o-glycans chapter 8 april 6, 2004 jeff esko jesko@ucsd
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
-elimination
O
OH
H
H
HO
H
O
NAcHH
OH
NH2CHC
H2C
O Ser
GalNAc
Examples of O-Glycans?
Ser/Thr
GalNAc
Mucins
Ser/Thr
Man
Yeast mannoproteins-dystroglycan
Ser/Thr
Fuc
NotchCoagulation FactorsFibrinolytic Factors
Ser/Thr
GlcNAc
Nuclear ProteinsCytoplasmic Proteins
More O-glycans
Ser
Glc
Thr
Proteoglycans(Glycosaminglycans)
Ser
Xyl
Rho proteinsWorm CollagensDictyostelium proteins
Man Glc Gal GlcNAc
Even more….
HydroxyLys
Gal
CollagenC1q complement
Plant glycoproteinsDictyostelium
HydroxyPro
Ara Gal GlcNAc
TyrGlycogenin
Glc
Mucin-Type O-GalNAc Glycans• Major vertebrate O-glycan• Begins in cis-Golgi by
attachment of GalNAc in -linkage to specific Ser/Thr residues
• Assembly is simpler than N-linked chains - no lipid intermediate is used
• Always involves nucleotide sugars
• Always occurs by addition to non-reducing terminus or by branching
4
3
4
3
4
3
3
3
Ser/Thr
3 6
Polypeptide GalNAc Transferases
• 12 members of mammalian ppGalNAcT family• Estimated size of family = 24• Share structural features in active site• Some have lectin (ricin) domain
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).
Core 2GlcNAcT
Core 1GalT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 1 and Core 2 Synthesis
Core 4GlcNAcT
Core 3GlcNAcT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 3 and Core 4 Synthesis
Core 3
Ser/Thr
3
Core 4
Ser/Thr
3 6
Ser/Thr
3
Core 1
Ser/Thr
3 6
Core 2
Core 7
Ser/Thr
6
Core 6?
Ser/Thr
6
Core 5
Ser/Thr
3
Core 8
Ser/Thr
3
Unusual Core O-Glycan Structures
T-antigens
• Tumor associated antigens• First one designated Thomsen-Friedenreich (TF)
antigen, later renamed T-antigen• Precursor (GalNAc-O-Ser/Thr) is called Tn-
antigen
Ser/Thr
Tn-antigen
Ser/Thr
3
T-antigen
3GalT
Ser/Thr Ser/Thr
3
T-antigen
3GalT 6GlcNAcT 3 6
Ser/Thr
3
3
3 6sialyl Tn-
antigen6
3 6
Tn-antigen
disialylT-antigen
Core 2
sialylT-antigen
ST3Gal-I, II, IV
ST6GalNAcIII, IV, I, II
ST6GalNAc-I ST6GalNAc-II, I
Cosmc• Tn-antigens accumulate due to loss of 3Gal transferase
activity• No mutations in 3GalT; message is expressed• Missing Cosmc (Core 1 3GalT -specific molecular chaperone,
Xq23• 25% identity, >40% homology to 3GalT• Absence of Cosmc results in proteosome degradation of 3GalT
Cosmc
Ju & Cummings (2002) PNAS 99:16613-8
Ser/Thr
Tn-antigen
Ser/Thr
3
T-antigen
3GalT
Core 26GlcNAcT
Ser/Thr
3 6
Ser/Thr
3
Ser/Thr
Core 2 GlcNAc Transferases
• Three genes known, Core 2 6GlcNAcT I, II, III• Two isoforms, 6GlcNAcT I and III, in lymphocytes
and other non-epithelial cells• One isoform, 6GlcNAcT II, specific for mucin
secreting epithelia
Outer Chain Assembly• Sequential action of 4GalT and
3GlcNAcT gives rise to polylactosamine chains (Type II repeats)
• Type I repeats (Gal3GlcNAc4) also occur
4
3
4
3
4
3
3
3
Ser/Thr
3 6
• GlcNAc6Gal branches (I-antigen) can occur
• The ends of the chains are capped in -linked sugars, e.g. 3/4Fuc and 3/6sialic 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
• 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
Dekker et al. (2002) TIBS 27:126
Lung Epithelium
Goblet cells in intestinal crypts
Mucin Production
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
Vascular Injury
HeparanSulfate
Endothelial Cells
P-selectin
SelectinLigands
Chemokines
Rolling Chemokine Activation of Integrins Extravasation
Cytokines
Integrin-ICAML-selectin
Infiltration of leukocytes into sites of inflammation depends on multiple carbohydrate-protein interactions
Leukocyte Rolling
QuickTime™ and aSorenson Video decompressorare 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
cells
/ml
Neutrophils Lymphocytes EosinophilsMonocytes
wt/∆wildtype ∆/∆
Leukocytosis (Ellies et al. (1998) Immunity 9:881-90)
L-selectin chimeras were used to probe lymph node sections
Wildtype Null
Lymph Node High Endothelial Venules (HEVs)
Ellies et al. (1998) Immunity 9:881-90
CarcinomaCell
Tumor cells express mucins that define ligands for selectin adhesion receptors
Platelets: P-selectinLeukocytes: L-selectinEndothelia: E- and P-selectins
Leukocyte
L
P
Activated
RestingPlatelet
Platelet
ACTIVATED ENDOTHELIUM
Neoplastic emboli can lodge in the small vessels
P E
MembraneBound Mucin
P
P
P
Tumor markers: CA19-9 (sLeA), CA125 (MUC16) and others
Induced O-glycan Deficiencies in the Mouseand 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?
Core 1GalT
Ser/Thr
3
Ser/Thr
N-acetylgalactosaminides
O O
Core 1GalT