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.
Highlights
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
No strict sequence dependence is known for the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases,1 functionally expressed recombinant enzymes display a distinct selectivity for peptide motifs in the vicinity of putative glycosylation sites [1,2,3]
Evidence for negative effects on vicinal sites induced by mono- or disaccharide substituents has previously been reported for a series of glycopeptide substrates based on the MUC2 repeat peptide [12]
Summary
Glycopeptides and Peptides—The glycopeptides listed in Table I were synthesized as described previously [13] and were analyzed by 1H-NMR spectroscopy (400 MHz) [14] and by MALDI mass spectrometry [15]. The peptides A1 to A8 correspond to a 21-mer of the MUC1 tandem repeat domain starting with the AHG motif and carrying one to four O-linked disaccharides Gal1–3GalNAc␣ at various positions. Nonglycosylated peptide TAP25, corresponding to one repeat and five overlapping amino acids (starting with the TAP motif), was kindly provided by Dr Taylor-Papadimitriou (Imperial Cancer Research Fund, London, United Kingdom). Enzymes—Polypeptide GalNAc-transferases were obtained as follows: secreted, soluble recombinant forms of GalNAc-T1, -T2, and -T3 were expressed in insect cells as described previously [2, 3]. Enzymes were partially purified from serum-free culture supernatant of transfected High-FiveTM (Invitrogen) cells as described previously [3]. The ppGalNAc-transferases from human milk were enriched by ultracentrifugation as described previously and mixed with an equal volume of 0.9% NaCl, 0.4% Triton X-100, containing a series of protease inhibitors [7]
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