STRUCTURAL-ANALYSIS OF PEPTIDE-SUBSTRATES FOR MUCIN-TYPE O-GLYCOSYLATION

The structures of three nine-residue peptide substrates that show diff erential kinetics of O-linked glycosylation catalyzed by distinct reco mbinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetyl galactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techn iques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, Ga lNAc-T1 and GalNAc-T3. In the proposed model, the formation of an exte nded backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extende d structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand confor mation. The hydroxyl group of the threonine to be glycosylated is expo sed to solvent, and both the amide proton and carbonyl oxygen of the p eptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate effi ciency, leading us to hypothesize that this proton may serve as a dono r for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may a lso be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.