title: 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.
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 GalNAcalpha or Galbeta1-3GalNAcalpha 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 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 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.
authors: Institute of Biochemistry, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany. franz.hanisch@uni-koeln.de
authors: F G
authors: FG
authors: Hanisch
authors: S
authors: S
authors: Müller
authors: H
authors: H
authors: Hassan
authors: H
authors: H
authors: Clausen
authors: N
authors: N
authors: Zachara
authors: A A
authors: AA
authors: Gooley
authors: H
authors: H
authors: Paulsen
authors: K
authors: K
authors: Alving
authors: J
authors: J
authors: Peter-Katalinic
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D000116:Acetylgalactosamine; D018396:Mucin-1; D017350:N-Acetylgalactosaminyltransferases; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C116055:MUC1 tandem repeat peptide