Transcript Essentials of Glycobiology Lecture 31 May 23, 2000 Jeff Esko

Unicellular Organisms:
Eubacteria, Archea, Yeast
Lecture 27, Chapter 21
May 13, 2004
Jeff Esko
Overview
 General structure of bacterial cell walls
 Structure, function and assembly of
peptidoglycan (murein)
 Periplasmic -glucans (MDO)
 Lipopolysaccharide (LPS) - endotoxin
 Capsular polysaccharides - mimicry
 Archea glycoproteins - a new area
 Yeast glycosylation and cell walls
Capsule
LPS
PG
MDO
Gram-negative bacteria cell wall
Peptidoglycan
A
D
G
A
A
D
G
A
A
G
D
A
A
D
G
A
A
D
G
A
A
G
D
A
A
D
G
A
A
G
D
A
A
D
G
A
A
D
G
A
A
G
D
A
A
G
D
A
A
D
G
A
A
G
D
A
A
D
G
A
A
G
D
A
A
D
G
A
A
G
D
A
[GlcNAc1,4MurNAc1,4]n
A
G
D
A
A
G
D
A
A
D
G
A
A
G
D
A
A
G
D
A
Heijenoort (2001) Glycobiology 11:25R
Murein Glycopeptide
CH2 OH
CH2OH
O
O
O
HC CH3
O C
L-Ala
D-Glu
DAP
D-Ala
D-Ala
NHAc
Notice D-amino acids
O
O
O
O
NHAc
DAP = diaminopimelic acid
Peptidoglycan
A
D
G
A
A
D
G
A
A
G
D
A
A
A
D
G
A
A
D
G
A
A
G
D
A
A
D
G
A
A
G
D
A
A
A
D
G
A
A
D
G
A
A
G
D
A
A
G
D
A
A
D
G
A
A
G
D
A
A
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G
D
A
A
A
D
G
A
A
G
D
A
A
D
G
A
A
G
D
A
The final step involves
cleavage between the
D-Ala-D-Ala unit and
transpeptidation to the
amino group of DAP of
another unit
A
G
D
A
A
D
G
A
A
G
D
A
A
G
D
A
Vancomycin
Moenomycins
Peptidoglycan Biosynthesis
P
Undecaprenyl
phosphate
MDO
Gram-negative bacteria cell wall
Membrane Derived Oligosaccharides (MDO)
2
2
2
2
2
2
2
+ Phosphoethanolamine
+ Phosphoglycerol
+ Succinate
 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
Gram-negative bacteria cell wall
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
HO
OH
O
O
O
HO
HO
AcN
P
O
HO
O
O
OH
P
Uri din e
O
OH
O
O
O
O
NH
C
O
P
O
O
OH
P
Uri din e
O
OH
HO
 Initiates by acylation of UDP-GlcNAc at C3,
followed by N-deacetylation, and Nacylation
HO
Wyckoff et al. (1998) Trends Microbiol. 6:154
Lipid A Assembly
OH
OH
O
HO
O
O
O C
O
HO
HO
O
HO
NH P
O P O
O
OH
OH
Uridine
+
O
HO
O
O
O C
O
HO
OH
HO
NH P
O
O
OH
O
CH 2
O
O
O
HO
NH
O C
O
O
O
O C
O
HO
HO
HO
HO
Diacylglucosamine-1-P condenses with another
molecule of UDP-diacylglucosamine to form the
tetraacyl disaccharide core
NH P O
O OH
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 was
positionally cloned.
 lps turned out to be homologous to toll receptors in
Drosophila, which were known to be involved in innate
immunity to fungal infection
 lps turns out to beTlr4, a member of a family of signaling
receptors (10 members known).
- Tlr4 binds to Lipid A.
- Tlr2 apparently binds and responds to muramyldipeptide
Takeda & Akira (2001)Genes to Cells 6:733
LPS Structural and Functional Domains
Core region contains unusual sugars
CH2 OH
HOHC
O
OH
CH2 OH
CHOH
O
COOH
OH
OH
OH
3-deoxy-D-mannooctulosonic acid (KDO)



OH
OH
OH
L-glycero-D-mannoheptulose
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
LPS Structural and Functional Domains
O-antigens
O-antigen
Structure
O9
a3
O6
a3
O124
3
a2
2
a4
a2
4
3
6
a3
a2
a4
6 GlcLA
GlcLA = glucolactillic acid

O-antigens consist of 2-8 sugars,
repeated ≤50 times

O-antigens gives rise to different
serotypes and some are
correlated with disease
a3
Capsule
Gram-negative bacteria cell wall
Capsule type
K1, polysialic acid
K5, N-acetylheparosan
Group A Streptococcus
(hyaluronan)



Structure
a8
a8
a8
a8
a8
a4
4
a4
4
a4
4
3
4
3
4
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
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 Nlinked attachment
sites and
mechanism of
assembly of glycan
(dolichol pathway)
Burda & Aebi (1999) Biochim Biophys Acta 1426:239
Yeast make AsN-linked Glycoproteins
• Yeast make
membrane N-linked
glycoproteins
much in the same
way as higher
eukaryotes
• Serves as a model
for human genetic
diseases (CDG)
M Aebi (2001) Trends in Cell Biology 11:136
Yeast Mannans
a6
a6
a3
a2
a2
a3
a2
a2
a3
a2
a2
a3
a6
a3
a6
a3
a3
a3
a2
a2
a2
a2
a6
a6
-1-P
a2
a2
a4
a2
a2
a
a
Ser/Thr
Ser/Thr
-P-
a6
a3 a6
a6
a3
a6
4
4
Asn
Willer et al (2003) Curr Opin Struct Biol 13:621
a3
Vertebrate
a-dystroglycan
Yeast Cell Walls
GPI
proteins
1,6-glucans
Pir cell wall
proteins
GPI
proteins
GPI
proteins
1,6-glucans
1,6-glucans
1,3-glucans
Plasma
membrane
The cell wall is made of 60% -glucans,
40% mannoproteins, and ~1% chitin
Smits et al (2001) Microbiology 147:781
Summary
• Bacterial cell walls are complex layered structures
composed of multiple classes of glycans
• Bacterial wall glycans provide an exoskeleton, define shape,
protect against turgor pressure, create antigenic serotypes,
and provide a way to prevent immune recognition
• Glycosylated proteins are present in eubacteria and in
archaea
• Yeast produce many of the same glycans found in higher
eukaryotes, but the mecahnism of assembly differs in subtle
way
• Yeast cell walls are composed of glycans (glucans and
chitin)
• Yeast and bacteria provide powerful genetic systems for
understanding membrane and cell wall assembly