Structural analysis of peptide substrates for mucin-type O-glycosylation.
The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.
Kirnarsky
L
L
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Nomoto
M
M
Ikematsu
Y
Y
Hassan
H
H
Bennett
E P
EP
Cerny
R L
RL
Clausen
H
H
Hollingsworth
M A
MA
Sherman
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases; D009842:Oligopeptides; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase