UDPGALACTOSE-GLYCOPROTEIN-N-ACETYL-D-GALACTOSAMINE 3-BETA-D-GALACTOSYLTRANSFERASE ACTIVITY SYNTHESIZING O-GLYCAN CORE-1 IS CONTROLLED BY THE AMINO-ACID-SEQUENCE AND GLYCOSYLATION OF GLYCOPEPTIDE SUBSTRATES

In order to investigate the role of the peptide moiety of glycoprotein s in the control of O-glycan biosynthesis, UDPgalactose:glycoprotein-N -acetyl-D-galactosamine 3-beta-D-galactosyltransferase (core 1 beta 3- Gal-T) from rat liver was tested for its specificity towards GalNAc-co ntaining glycopeptide substrates. Series of glycopeptides have been sy nthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based o n variations of the repeat sequences of human intestinal mucin. Most g lycopeptides were excellent substrates for core 1 beta 3-Gal-T compare d to benzyl alpha-D-galactosamine as indicated by their relatively hig h V-max/K-m. The enzyme preferred threonine alpha-D-galactosamine Thr( GalNAc) to serine alpha-D-galactosamine. Pro on the carboxy-terminal s ide adjacent to Thr(GaLNAc) was inhibitory. Negatively charged amino a cids on either side showed a low K-m; substrates with negatively charg ed amino acids on the amino-terminal side were highly efficient substr ates, suggesting charge- charge interactions between enzyme and substr ate. Gal beta l-3GalNAc alpha residues adjacent to Thr(GalNAc) reduced the activity. Product analysis using glycopeptide substrates with thr ee adjacent GalNAc residues showed incorporation of one, two and a sma ll amount of three Gal residues per molecule with an uneven distributi on of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the gly cosylation pathways of O-glycans is also controlled by the structure a nd glycosylation of the peptide core of substrates.