Structural analysis of peptide substrates for mucin-type O- glycosylation

Leonid Kirnarsky, Mitsuharu Nomoto, Yoshito Ikematsu, Helle Hassan, Eric P. Bennett, Ronald Cerny, Henrik Clausen, Michael A Hollingsworth, Simon Sherman

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Abstract

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 β-like or an inverse γ-turn conformation and flanking residues in a β-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-TI and -T3 transferases.

Original languageEnglish (US)
Pages (from-to)12811-12817
Number of pages7
JournalBiochemistry
Volume37
Issue number37
DOIs
StatePublished - Sep 15 1998

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Kirnarsky, L., Nomoto, M., Ikematsu, Y., Hassan, H., Bennett, E. P., Cerny, R., Clausen, H., Hollingsworth, M. A., & Sherman, S. (1998). Structural analysis of peptide substrates for mucin-type O- glycosylation. Biochemistry, 37(37), 12811-12817. https://doi.org/10.1021/bi981034a