Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates

TY - JOUR

T1 - Elucidation of the sugar recognition ability of the lectin domain of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 by using unnatural glycopeptide substrates

AU - Yoshimura, Yayoi

AU - Nudelman, Aaron S

AU - Levery, Steven B

AU - Wandall, Hans H

AU - Bennett, Eric P

AU - Hindsgaul, Ole

AU - Clausen, Henrik

AU - Nishimura, Shin-Ichiro

PY - 2012/3

Y1 - 2012/3

N2 - 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.

AB - 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.

U2 - 10.1093/glycob/cwr159

DO - 10.1093/glycob/cwr159

M3 - Journal article

C2 - 22042768

SN - 0959-6658

VL - 22

SP - 429

EP - 438

JO - Glycobiology

JF - Glycobiology

IS - 3

ER -