Purification and characterization of fetal bovine serum β-N-acetyl-D- galactosaminyltransferase and β-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis

Abstract

β-N-Acetylgalactosaminyltransferase II and β-glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N- acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through β-linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500-fold and 850-fold, respectively, by sequential chromatographies on Red A-agarose, phenyl- Sepharose, S-Sepharose and wheat germ agglutinin-agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; β-N- acetylgalactosaminyltransferase and β-glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin- synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both β-N- acetylgalactosaminyltransferase II and β-glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.