Studies of acceptor site specificities for three members of UDP-GalNAc : N-acetylgalactosaminyltransferases by using a synthetic peptide mimicking the tandem repeat of MUC5AC

The acceptor specificity of three major isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyltranferases (murine recombinant proteins GaNTase-T1, -T2 and -T3) was investigated using the synthetic peptide (GTTPSPVPTTSTTSAP ) containing clusters of threonine residues mimicking the mucin tandem repe at unit of MUC5AC. The O-glycosylated products obtained after in vitro reac tions were fractionated by capillary electrophoresis and the purified glyco peptides were characterized by MALDI mass spectrometry (number of O-GalNAc residues) and by Edman degradation (site location). A maximum of three GalN Ac residues was transferred into the MUC5AC motif peptide and the preferent ial order of incorporation for each GaNTase isoform was determined. Our res ults suggest that clusters of threonine appear to be essential for site rec ognition of peptide backbone by the ubiquitous GaNTases and also support th e notion that the different GaNTase isoforms with varying substrate specifi cities are involved in a hierarchical order of O-glycosylation processing o f the mucin-type O-glycoproteins. (C) 2001 Elsevier Science Ltd. All rights reserved.