Essentials of Glycobiology

Lecture 33

May 28, 2002

Jeff Esko

Bacterial Polysaccharides: Structure, Biosynthesis, Biological Functions and Significance

…or I never met a glycan I didn’t like

Overview

General structure of bacterial cell walls

Structure, function and assembly of peptidoglycan (murein)

Periplasmic -glucans (MDO)

Lipopolysaccharide (LPS) - endotoxin

Capsular polysaccharides - mimicry

Bacterial glycoproteins - a new area

LPS

MDO

PG

Capsule

Peptidoglycan

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

A A

AGDA

AGD

AGDA

AGDA

AGD

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

[GlcNAc1,4MurNAc1,4]n

Heijenoort (2001) Glycobiology 11:25R

Murein Glycopeptide

O

NHAc

O

CH2OH

HC CH3

C

O

NHAc

O

CH2OH

O OO

L-Ala

D-Glu

DAP

D-Ala

D-Ala

O

Notice D-amino acids

DAP = diaminopimelic acid

Peptidoglycan

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

A A

AGDA

AGD

AGDA

AGDA

AGD

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

AGDA

The final step involves cleavage between the D-Ala-D-Ala unit and transpeptidation to the amino group of DAP of another unit

A AA A

Vancomycin

Monenomycins

Peptidoglycan Biosynthesis

P

Undecaprenylphopsphate

MDO

Membrane Derived Oligosaccharides (MDO)

Branched -glucans Represent about 1-5%

of dry weight Charged substituents

act as an osmolyte and protects the inner membrane against the large difference in osmolarity inside the cell compared to outside the cell

LPS

Lipopolysaccharide (LPS)

LPS consists of three domains

– Lipid A, otherwise known as endotoxin

– Core region composed of KDO (K), heptoses (H), and hexoses (open hexagons

– Highly variable outer O-antigen region

Initiates by acylation of UDP-GlcNAc at C3, followed by N-deacetylation, and N-acylation

HOO

ONH

O H

CO

HO

OO

P

O

OO H

HO

P

O

OO H

Uridin e

HO

O

HOAcN

HO

OP

O

OO H

P

O

OO H

Uridin e

Wyckoff et al. (1998) Trends Microbiol. 6:154

Lipid A Assembly

HOO

ONH

OH

CO

HO

OO

P

O

OOH

HO

P

O

OOH

UridineHO

O

ONH

OH

CO

HO

O OP

O

OOH

HO

HOO

ONH

OH

CO

HO

O

O

HO

HOO

ONH

CH2

CO

HO

O OP

O

OOH

HO

+

Diacylglucosamine-1-P condenses with another molecule of UDP-diacylglucosamine to form the tetraacyl disaccharide core

Lipid A Assembly

KDO transferases initiate the formation of the core

Additional C12 fatty acids added to -hydroxy groups (wax)

Lipid A translocates to the outer leaflet of the outer membrane by msbA (ABC transporter)

Doerrler et al. (2001) J Biol Chem. 276:11461

Lipid A Biology

Lipid A, the heat stable endotoxin of gram negatives

Resistant strains of mice defined a locus, lps, which as positionally cloned. This turned out to be homologous to toll receptors in Drosophila, which were known to be involved in innate immunity to fungal infection (anitmicrobial peptides)

lps turns out to beTlr4, a member of a family of signaling receptors (10 members known) that induce . Tlr4 binds to Lipid A. Tlr2 apparently binds and responds to muramyl-dipeptide

Takeda & Akira(2001)Genes to Cells 6:733

LPS Structural and Functional Domains

Core region contains unusual sugars

The inner core contains 1-4 KDO residues, which look like an analog of sialic acid.

The core also contains heptopyranoses, which can vary stereochemically

The rest of the core consists of various combinations of Glc and Gal

OH O COOH

OHOH

HOHCCH2OH

3-deoxy-D-manno-octulosonic acid (KDO)

L-glycero-D-manno-heptulose

O

OHOH

CHOHCH2OH

OHOH

LPS Structural and Functional Domains

O-antigens

O-antigens consist of 2-8 sugars, repeated ≤50 times

O-antigens gives rise to different serotypes and some are correlated with disease

Capsule

Mucoid strains contain a polysaccharide capsule >80 different capsules types are known just in E. coli Extraordinary diversity of structure

Mycobacteria

Crick et al. (2001) Glycobiology 11:107R

Crick et al. (2001) Glycobiology 11:107R

Bacterial Glycoproteins

Surface-layer (S-layer) glycoproteins

Prevalent in Bacteria and Archaea, but structures differ, e.g., N-linked glycosylation only in Archaea

Structural analyses have revealed unusual carbohydrate-linkage regions….

….and unusual nucleoside diphosphate-linked oligosaccharides

Schaffer et al (2001) Proteomics 1:248

Notice diversity of linkages

Similarity in sequence of N-linked attachment sites and mechanism of assembly of glycan (dolichol pathway)

Burda &Aebi (1999) Biochim Biophys Acta 1426:239