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

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

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

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

Mucin-Type O-GalNAc Glycans b4 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

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 GalT Core 2 GlcNAcT Ser/Thr b3 Ser/Thr b3 b6 Ser/Thr

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

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

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

disialyl T-antigen sialyl Tn-antigen sialyl T-antigen b3 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

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 99:16613-8

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 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

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

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.

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

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

Leukocyte Rolling Tim Springer, Harvard

Cell Surface Mucin: PSGL-1 www.imech.ac.cn/mianlong/ ppt/ppt3.htm

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

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)

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

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

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 Ser/Thr Core 1 GalT O O