Kong, Yun2; Joshi, Hiren J5; Schjoldager, Katrine Ter-Borch Gram5; Madsen, Thomas Daugbjerg5; Gerken, Thomas A4; Vester-Christensen, Malene B5; Wandall, Hans H5; Bennett, Eric Paul6; Levery, Steven B5; Vakhrushev, Sergey Y5; Clausen, Henrik5
1 Section III. Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet2 Glycomics Program, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet3 Section 01 - Prosthetics, Department of Odontology, Faculty of Health and Medical Sciences, Københavns Universitet4 unknown5 Section III. Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet6 Section 01 - Prosthetics, Department of Odontology, Faculty of Health and Medical Sciences, Københavns Universitet
N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.