8 Siglec-10 (low) Eosin- ophil Baso- phil CD22? Siglec-8 (low) Monocyte CD33 Siglec-5 Siglec-7 Siglec-9 Siglec-10 (low) Adapted from: Crocker P. and Varki, A. Trends in Immunology 22: 337, 2001

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) CarbonOxygenNitrogen 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 NH 2 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 the Mechanistic 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 Mild periodate oxidation 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 :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? Constitutively Unmasked Siglec expressing cell Ligand expressing cell Masked Cell signalling (e.g., CD22) CD33-related Siglecs? Siglec expressing cell Ligand expressing cell ? i ii Unmasked on activation or exposure to sialidase (e.g., viral infection) Adhesion/Signalling? (e.g., CD22) CD33-related Siglecs? Siglec expressing cell Ligand expressing cell ? iii Reduced Adhesion/Signalling? CD33-related Siglecs? Unmasked on activation or exposure to Sialidase (e.g., viral infection) Siglec expressing cell Virally infected 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 for Intrinsic Receptors Siglecs Factor H Selectins Uterine Agglutinin Laminins INTRINSIC RECEPTOR SIALYLATED GLYCAN = M = Micro-organism/Toxin SELF M EXTRINSIC RECEPTOR Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Ligands for Extrinsic Receptors Biological Roles of Sialic Acids Structural/Physical Roles Molecular Mimicry E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. ?

Possible interactions of Siglecs with Sialylated pathogens Sialoadhesin expressing macrophage Increased pathogen uptake? Decreased pathogen survival? Siglec expressing cell Siglec expressing cell unmasking Decreased immune cell activation? Increased pathogen survival? Sialylated pathogen Adapted from: Crocker P. and Varki, A. Immunology 103: , 2001 Sialic-acid- containing glycan Siglecs Desialylated glycan

“Great Apes” Evolutionary Relationships amongst Humans and the Great Apes 10 5 Millions of Years Before Present * 0 * Precise Timing Uncertain Gorilla gorilla Gorilla Pan paniscus Bonobo Pan troglodytes Chimpanzee MEAN Amino Acid Difference ~0.5% <1.0% H omo sapiens Human Pongo pygmaeus Orangutan Neu5Ac Neu5Gc Genetic Mutation Causing loss Of Neu5Gc

Alignment of Siglec Gene Clusters from 5 Species 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 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

CD83 P0 1 Siglec-3-related subfamily Siglecs Other I-type lectins L1 NCAM ICAM-1 Hemolin CD2 2 Sn CD22 MAG CD33 S2V L1 Ig V-set Ig C2-set FNIII Transmembrane ITAM ITIM putative tyrosine-based motif ? AIRM1 OB-BP1

Tissue distribution and glycan recognition of Non-Siglec I-type lectins CRD not defined in any of these