CHARACTERIZATION OF SERUM BETA-GLUCURONOSYLTRANSFERASE INVOLVED IN CHONDROITIN SULFATE BIOSYNTHESIS

We studied a glucuronyltransferase involved in chondroitin sulfate (CS ) biosynthesis in a preparation obtained from fetal bovine serum by he parin-Sepharose affinity chromatography, This enzyme transferred GlcA from UDP-GlcA to the nonreducing GalNAc residues of polymeric chondroi tin, It required Mn2+ for maximal activity and showed a sharp pH optim um between pH 5.5 and 6.0, The apparent Km value of the glucuronyltran sferase for UDP-GlcA was 51 mu M The specificity was investigated usin g structurally defined acceptor substrates, which consisted of chemica lly synthesized tri-, penta-, and heptasaccharide-serines and various odd-numbered oligosaccharides with a GalNAc residue at the nonreducing terminus, prepared from chondroitin and CS by chondroitinase ABC dige stion followed by mercuric acetate treatment, The enzyme utilized a he ptasaccharide-serine GalNAc beta 1-4GlcA beta 1-3GalNAc beta 1-4GlcA b eta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and a pentasaccharide- serine GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1- O-Ser as accepters, In contrast, neither a trisaccharide-serine Gal be ta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor an alpha-GalNAc-capped pentasac charide-serine GalNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xy l beta 1-O-Ser that is a model compound of the reaction product formed by the action of the alpha-GalNAc transferase recently demonstrated i n fetal bovine serum (Kitagawa et al., J, Biol. Chern., 270, 22190-221 95, 1995) was utilized as an acceptor, Besides, all nonsulfated odd-nu mbered oligosaccharides except for the trisaccharide GalNAc beta 1-4Gl cA beta 1-3GalNAc served as acceptors and the transfer rates roughly i ncreased with increasing chain length, Moreover, 6-O-sulfation of nonr educing terminal GalNAc markedly enhanced GlcA transfer, whereas 4-O-s ulfation had little effect on it, These results indicated that at leas t two glucuronyltransferases are involved in the biosynthesis of CS an d that sulfation reactions may play important roles in chain elongatio n.