O GalNAc Glycans Dr Lianchun Wang Outline O GalNAc Glycans Mucin Glycoproteins Function of Mucin Glycoproteins O GalNAc Glycan Structures and Tissue Distribution Modification of O GalNAc Glycans O GalNAc Glycan Analysis O GalNAc Glycan Biosynthesis Functions of O GalNAc Glycans Common Classes of Animal Glycans O GalNAc Glycans O glycosylation is a common covalent modification of serine and threonine residues of mammalian glycoproteins O GalNAc glycans are covalently linked via an N acetylgalactosamine GalNAc moiety to the OH of serine or threonine by an O glycosidic bond Core structure linked N GalNAc residue linked to serine or threonine The further addition of Gal GlcNAc or GalNAc defines Cores 1 8 O GalNAc glycans are antigenic Modification of O GalNAc glycans Oacetylation of sialic acid and O sulfation of galactose and N acetylglucosamine leading to structural heterogeneicity Other O glycans linked O fucose linked Oxylose linked O mannose linked O GlcNAc or linked O galactose and or linked Oglucose glycans Shield the epithelial surfaces against physical and chemical damage and protect against infection by pathogens O GalNAc Glycans O glycosylation is a common covalent modification of serine and threonine residues of mammalian glycoproteins O GalNAc glycans are covalently linked via an N acetylgalactosamine GalNAc moiety to the OH of serine or threonine by an O glycosidic bond Core structure linked N GalNAc residue linked to serine or threonine The further addition of Gal GlcNAc or GalNAc defines Cores 1 8 O GalNAc glycans are antigenic Modification of O GalNAc glycans Oacetylation of sialic acid and O sulfation of galactose and N acetylglucosamine leading to structural heterogeneicity Other O glycans linked O fucose linked Oxylose linked O mannose linked O GlcNAc or linked O galactose and or linked Oglucose glycans Shield the epithelial surfaces against physical and chemical damage and protect against infection by pathogens Mucin Glycoproteins Mucin Large glycoprotein with a high content of serine threonine and proline residues and numerous O GalNAc linked saccharides often occurring in clusters on the polypeptide In mucous secretions and as transmembrane glycoproteins Gel forming mucins large mainly produced in the goblet or mucous cells of the tracheobronchial gastrointestinal and genitourinary tracts In goblet cells mucins are stored intracellularly in mucin granules and can be quickly secreted upon external stimuli Hallmark Repeated peptide stretches Variable number of tandem repeat VNTR Rich in serine and or threoninn Abundant O GalNAc glycans bottle brush conformation Rich in proline that facilitates O GalNAc glycosylation Secreted mucins have cysteine rich regions and cystine knots that are responsible for their polymerization and the formation of extremely large molecules of several million daltons Cell surface mucins contain an extracellular domain with a central VNTR region that carries O GalNAc glycan chains a single transmembrane domain and a small cytoplasmic tail at the carboxyl terminus About 20 mucin genes express in tissue specific fashion and vary in the number and composition of the peptide repeats in their VNTR regions Within the same mucin the repeats usually vary in their amino acid sequences The expression of mucin genes is regulated by a large number of cytokines and growth factors differentiation factors and bacterial products Function of Mucin Glycoproteins Viscoelastic properties that contribute to the high viscosity of mucous secretions Hydrophilic and contain charges that attract water and salts Trap Bacteria viruses and other microbes sometimes specific O GalNAc glycans serve as receptors Mucins regulate signal transduction and cell adhesion Immune response GlyCAM 1 CD34 and PSGL 1 fertilization blastocyst implantation Abnormal structure with human diseases O GalNAc Glycan Structures and Tissue Distribution Tn antigen GalNAc Ser Thr Core 1 O GalNAc glycan T antigen Gal 13GalNAc Ser Thr found in many glycoproteins and mucins Core 2 O GalNAc glycans contains a branching Nacetylglucosamine attached to core 1 found in both glycoproteins and mucins from a variety of cells and tissues Cores 3 and 4 O GalNAc glycans found only in secreted mucins of certain mucin secreting tissues such as bronchi colon and salivary glands Core 5 8 O GalNAc glycans Extremely restricted occurrence Core 5 human meconium and intestinal adenocarcinoma tissue Core 6 human intestinal mucin and ovarian cyst mucin Core 7 bovine submaxillary mucin Core 8 human respiratory mucin Modification of O GalNAc Glycans Sialylation All cores Only Cores 1 4 6 occur as extended complex O glycans The terminal structures of O GalNAc glycans may contain fucose galactose N acetylglucosamine and sialic acid in linkages Nacetylgalactosamine in both and linkages and sulfate Many of these terminal sugar structures are antigenic or represent recognition sites for lectins the sialylated and sulfated Lewis antigens are ligands for selectins O GalNAc Glycan Analysis Release elimination N acetylgalactosamine to N acetylgalactosaminitol N acetylgalactosaminidase Unsubstituted N acetylgalactosamine residues O glycanase unsubstituted Gal 1 3GalNAc core 1 Purification gel filtration anion exchange chromatography HPLC Analysis composition linkages and structure MS NMR Exoglycosidase Antibody N acetylgalactosamine anti Tn and core 1 anti T Lectin Prediction based on presence of active glycosyltransferase O GalNAc Glycan Biosynthesis Polypeptide N Acetylgalactosaminyltransferases ppGalNAcT O GalNAc Glycan Biosynthesis Polypeptide N Acetylgalactosaminyltransferases ppGalNAcT ppGalNAcT transfer N acetylgalactosamine from UDG GalNAc to Ser Thr residues 21 members ppGalNAT 1 to 21 Localize in Golgi Type type II membrane protein may have a distinct lectin like domain at the carboxyl terminus The subcellular localization of ppGalNAcTs and other glycosyltransferases involved in Oglycosylation has a critical role in determining the range of O glycans synthesized by a cell ppGalNAcT expression levels vary considerably between cell types and mammalian tissues All ppGalNAcTs bind UDP GalNAc the donor of N acetylgalactosamine but they often differ in the protein substrates to which they transfer N acetylgalactosamine Such differences allow ppGalNAcTs to be distinguished Many ppGalNAcTs appear to have a hierarchical relationship with one another such that one enzyme cannot attach an N acetylgalactosamine until an adjacent