TY - JOUR
T1 - Dynamic epigenetic regulation of initial O-glycosylation by UDP-N- acetylgalactosamine
T2 - Peptide N-acetylgalactosaminyltransferases. Site- specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions
AU - Hanisch, Franz Georg
AU - Müller, Stefan
AU - Hassann, Helle
AU - Clausen, Henrik
AU - Zachara, Natasha
AU - Gooley, Andrew A.
AU - Paulsen, Hans
AU - Alving, Kim
AU - Peter-Katalinic, Jasna
PY - 1999/4/9
Y1 - 1999/4/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0039374466&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0039374466&partnerID=8YFLogxK
U2 - 10.1074/jbc.274.15.9946
DO - 10.1074/jbc.274.15.9946
M3 - Article
C2 - 10187769
AN - SCOPUS:0039374466
SN - 0021-9258
VL - 274
SP - 9946
EP - 9954
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 15
ER -