Control of O-glycan synthesis: specificity and inhibition of O-glycan core 1 UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase from rat liver

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase (β3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, β4-galactosyltransferase, β-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the β3-Gal-T. β3-Gal-T activity is sensitive to changes in the R-group of the GalNAcα-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAcα-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished β3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAcα-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. Derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAcα-benzyl, which inhibited Gal incorporation into GalNAcα-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAcβ6[Galβ3]GalNAcα-) is formed from core 1 (Galβ3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.Key words: β3-Gal-transferase, mucin synthesis, O-glycan core 1, enzyme specificity, enzyme inhibition.