Dynamic epigenetic regulation of initial O-glycosylation by UDP-N- acetylgalactosamine: Peptide N-acetylgalactosaminyltransferases. Site- specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions

Franz Georg Hanisch, Stefan Müller, Helle Hassann, Henrik Clausen, Natasha Zachara, Andrew A. Gooley, Hans Paulsen, Kim Alving, Jasna Peter-Katalinic

Research output: Contribution to journalArticlepeer-review

Abstract

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N- acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitro addition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcα or Galβ1-3GalNAcα at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 doses not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disaccharide at position -11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.

Original languageEnglish (US)
Pages (from-to)9946-9954
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number15
DOIs
StatePublished - Apr 9 1999
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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