O-GalNAc Glycans! Dr. Lianchun WangOutline! 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 GlycansCommon Classes of Animal Glycans • O-glycosylation is a common covalentmodification of serine and threonine residues ofmammalian glycoproteins.• O-GalNAc glycans are covalently !-linked viaan N-acetylgalactosamine (GalNAc) moiety tothe -OH of serine or threonine by an O-glycosidicbond• Core structure: !-linked N-GalNAc residuelinked to serine or threonine. The further additionof Gal, GlcNAc or GalNAc defines Cores 1-8.• O-GalNAc glycans are antigenic• Modification of O-GalNAc glycans: O-acetylation of sialic acid and O-sulfation ofgalactose and N-acetylglucosamine, leading tostructural heterogeneicity.•Other O-glycans: !-linked O-fucose, "-linked O-xylose, !-linked O-mannose, "-linked O-GlcNAc,!- or "-linked O-galactose, and !- or "-linked O-glucose glycans•Shield the epithelial surfaces against physicaland chemical damage and protect againstinfection by pathogensO-GalNAc Glycans• O-glycosylation is a common covalentmodification of serine and threonine residues ofmammalian glycoproteins.• O-GalNAc glycans are covalently !-linked viaan N-acetylgalactosamine (GalNAc) moiety tothe -OH of serine or threonine by an O-glycosidicbond• Core structure: !-linked N-GalNAc residuelinked to serine or threonine. The further additionof Gal, GlcNAc or GalNAc defines Cores 1-8.• O-GalNAc glycans are antigenic• Modification of O-GalNAc glycans: O-acetylation of sialic acid and O-sulfation ofgalactose and N-acetylglucosamine, leading tostructural heterogeneicity.•Other O-glycans: !-linked O-fucose, "-linked O-xylose, !-linked O-mannose, "-linked O-GlcNAc,!- or "-linked O-galactose, and !- or "-linked O-glucose glycans•Shield the epithelial surfaces against physicaland chemical damage and protect againstinfection by pathogensO-GalNAc GlycansMucin Glycoproteins• Mucin: Large glycoprotein with a high contentof serine, threonine, and proline residues andnumerous O-GalNAc-linked saccharides, oftenoccurring in clusters on the polypeptide.• In mucous secretions and as transmembraneglycoproteins.• Gel-forming mucins: large, mainly produced in the goblet or mucous cells of thetracheobronchial, gastrointestinal, and genitourinary tracts.• In goblet cells, mucins are stored intracellularly in mucin granules and can be quicklysecreted 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 theirpolymerization and the formation of extremely large molecules of several million daltons.• Cell-surface mucins contain an extracellular domain with a central VNTR region that carriesO-GalNAc glycan chains, a single transmembrane domain, and a small cytoplasmic tail at thecarboxyl terminus.• About 20 mucin genes, express in tissue-specific fashion and vary in the number andcomposition of the peptide repeats in their VNTR regions. Within the same mucin, the repeatsusually vary in their amino acid sequences.• The expression of mucin genes is regulated by a large number of cytokines and growthfactors, differentiation factors, and bacterial products.Function of Mucin Glycoproteins• Viscoelastic properties that contribute to the high viscosity ofmucous 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 diseasesO-GalNAc Glycan Structures and Tissue Distribution• Tn antigen: GalNAc!-Ser/Thr.• Core 1 O-GalNAc glycan (T antigen): Gal"1-3GalNAc-Ser/Thr, found in many glycoproteins andmucins• Core 2 O-GalNAc glycans: contains a branching N-acetylglucosamine attached to core 1 & found in bothglycoproteins and mucins from a variety of cells and tissues• Cores 3 and 4 O-GalNAc glycans: found only insecreted mucins of certain mucin-secreting tissues,such as bronchi, colon, and salivary glands• Core 5-8 O-GalNAc glycans: Extremely restrictedoccurrence- Core 5 : human meconium and intestinaladenocarcinoma tissue- Core 6 : human intestinal mucin and ovarian cystmucin- Core 7: bovine submaxillary mucin- Core 8: human respiratory mucinModification of O-GalNAc Glycans• Sialylation: All cores.• Only Cores 1-4 & 6 occur asextended complex O-glycans.• The terminal structures of O-GalNAcglycans may contain fucose,galactose, N-acetylglucosamine, andsialic acid in !-linkages, N-acetylgalactosamine in both !- and "-linkages, and sulfate.• Many of these terminal sugarstructures are antigenic or representrecognition sites for lectins.- the sialylated and sulfated Lewisantigens are ligands for selectinsO-GalNAc Glycan Analysis• Release– "-elimination: N-acetylgalactosamine to N-acetylgalactosaminitol– N-acetylgalactosaminidase:Unsubstituted N-acetylgalactosamineresidues– 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 glycosyltransferaseO-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 carboxylterminus• The subcellular localization of ppGalNAcTs and other glycosyltransferases involved in O-glycosylation has a critical role in determining the range of O-glycans synthesized by acell• ppGalNAcT expression levels vary considerably between cell types and mammaliantissues• All ppGalNAcTs bind UDP-GalNAc (the donor