title: Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Yun
authors.initials: Y
authors.lastname: Kong
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Hiren J
authors.initials: HJ
authors.lastname: Joshi
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Katrine Ter-Borch Gram
authors.initials: KT
authors.lastname: Schjoldager
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Thomas Daugbjerg
authors.initials: TD
authors.lastname: Madsen
authors.affiliation: Department of Pediatrics Department of Biochemistry and Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Gerken
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Malene B
authors.initials: MB
authors.lastname: Vester-Christensen
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Hans H
authors.initials: HH
authors.lastname: Wandall
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennett
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Vakhrushev
authors.affiliation: Department of Cellular and Molecular Medicine and Odontology, Copenhagen, Center for Glycomics, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark hclau@sund.ku.dk.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D002240:Carbohydrate Sequence; D057075:Enzyme Assays; D005786:Gene Expression Regulation; D054794:Glycomics; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D007527:Isoenzymes; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides; D011134:Polysaccharides; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase
keywords: GALNT; ISOGlyP; MALDI-TOF; NetOGlyc4.0; SimpleCell