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O-Glycans Chapter 8 April 6, 2004 Jeff Esko

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1 O-Glycans Chapter 8 April 6, 2004 Jeff Esko

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) -elimination Ser GalNAc

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

5 More O-glycans Ser Glc Thr Proteoglycans (Glycosaminglycans) Ser Xyl Rho proteins Worm Collagens Dictyostelium proteins ManGlcGalGlcNAc

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

7 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 Ser/Thr 3 6

8 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).

9 Core 2 GlcNAcT Core 1 GalT Ser/Thr Core 1 and Core 2 Synthesis

10 Core 4 GlcNAcT Core 3 GlcNAcT Ser/Thr Core 3 and Core 4 Synthesis

11 Core 3 Ser/Thr 3 Core 4 Ser/Thr 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

12 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

13 Ser/Thr 3 T-antigen 3GalT 6GlcNAcT 3 6 Ser/Thr sialyl Tn- antigen Tn-antigen disialyl T-antigen Core 2 sialyl T-antigen ST3Gal-I, II, IV ST6GalNAc III, IV, I, II ST6GalNAc-I ST6GalNAc-II, I

14 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: Ser/Thr Tn-antigen Ser/Thr 3 T-antigen 3GalT

15 Core 2 6GlcNAcT 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

16 Outer Chain Assembly Sequential action of 4GalT and 3GlcNAcT gives rise to polylactosamine chains (Type II repeats) Type I repeats (Gal 3GlcNAc 4) also occur Ser/Thr 3 6 GlcNAc 6Gal 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

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

18 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

19 Lung Epithelium Goblet cells in intestinal crypts Mucin Production

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] n40 with Core 1 disaccharides

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

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

26 L-selectin chimeras were used to probe lymph node sections WildtypeNull Lymph Node High Endothelial Venules (HEVs) Ellies et al. (1998) Immunity 9:881-90

27 Carcinoma Cell Tumor cells express mucins that define ligands for selectin adhesion receptors Platelets: P-selectin Leukocytes: L-selectin Endothelia: E- and P- selectins Leukocyte L P Activated Resting Platelet ACTIVATED ENDOTHELIUM Neoplastic emboli can lodge in the small vessels P E Membrane Bound Mucin P P P Tumor markers: CA19-9 (sLe A ), CA125 (MUC16) and others

28 Induced O-glycan Deficiencies in the Mouse and Biological Effects EnzymePhenotype Polypeptide GalNAcT-1B-lymphocyte deficiency in nodes Polypeptide GalNAcT-8None so far Core 2 GlcNAc T-ILeukocytosis 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 Core 1 GalT Ser/Thr 3 N-acetylgalactosaminides O O Core 1 GalT


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