Download Ser/Thr

O-Glycans
Chapter 8
April 6, 2004
Jeff Esko
[email protected]
Overview
• Types of O-linked glycans
• T and Tn antigens
• Glycosyltransferases involved in O-glycan
assembly
• Mucins
• Function of leukocyte membrane mucin
• Tumor mucins
O-Glycosidic Linkage
OH
OH
GalNAc
H
O
H
H
HO
H
a
NAc
H
b-elimination
O
H 2C
C
CH
O
Ser
O-glycosidic
linkage is sensitive
to alkali (regardless
of stereochemistry)
NH2
Examples of O-Glycans?
Yeast mannoproteins
a-dystroglycan
Nuclear Proteins
Cytoplasmic Proteins
GalNAc
Man
Fuc
GlcNAc
Ser/Thr
Ser/Thr
Ser/Thr
Ser/Thr
Mucins
Notch
Coagulation Factors
Fibrinolytic Factors
More O-glycans
Glc
Xyl
Ser
Ser
Proteoglycans
(Glycosaminglycans)
Man
Glc
Gal GlcNAc
Thr
Rho proteins
Worm Collagens
Dictyostelium proteins
Even more….
Gal
Ara
Gal GlcNAc
Glc
HydroxyLys
HydroxyPro
Tyr
Collagen
C1q complement
Plant glycoproteins
Dictyostelium
Glycogenin
Mucin-Type O-GalNAc Glycans
a3
b4
a3
b3
b4
b3
b4
a3
b3
b6
a
Ser/Thr
• Major vertebrate O-glycan
• Begins in cis-Golgi by
attachment of GalNAc in alinkage to specific Ser/Thr
residues
• Assembly is simpler than Nlinked chains - no lipid
intermediate is used
• Always involves nucleotide
sugars
• Always occurs by addition to
non-reducing terminus or by
branching
Polypeptide GalNAc Transferases
Regions in white, pink, red, and black represent, respectively, 0–29%, 30–69%, 70–
99%, and 100% sequence identity (Hagen et al. (2003) Glycobiology 13:1R-16R).
•
•
•
•
12 members of mammalian ppGalNAcT family
Estimated size of family = 24
Share structural features in active site
Some have lectin (ricin) domain
Core 1 and Core 2 Synthesis
Core 1
GalT
Ser/Thr
Core 2
GlcNAcT
b3
b3
b6
Ser/Thr
Ser/Thr
Core 3 and Core 4 Synthesis
Core 3
GlcNAcT
Ser/Thr
Core 4
GlcNAcT
b3
b3
b6
Ser/Thr
Ser/Thr
Unusual Core O-Glycan Structures
Core 1
Core 2
Core 3
Core 4
b3
b3
b3
b3
Ser/Thr
Ser/Thr
Ser/Thr
Ser/Thr
Core 5
Core 6?
Core 7
Core 8
a3
Ser/Thr
b6
b6
a6
Ser/Thr
Ser/Thr
b6
a3
Ser/Thr
T-antigens
• Tumor associated antigens
• First one designated Thomsen-Friedenreich (TF)
antigen, later renamed T-antigen
• Precursor (GalNAca-O-Ser/Thr) is called Tnantigen
b3GalT
Ser/Thr
Tn-antigen
b3
Ser/Thr
T-antigen
ST6GalNAc
III, IV, I, II
a3
b3
b3
a6
disialyl
T-antigen
ST3Gal-I, II, IV
b3GalT
b3
b6GlcNAcT
b3
b6
Ser/Thr
Ser/Thr
Ser/Thr
Tn-antigen
T-antigen
Core 2
ST6GalNAc-II, I
ST6GalNAc-I
a6
sialyl
Tnantigen
b3
a6
sialyl
T-antigen
Cosmc
• Tn-antigens accumulate due to loss of b3Gal transferase
activity
• No mutations in b3GalT; message is expressed
• Missing Cosmc (Core 1 b3GalT -specific molecular chaperone,
Xq23
• 25% identity, >40% homology to b3GalT
• Absence of Cosmc results in proteosome degradation of
b3GalT
b3GalT
Ser/Thr
Cosmc
Tn-antigen
b3
Ser/Thr
T-antigen
Ju & Cummings (2002) PNAS 99:16613-8
Core 2 GlcNAc Transferases
• Three genes known, Core 2 b6GlcNAcT I, II, III
• Two isoforms, b6GlcNAcT I and III, in
lymphocytes and other non-epithelial cells
• One isoform, b6GlcNAcT II, specific for mucin
secreting epithelia
Core 2
b6GlcNAcT
Ser/Thr
b3
b3
b6
Ser/Thr
Ser/Thr
Outer Chain Assembly
a3
b4
