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

In search of possible epigenetic regulatory mechanisms ruling the initiatio n of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopepti de substrates on the site-specific in vitro addition of N-acetylgalactosami ne (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3), T he substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem re peat peptide carrying GalNAc alpha or Gal beta 1-3GalNAc alpha at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Dis accharide placed on the first position of the diad Ser-16-Thr-17 prevents g lycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does 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 glyc osylation at position -6 (Thr-10) or -10 (Ser-6) and is inhibited by disacc haride at position -11 (Thr-5), suggesting the occurrence of glycosylation- induced effects on distant acceptor sites. Kinetic studies revealed the acc elerated addition of GalNAc to Ser-16 adjacent Do GalNAc-substituted Thr-17 , demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharide s could underlie a postulated regulatory mechanism.