essentials of glycobiology may 14, 2002 jeff esko

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

Microbe

Lining of gastrointestinal, urinary, respiratory, or reproductive tract

MucinMicrobe

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

Bin

din

g

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 GM1

Many bacteria secrete exotoxins that bind to carbohydrates

Cholera toxin consists of two types of subunits, AB5, which

bind to ganglioside GM1

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

BindingCatalytic Translocation

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

OSer/Thr

NAsn

N-LINKED CHAINN-LINKED CHAIN

O-LINKED CHAINO-LINKED CHAIN

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

OUTSIDE

INSIDE

S

= Sialic acid

CELLMEMBRANE

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 Fuc

DHAP +Lacaldehyde

FF

FF csp

Pcsp

pmFuc R, I, A, K

PRIAK Ppm

2

2

2

2

2

Induction of Fuc1,2

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