CONTROL OF MUCIN SYNTHESIS - THE PEPTIDE PORTION OF SYNTHETIC O-GLYCOPEPTIDE SUBSTRATES INFLUENCES THE ACTIVITY OF O-GLYCAN CORE-1 UDPGALACTOSE-N-ACETYL-ALPHA-GALACTOSAMINYL-R BETA-3-GALACTOSYLTRANSFERASE

Synthetic O-glycopeptides containing one or two GalNAc residues attached to Ser or Thr were used as substrates to investigate the effect of peptide structure on the activity of crude preparations of UDP-Gal:GalNAcα-R β33-Gal-transferase from pig stomach and pig and rat colonic mucosa and of a partially purified enzyme preparation from rat liver. High-performance liquid chromatography used to separate enzyme products revealed that uncharged glycopeptides with an acetyl group at the amino-terminal end and a tertiary butyl or an amide group at the carboxy-terminal end were resistant to proteolysis in crude preparations. The activity of β3-Gal-transferase varied with the sequence and length of the peptide portion of the substrate, the presence of protecting groups, the attachment site of GalNAc, and the number of GalNAc residues in the substrate. The presence and position of Pro had little effect on enzyme activity; ionizing groups near the GalNAc unit interfered with enzyme activity. Since the GalNAc-Thr moieties in many of these O-glycopeptides have been shown to assume similar rigid conformations, the variation in enzyme activity indicates that the β3-Gal-transferase recognizes both the peptide and carbohydrate moieties of the substrate. Rat and pig colonic mucosal homogenates contain β3- and β6-GlcNAc-transferases that synthesize respectively O-glycan core 3 (GlcNAcβ3GalNAcα-R) and core 4 [GlcNAcβ6(GlcNAcβ3)GalNAcα-R], These enzymes also showed variations in activity with different peptide structures; these effects did not parallel those observed with β3-Gal-transferase. The results show that the peptide portion in glycoproteins affects the activities of three glycosyltransferases which act on GalNAcα-Ser(Thr)-X. It is predicted that individual O-glycosylation sites carry characteristic O-glycan core structures. © 1990, American Chemical Society. All rights reserved.