a3
b3
b4
b3
b4
a3
b3
b6
Ser/Thr
• Sequential action of b4GalT and
b3GlcNAcT gives rise to
polylactosamine chains (Type II repeats)
• Type I repeats (Galb3GlcNAcb4) also
occur
• GlcNAcb6Gal branches (Iantigen) can occur
• The ends of the chains are
capped in a-linked sugars,
e.g. a3/4Fuc and a3/6sialic
acids
• Terminal structures make up
important blood group
determinants, e.g. the Lewis
antigens
Mucins are Heavily O-glycosylated
• Apomucin contain tandem repeats (8-169 amino
acids) rich in proline, threonine, and serine (PTS
domains)
• Glycosylation constitutes as much as 80% of mass
and tend clustered - bottle brush
• Expressed by epithelial cells that line the
gastrointestinal, respiratory, and genito-urinary tracts
Mucins
Dekker et al. (2002) TIBS 27:126
•
•
11p15 family (MUC2, MUC5AC, MUC5B, MUC6) probably
responsible for the formation of mucus layers
7q22 family (MUC3A, MUC3B, MUC12), 1q21 (MUC1), and
3q (MUC4, MUC13) are membrane mucins
Mucin Production
Lung Epithelium
Goblet cells in
intestinal crypts
Mucins: Protective Barriers for
Epithelial Cells
• Lubrication for epithelial surfaces
• Modulate infection:
– Receptors for bacterial adhesins
– Secreted mucins can act as decoys
• Barrier against freezing:
– Antifreeze glycoproteins
– [Ala-Ala-Thr]n≤40 with Core 1
disaccharides
Leukocyte Trafficking
Infiltration of leukocytes into sites of inflammation depends on
multiple carbohydrate-protein interactions
Leukocyte Rolling
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
Tim Springer, Harvard
Cell Surface Mucin: PSGL-1
www.imech.ac.cn/mianlong/ ppt/ppt3.htm
Lappänen et al. (2000) JBC 275:39569
Leukocyte Trafficking Defects in Mice
Lacking Core 2 GlcNAc Transferase
(Ellies et al. (1998) Immunity 9:881-90)
Leukocytosis
wt/∆
∆/∆
cells/ml
wildtype
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Lymph Node High Endothelial Venules
(HEVs)
Wildtype
Null
L-selectin chimeras were used
to probe lymph node sections
Ellies et al. (1998) Immunity 9:881-90
Tumor cells express mucins
that define ligands for
selectin adhesion receptors
Resting
Platelet
Leukocyte
Activated
Platelet
Carcinoma
Cell
P
Platelets: P-selectin
Leukocytes: L-selectin
Endothelia: E- and Pselectins
L
Neoplastic emboli
can lodge in the
small vessels
P
Membrane
Bound Mucin
P
Tumor markers:
CA19-9 (sLeA),
CA125 (MUC16) and
others
ACTIVATED
ENDOTHELIUM
E
Induced O-glycan Deficiencies in the Mouse
and Biological Effects
Enzyme
Phenotype
Polypeptide GalNAcT-1
B-lymphocyte deficiency in
nodes
Polypeptide GalNAcT-8
None so far
Core 2 GlcNAc T-I
Leukocytosis and defect in
inflammation
Questions
• What is the function of multiple polypeptide
GalNAc transferases?
• Do various transferases within a family act on the
same or different substrates?
• How is tissue specific expression of transferases
regulated?
• How does competition of transferases for
substrates determine the glycoforms expressed
by cells and tissues?
• Would small molecule inhibitors of O-glycan
formation prove therapeutically useful?
N-acetylgalactosaminides
Core 1
GalT
b3
Ser/Thr
O
Ser/Thr
Core 1
GalT
O