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

O-Glycans Chapter 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.

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

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  1. O-GlycansChapter 8April 6, 2004Jeff Eskojesko@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. Man Ser/Thr Ser/Thr Ser/Thr Ser/Thr Examples of O-Glycans? Yeast mannoproteins a-dystroglycan Nuclear Proteins Cytoplasmic Proteins GalNAc Fuc GlcNAc Mucins Notch Coagulation Factors Fibrinolytic Factors

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

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

  7. a3 b4 a3 b3 b4 b3 b4 a3 b3 b6 Ser/Thr Mucin-Type O-GalNAc Glycans • 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 • 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. b3 b6 b3 Ser/Thr Ser/Thr Ser/Thr Core 1 and Core 2 Synthesis Core 1 GalT Core 2 GlcNAcT

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

  11. Core 1 Core 2 Core 3 Core 4 b3 b6 Ser/Thr Core 6? Core 7 Core 8 Core 5 b6 a6 a3 b3 a3 b3 b3 b6 Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Ser/Thr Unusual Core O-Glycan Structures

  12. b3GalT Ser/Thr b3 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 Tn-antigen Tn-antigen T-antigen

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

  14. b3GalT Ser/Thr b3 Ser/Thr 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 Cosmc Tn-antigen T-antigen Ju & Cummings (2002) PNAS 99:16613-8

  15. b3 b6 b3 Ser/Thr Ser/Thr Ser/Thr 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

  16. a3 b4 a3 b3 b4 b3 b4 a3 b3 b6 Ser/Thr Outer Chain Assembly • Sequential action of b4GalT and b3GlcNAcT gives rise to polylactosamine chains (Type II repeats) • Type I repeats (Galb3GlcNAcb4) also occur • 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

  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. 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

  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 www.imech.ac.cn/mianlong/ ppt/ppt3.htm

  24. Lappänen et al. (2000) JBC 275:39569

  25. Leukocytosis wildtype wt/∆ ∆/∆ cells/ml Neutrophils Lymphocytes Monocytes Eosinophils Leukocyte Trafficking Defects in Mice Lacking Core 2 GlcNAc Transferase (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. Resting Platelet Activated L e u k o c y t e Platelet P L P P Membrane P Bound Mucin E P A C T I V A T E D E N D O T H E L I U M Tumor cells express mucins that define ligands for selectin adhesion receptors Platelets: P-selectin Leukocytes: L-selectin Endothelia: E- and P-selectins C a r c i n o m a C e l l Neoplastic emboli can lodge in the small vessels Tumor markers: CA19-9 (sLeA), CA125 (MUC16) and others

  28. Induced O-glycan Deficiencies in the Mouse and Biological Effects

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

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