Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 serine or threonine is glycosylated by a different ppGalNAcT • Defined amino acid sequons that accept N-acetylgalactosamine have not been identified, certain amino acids are preferred in the substrate. Proline residues near the site of Nacetylgalactosamine addition are usually favorable to mucin O-glycosylation, whereas charged amino acids may interfere with ppGalNAcT activity. O-GalNAc Glycan Biosynthesis: Core 1 and 2 • Initiated by ppGalNAcT transfering the first sugar from UDP-GalNAc directly to serine or threonine in a protein, creating the Tn antigen. • Tn antigen is uncommon in normal mucins, but is often found in mucins derived from tumors. • Sialyl-Tn antigen: contains a sialic acid residue linked to C-6 of N-GalNAc of Tn, commonly associated with cancer. • The subsequent sugar addition determines the formation of different Oglycan core structures. • Core 1: core 1 "1-3 galactosyltransferase (T synthase or C1GalT-1) transfers a Gal from Gal-UDP to from Gal"1-3GalNAc-Ser/Thr (T antigen). - Present in most cell type - Requires ER protein Cosmc for full activity in Golgi - Sialic acid modification at C-3 of Gal and C6 of N-GalNAc, prohibiting further modification - Leukemia and tumor cells contain large number of sialylated core 1 glycan. - Unsubstituted core 1 T antigen seen in cancer and inflammatory bowel disease O-GalNAc Glycan Biosynthesis: Core 1 and 2 • Core 2: core 2 "1-6 Nacetylglucosaminyltransferase ( C2GnT) transfers a GlcNAc from GlcNAc-UDP to from GlcNAc"1-6(Gal "1-3)GalNAcSer/Thr • Produced in many tissues • Synthesis is regulated during lymphocyte activation, cytokine stimulation, and embryonic development. • Altered in leukemia, cancer and other diseases • Synthesis is correlated with tumor progression • Block the exposure of mucin peptide epitopes • C2GnT1-3. - L type (leukocyte type, C2GnT-1 & -3); synthesize only the core 2 structure, active in many cell types and tissues - M type (mucin type, C2GnT-2): Also involved in the synthesis of core 4 and other GlcNAc "1-6-linked branches, active only in mucin-secreting cell type - The expression and activity of both the L-a nd M enzymes are altered in certain tumors O-GalNAc Glycan Biosynthesis: Core 3 and 4 • Core 3: core 3 "1-3 Nacetylglucosaminyltransferase (C3GnT) transfers a GlcNAc from GlcNAc-UDP to from GlcNAc"13GalNAc-Ser/Thr . • Restricted mostly to mucous epithelia from gastrointestinal, respiratory tracts and salivary glands • The activity of C3GnT is especially low in colonic tumors and absent in tumor cells in culture. • Core 4: synthesis by the M-type "1-6 Nacetylglucosaminyltransferase (C2GnT-2) and requires the prior synthesis of a core 3 O-GalNAc glycan Synthesis of Complex O-GalNAc Glycans • Elongation – Elongation of Gal residue of Core 1 and Core 2 by O-GlcNAC specific "1-3 Nacetylglucosaminyltransferase. – Extended by N-acetylglucosaminyltransferases and galactosyltransferases to form repeated GlcNAc"1-3Gal"1-4 (poly-N-acetyllactosamine) sequences that represent the little i antigen. – Linear poly-N-acetyllactosamine units can be branched by members of the "1-6 Nacetylglucosaminyltransferase family, resulting in the large I antigen – Less common elongation reactions are the formation of GalNAc"1-4GlcNAc- (LacdiNAc) and Gal"1-3GlcNAc- sequences • Terminal structures in mucin – ABO and other glycan-based blood groups – Sialic acid – Fucose – Sulfate Control of O-GalNAc Glycan Synthesis • The acceptor specificities of glycosyltransferases and sulfotransferases are the main factors determining the structures of O-GalNAc glycans found in mucins, and these specificities restrict the high number of theoretically possible O-glycans to “only” a few hundred. • The specificities also direct the pathways that are feasible •The relative activities of the glycosyltransferases determine the relative amounts of OGalNAc glycans in mucin – C2GnT-1 and !2-3 sialyltransferase both located in cis- and medial-Golgi compartments, and competes for the common core 1 substrate •The activities of transferases are controlled by many factors such as metal ions and membrane components – "1-4 galactosyltransferase 1: !-lactalbumin – "1-3 galactosyltransferase: coexpression of chaperone Cosmc. • The first step of O-GalNAc glycosylation is clearly regulated by the amino acid sequence of the acceptor substrate. • Intracellular localization • Donor substrate Functions of O-GalNAc Glycans • Essential to hydrate and protect the underlying epithelium. •Physical barrier between lumen and epithelium, protection from protease degradation • Trap and remove bacteria, virus and other pathogens • Serve as “decoys,” masking underlying antigens or receptors • Affect the conformation of the attached protein • O-glycans of cell-surface receptors regulate receptor stability and expression levels • O-glycans altered during during lymphocyte activation and in leukemia • Sialyl Lewisx epitopes attached to core 2 O-GalNAc glycans interacts with selectins to mediate leukocyte trafficking. • Sialyl Lewisx-selectin is involved in tumor metastasis • Fertilization