Essentials of Glycobiology May 14, 2002 Jeff Esko
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Transcript of Essentials of Glycobiology May 14, 2002 Jeff Esko
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Essentials of Glycobiology
May 14, 2002
Jeff Esko
Bacterial and viral carbohydrate-binding proteins
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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
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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
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To colonize tissues and cause infection, microbes must first adhere
Epithelial Cell
Microbe
Lining of gastrointestinal, urinary, respiratory, or reproductive tract
MucinMicrobe
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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
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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
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Bacterial Adhesins
Many bacterial adhesins are found on pili (hairs) or fimbrae (threads)
Velcro effect due to lateral mobility of adhesin in membrane
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Pilus: An organelle
Adhesin subunit (G) usually found on the tip
Sauer et al. (2000) COSB 10:548
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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
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Choudhury et al. (1999) Science 285:1061
FimH
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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
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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
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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
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Glycosphingolipid Receptors for Toxins
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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
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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
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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
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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)
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Influenza Virus
Viral envelope contains a sialic acid hemagglutinin and a neuraminidase (sialidase)
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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
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GAG-Binding Adhesins
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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
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Flavivirus Adhesin Model
Example of a relatively non-specific binding site, which interacts with many heparan sulfate sequences with variable affinity
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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
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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
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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
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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