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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 OH OH GalNAc H O H H HO H a NAc H b-elimination O H 2C C CH O Ser O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry) NH2 Examples of O-Glycans? Yeast mannoproteins a-dystroglycan Nuclear Proteins Cytoplasmic Proteins GalNAc Man Fuc GlcNAc Ser/Thr Ser/Thr Ser/Thr Ser/Thr Mucins Notch Coagulation Factors Fibrinolytic Factors More O-glycans Glc Xyl Ser Ser Proteoglycans (Glycosaminglycans) Man Glc Gal GlcNAc Thr Rho proteins Worm Collagens Dictyostelium proteins Even more…. Gal Ara Gal GlcNAc Glc HydroxyLys HydroxyPro Tyr Collagen C1q complement Plant glycoproteins Dictyostelium Glycogenin Mucin-Type O-GalNAc Glycans a3 b4 a3 b3 b4 b3 b4 a3 b3 b6 a Ser/Thr • Major vertebrate O-glycan • Begins in cis-Golgi by attachment of GalNAc in alinkage to specific Ser/Thr residues • Assembly is simpler than Nlinked chains - no lipid intermediate is used • Always involves nucleotide sugars • Always occurs by addition to non-reducing terminus or by branching 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 Core 1 and Core 2 Synthesis Core 1 GalT Ser/Thr Core 2 GlcNAcT b3 b3 b6 Ser/Thr Ser/Thr Core 3 and Core 4 Synthesis Core 3 GlcNAcT Ser/Thr Core 4 GlcNAcT b3 b3 b6 Ser/Thr Ser/Thr Unusual Core O-Glycan Structures Core 1 Core 2 Core 3 Core 4 b3 b3 b3 b3 Ser/Thr Ser/Thr Ser/Thr Ser/Thr Core 5 Core 6? Core 7 Core 8 a3 Ser/Thr b6 b6 a6 Ser/Thr Ser/Thr b6 a3 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 Tnantigen b3GalT Ser/Thr Tn-antigen b3 Ser/Thr T-antigen ST6GalNAc III, IV, I, II a3 b3 b3 a6 disialyl T-antigen ST3Gal-I, II, IV b3GalT b3 b6GlcNAcT b3 b6 Ser/Thr Ser/Thr Ser/Thr Tn-antigen T-antigen Core 2 ST6GalNAc-II, I ST6GalNAc-I a6 sialyl Tnantigen b3 a6 sialyl T-antigen 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 b3GalT Ser/Thr Cosmc Tn-antigen b3 Ser/Thr T-antigen Ju & Cummings (2002) PNAS 99:16613-8 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 Ser/Thr b3 b3 b6 Ser/Thr Ser/Thr Outer Chain Assembly a3 b4 a3 b3 b4 b3 b4 a3 b3 b6 Ser/Thr • Sequential action of b4GalT and b3GlcNAcT gives rise to polylactosamine chains (Type II repeats) • Type I repeats (Galb3GlcNAcb4) also occur • GlcNAcb6Gal branches (Iantigen) 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 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 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 Mucin Production Lung Epithelium Goblet cells in intestinal crypts 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 Infiltration of leukocytes into sites of inflammation depends on multiple carbohydrate-protein interactions Leukocyte Rolling QuickTime™ and a Sorenson Video decompressor are 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 (Ellies et al. (1998) Immunity 9:881-90) Leukocytosis wt/∆ ∆/∆ cells/ml wildtype Neutrophils Lymphocytes Monocytes Eosinophils 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 Tumor cells express mucins that define ligands for selectin adhesion receptors Resting Platelet Leukocyte Activated Platelet Carcinoma Cell P Platelets: P-selectin Leukocytes: L-selectin Endothelia: E- and Pselectins L Neoplastic emboli can lodge in the small vessels P Membrane Bound Mucin P Tumor markers: CA19-9 (sLeA), CA125 (MUC16) and others ACTIVATED ENDOTHELIUM E 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 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? N-acetylgalactosaminides Core 1 GalT b3 Ser/Thr O Ser/Thr Core 1 GalT O