Download Essentials of Glycobiology Lecture 27 May 14,1998 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
Microb
e
Mucin
Microbe
Epithelial Cell
Lining of gastrointestinal, urinary, respiratory, or reproductive tract
Tropism: Modification of the direction of growth
3 Adhesion occurs in a tissue specific manner
due to specific CHO-protein interactions
3 Can also help establish the normal flora
(e.g., commensal or symbiotic bacteria in
the gastrointestinal tract)
3 Colonization can lead to infection (e.g.,
uropathogenic infections)
Adhesins: Proteins that Mediate Adhesion
Adhesins
3 Many adhesins are
lectins
3 Some bind to terminal
sugars, others bind to
internal carbohydrate
sequences
3 Most microorganisms
express more than one
type of adhesin
Bacterial Adhesins
3 Many bacterial adhesins
are found on pili (hairs) or
fimbrae (threads)
3 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
FimH
Choudhury et al. (1999) Science 285:1061
.
Measuring Adhesin-Receptor Interactions
Direct Binding:
Binding
Hemagglutination:
Bacteria
•
•
•
•
Use mutant cells or nutritionally manipulate composition
Competition experiments with soluble carbohydrates
Remove receptor with exoglycosidases
Regenerate different receptor with glycosyltransferase
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
Large Clostridial Cytotoxins
Toxins A and B from Clostridium difficile (antibioticassociated diarrhea, pseudomembranous colitis)
Hemorrhagic and lethal toxins of C. sordellii and atoxin of C. novyi (enterotoxemia and gas gangrene)
These toxins turn out to be glucosyltransferases
Catalytic
Translocation
Binding
Large Clostridial Cytotoxins
Modification of
target proteins by
glucosylation
Targets include
Rho (cytoskeletal
organization),
Ras (growth
control), Rac,
cdc42 and other
GTPases
Busch & Aktories (2000) COSB 10:528
Microbial Binding to Glycoproteins
= Sialic
acid
N-LINKED CHAIN
O-LINKED CHAIN
GLYCOSPHINGOLIPID
S
O
Ser/Thr
N
Asn
OUTSIDE
CELL
MEMBRANE
INSIDE
Glycoprotein glycans are displaced from the membrane
compared to glycolipids, which may make it less effective as
a receptor
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 a2,6 sialic acids
– Flu B binds to a2,3 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
a2
a2
F
Fuc
PM
F
a2
Fuc
Fuc R, I, A, K
PRIAK
pm
Ppm
F
F
csp
Pcsp
a2
DHAP +
Lacaldehyde
Hooper & Gordon (2001) Glycobiology 11:1R
a2
Induction of
Fuca1,2
transferase
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