O-GlycansChapter 8

April 6, 2004

Jeff Esko

jesko@ucsd.edu

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

Tyr

Glycogenin

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 6

sialyl Tn-

antigen6

36

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