title: Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3.
abstract: Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.
authors.affiliation: School of Dentistry, University of Copenhagen, Norre Allé 20, 2200 Copenhagen N, Denmark.
authors.forename: H H
authors.initials: HH
authors.lastname: Wandall
authors.forename: H
authors.initials: H
authors.lastname: Hassan
authors.forename: E
authors.initials: E
authors.lastname: Mirgorodskaya
authors.forename: A K
authors.initials: AK
authors.lastname: Kristensen
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
authors.forename: E P
authors.initials: EP
authors.lastname: Bennett
authors.forename: P A
authors.initials: PA
authors.lastname: Nielsen
authors.forename: M A
authors.initials: MA
authors.lastname: Hollingsworth
authors.forename: J
authors.initials: J
authors.lastname: Burchell
authors.forename: J
authors.initials: J
authors.lastname: Taylor-Papadimitriou
authors.forename: H
authors.initials: H
authors.lastname: Clausen
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D017350:N-Acetylgalactosaminyltransferases