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 glycoproteins 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-containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 beta 3-Gal-T compared to benzyl alpha-D-galactosamine as indicated by their relatively high Vmax/Km. The enzyme preferred threonine alpha-D-galactosamine Thr(GalNAc) to serine alpha-D-galactosamine. Pro on the carboxy-terminal side adjacent to Thr(GalNAc) was inhibitory. Negatively charged amino acids on either side showed a low Km; substrates with negatively charged amino acids on the amino-terminal side were highly efficient substrates, suggesting charge-charge interactions between enzyme and substrate. Gal beta 1-3GalNAc alpha residues adjacent to Thr(GalNAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of O-glycans is also controlled by the structure and glycosylation of the peptide core of substrates.
Granovsky
M
M
Biochemistry Department, Research Institute, Hospital for Sick Children, Toronto, Canada.
Bielfeldt
T
T
Peters
S
S
Paulsen
H
H
Meldal
M
M
Brockhausen
J
J
Brockhausen
I
I
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006020:Glycopeptides; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; C076385:glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase