1. O-Glycans Chapter 8 April 6, 2004 Jeff Esko jesko@ucsd.edu

2. Overview Types of O-linked glycans T and Tn antigens
Glycosyltransferases involved in O-glycan assembly
Mucins
Function of leukocyte membrane mucin
Tumor mucins

3. O-Glycosidic Linkage
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
b-elimination
GalNAc a
Ser

4. Examples of O-Glycans? GalNAc Ser/Thr Man Fuc GlcNAc Ser/Thr Ser/Thr
Yeast mannoproteins
a-dystroglycan
Nuclear Proteins
Cytoplasmic Proteins
GalNAc
Ser/Thr
Man
Fuc
GlcNAc
Ser/Thr
Ser/Thr
Ser/Thr
Mucins
Notch
Coagulation Factors
Fibrinolytic Factors

5. More O-glycans Ser Glc Ser Xyl Man Glc Gal GlcNAc Thr Rho proteins
Worm Collagens
Dictyostelium proteins
Proteoglycans
(Glycosaminglycans)

6. Even more…. HydroxyLys Gal HydroxyPro Ara Gal GlcNAc Tyr Glc Collagen
C1q complement
Plant glycoproteins
Dictyostelium
HydroxyPro
Ara
Gal
GlcNAc
Tyr
Glycogenin
Glc

7. Mucin-Type O-GalNAc Glycansb4 b3 a3
Ser/Thr b6
Major vertebrate O-glycan
Begins in cis-Golgi by attachment of GalNAc in a-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 a

8. 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

9. Core 1 and Core 2 Synthesis
GalT
Core 2
GlcNAcT
Ser/Thr b3
Ser/Thr b3 b6
Ser/Thr

10. Core 3 and Core 4 Synthesis
GlcNAcT
Core 4
GlcNAcT b3 b3 b6
Ser/Thr
Ser/Thr
Ser/Thr

11. Unusual Core O-Glycan Structures
Ser/Thr b3
Core 4 b6
Core 1
Core 2
Core 7
Ser/Thr a6
Core 6? b6
Core 5 a3
Core 8

12. T-antigens Tumor associated antigens
First one designated Thomsen-Friedenreich (TF) antigen, later renamed T-antigen
Precursor (GalNAca-O-Ser/Thr) is called Tn-antigen
Ser/Thr b3
Ser/Thr
b3GalT
Tn-antigen
T-antigen

13. disialyl T-antigen sialyl Tn-antigen sialyl T-antigenb3 a3 b3 a6
ST6GalNAc
III, IV, I, II
disialyl
T-antigen
ST3Gal-I, II, IV
Ser/Thr b3 b3 b6
Ser/Thr
b3GalT
b6GlcNAcT
Ser/Thr
Tn-antigen
T-antigen
Core 2
ST6GalNAc-II, I
ST6GalNAc-I a6
sialyl Tn-antigen b3 a6
sialyl
T-antigen

14. 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
Ser/Thr b3
Ser/Thr
b3GalT
Cosmc
Tn-antigen
T-antigen
Ju & Cummings (2002) PNAS 99:

15. 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
b6GlcNAcT II also called M form
Will transfer to core 1, core 3, and to Gal residues to form I-antigen
Ser/Thr b3
Ser/Thr b3 b6
Ser/Thr

16. Outer Chain Assembly
Sequential action of b4GalT and b3GlcNAcT gives rise to polylactosamine chains (Type II repeats)
Type I repeats (Galb3GlcNAcb4) also occur b4 b3 a3
Ser/Thr b6
GlcNAcb6Gal branches (I-antigen) 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
b6GlcNAcT II also called M form
Will transfer to core 1, core 3, and to Gal residues to form I-antigen

17. 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
Gelation fun cation mediated by interchain disulfide bonding
Note that O-GalNAc addition can occur readily at adjacent Ser/Thr residues,allowing clustering to occur. Can't happen with N-linked chains since large precursor added en bloc.

18. 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

19. Mucin Production
Lung Epithelium
Goblet cells in intestinal crypts

20. 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
Individuals lacking salivary glands have abnormal mucosal surfaces and chronic low level inflammation
Mucins can agglutinate certain pathogenic bacteria (oral streptococci)

21. Leukocyte Trafficking
Infiltration of leukocytes into sites of inflammation depends on multiple carbohydrate-protein interactions

22. Leukocyte Rolling
Tim Springer, Harvard

23. Cell Surface Mucin: PSGL-1
ppt/ppt3.htm

24. Lappänen et al. (2000) JBC 275:39569
Heparin effects may be mediated by blocking the TyrS sites/

25. Leukocyte Trafficking Defects in Mice Lacking Core 2 GlcNAc Transferase
cells/ml
Neutrophils
Lymphocytes
Eosinophils
Monocytes
wt/∆
wildtype
∆/∆
Leukocytosis
(Ellies et al. (1998) Immunity 9:881-90)

26. 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

27. Platelets: P-selectin Leukocytes: L-selectin
Tumor cells express mucins that define ligands for selectin adhesion receptors
Platelets: P-selectin
Leukocytes: L-selectin
Endothelia: E- and P-selectins
Activated
Resting
Platelet L e u k o c y t C a r c i n o m a C e l l L P
Neoplastic emboli can lodge in the small vessels P E
Membrane
Bound Mucin
Tumor markers: CA19-9 (sLeA), CA125 (MUC16) and others A C T I V E D N O H L U M

28. 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

29. 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?

30. N-acetylgalactosaminides
Core 1
GalT b3
Ser/Thr
Ser/Thr
Core 1
GalT O O