Essentials of Glycobiology May 14, 2002 Jeff Esko Bacterial and viral carbohydrate-binding proteins

Types of Carbohydrate-Binding Proteins Glycosyltransferases and modifying enzymes Antibodies induced by carbohydrate antigens Plant Lectins: Con A, PHA, WGA, and many others Animal Lectins: P, C, S, R, L, and I-type, chaperone proteins Glycosaminoglycan-binding proteins Bacterial adhesins Viral hemagglutinins

Overview 1. Bacterial adhesion - adhesins and receptors 2. Binding and attachment to glycolipids 3. Measuring bacterial binding and adhesion 4. Toxins 5. Binding to glycoproteins 6. Binding to glycosaminoglycans 7. Host-responses 8. Therapeutic strategies

To colonize tissues and cause infection, microbes must first adhere Epithelial Cell Microb e Lining of gastrointestinal, urinary, respiratory, or reproductive tract Mucin Microbe

 Adhesion occurs in a tissue specific manner due to specific CHO-protein interactions  Can also help establish the normal flora (e.g., commensal or symbiotic bacteria in the gastrointestinal tract)  Colonization can lead to infection (e.g., uropathogenic infections) Tropism: Modification of the direction of growth

Adhesins: Proteins that Mediate Adhesion  Many adhesins are lectins  Some bind to terminal sugars, others bind to internal carbohydrate sequences  Most microorganisms express more than one type of adhesin Adhesins

Bacterial Adhesins  Many bacterial adhesins are found on pili (hairs) or fimbrae (threads)  Velcro effect due to lateral mobility of adhesin in membrane

Pilus: An organelle Adhesin subunit (G) usually found on the tip Sauer et al. (2000) COSB 10:548

Receptors Animal cells express “receptors” (carbohydrate ligands) for adhesins Receptors can be glycolipids, glycoproteins, or proteoglycans Tissue tropism is determined by the array of adhesin-receptor pairs Bacterium

Choudhury et al. (1999) Science 285:1061 FimH

Measuring Adhesin-Receptor Interactions Hemagglutination: Direct Binding: Use mutant cells or nutritionally manipulate composition Competition experiments with soluble carbohydrates Remove receptor with exoglycosidases Regenerate different receptor with glycosyltransferase. Bacteria Binding

Binding Measurements  Overlay methods: Challenge microorganisms to bind immobilized carbohydrate receptors  Can use tissue sections, TLC plates, PAGE blots  Using a centrifuge, you can measure the strength of binding in g-force

Cholera Toxin Binds to GM 1  Many bacteria secrete exotoxins that bind to carbohydrates  Cholera toxin consists of two types of subunits, AB 5, which bind to ganglioside GM 1

Glycosphingolipid Receptors for Toxins

Toxins A and B from Clostridium difficile (antibiotic- associated diarrhea, pseudomembranous colitis) Hemorrhagic and lethal toxins of C. sordellii and  - toxin of C. novyi (enterotoxemia and gas gangrene) These toxins turn out to be glucosyltransferases Large Clostridial Cytotoxins BindingCatalyticTranslocation

Modification of target proteins by glucosylation Targets include Rho (cytoskeletal organization), Ras (growth control), Rac, cdc42 and other GTPases Large Clostridial Cytotoxins Busch & Aktories (2000) COSB 10:528

Microbial Binding to Glycoproteins Glycoprotein glycans are displaced from the membrane compared to glycolipids, which may make it less effective as a receptor O Ser/Thr N Asn N-LINKED CHAIN O-LINKED CHAIN GLYCOSPHINGOLIPID OUTSIDE INSIDE S = Sialic acid CELL MEMBRANE

Microbial Binding to Glycoproteins  Entamoeba binds to terminal Gal residues on glycoproteins. –Residence in intestinal epithelium and perhaps it’s a food source  Trypanosoma cruzi produces a trans-sialidase that transfers sialic acid from serum proteins to glycoproteins on its own surface –Trans-sialidase could also act as lectin –After trans-sialylation, T. cruzi might bind to sialic acid binding proteins of the host (e.g., siglecs)

Influenza Virus Viral envelope contains a sialic acid hemagglutinin and a neuraminidase (sialidase)

Microbial Binding to Glycoproteins  Hemagglutinin binds to sialic acids on glycoproteins (crystal structure) –Flu A binds to  2,6 sialic acids –Flu B binds to  2,3 sialic acids –Flu C prefers 9-O-acetylated sialic acids  Neuraminidases coevolve with hemagglutinin –Dissociation of virus after budding –Prevents aggregation of virus –Facilitate spread of virus by modifying mucin

GAG-Binding Adhesins

Herpes Simplex Entry Herpes simplex uses heparan sulfate as a coreceptor, infection requires both proteoglycan and a protein receptor of the HVE class Fusion of the viral envelope with the host membrane also requires heparan sulfate and other viral proteins

Flavivirus Adhesin Model Example of a relatively non-specific binding site, which interacts with many heparan sulfate sequences with variable affinity

FMDV Depression that defines binding site for heparin is made up of segments from all three major capsid proteins Fry et al. (1999) EMBO J 18:543

Cellular Responses to Binding  Fusion reactions —Herpes simplex virus (HSV) fuses with the plasma membrane —Influenza virus fuses with the endosomal membrane  Immune response triggered by secreted interleukins  Some bacteria in the gut induce glycosylation in the mucosal epithelium

Gut Commensal Bacteroides Fuc DHAP + Lacaldehyde FF FF csp P csp pm Fuc R, I, A, K P RIAK P pm 22 22 22 22 22 Induction of Fuc  1,2 transferase PM Hooper & Gordon (2001) Glycobiology 11:1R

Therapeutics  Need to establish a causal relationship between adhesin/receptor interaction and pathogenesis –knock-outs of glycosyltransferases will be important  Antibodies to adhesins and soluble adhesins  Competitive ligands (carbohydrate receptors) –valency issues –delivery problems –analogs and derivatives