title: Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates.
abstract: Mucin-type glycosylation [α-N-acetyl-D-galactosamine (α-GalNAc)-O-Ser/Thr] on proteins is initiated biosynthetically by 16 homologous isoforms of GalNAc-Ts (uridine diphosphate-GalNAc:polypeptide N-acetylgalactosaminyltransferases). All the GalNAc-Ts consist of a catalytic domain and a lectin domain. Previous reports of GalNAc-T assays toward peptides and α-GalNAc glycopeptides showed that the lectin domain recognized the sugar on the substrates and affected the reaction; however, the details are not clear. Here, we report a new strategy to give insight on the sugar recognition ability and the function of the GalNAc-T3 lectin domain using chemically synthesized natural-type (α-GalNAc-O-Thr) and unnatural-type [β-GalNAc-O-Thr, α-Fuc-O-Thr and β-GlcNAc-O-Thr] MUC5AC glycopeptides. GalNAc-T3 is one of isoforms expressed in various organs, its substrate specificity extensively characterized and its anomalous expression has been identified in several types of cancer (e.g. pancreas and stomach). The glycopeptides used in this study were designed based on a preliminary peptide assay with a sequence derived from the MUC5AC tandem repeat. Through GalNAc-T3 and lectin-inactivated GalNAc-T3, competition assays between the glycopeptide substrates and product analyses (MALDI-TOF MS, RP-HPLC and ETD-MS/MS), we show that the lectin domain strictly recognized GalNAc on the substrate and this specificity controlled the glycosylation pathway.
authors: Graduate School of Life Science and Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo 001-0021, Japan. yayoi.yoshimura@carlsberglab.dk
authors: Yayoi
authors: Y
authors: Yoshimura
authors: Aaron S
authors: AS
authors: Nudelman
authors: Steven B
authors: SB
authors: Levery
authors: Hans H
authors: HH
authors: Wandall
authors: Eric P
authors: EP
authors: Bennett
authors: Ole
authors: O
authors: Hindsgaul
authors: Henrik
authors: H
authors: Clausen
authors: Shin-ichiro
authors: S
authors: Nishimura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays; D005643:Fucose / Q000737:chemistry*; D005688:Galactosamine / Q000737:chemistry*; D006020:Glycopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D055271:Mucin 5AC / Q000737:chemistry; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D005643:Fucose; D005688:Galactosamine; D006020:Glycopeptides; D055271:Mucin 5AC; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C524678:MUC5AC protein, human