Characterization of serum β-glucuronyltransferase involved in chondroitin sulfate biosynthesis

Abstract

We studied a glucuronyltransferase involved in chondroitin sulfate (CS) biosynthesis in a preparation obtained from fetal bovine serum by heparin-Sepharose affinity chromatography. This enzyme transferred GlcA from UDP-GlcA to the nonreducing GalNAc residues of polymeric chondroitin. It required Mn²⁺ for maximal activity and showed a sharp pH optimum between pH 5.5 and 6.0. The apparent Km value of the glucuronyltransferase for UDP-GlcA was 51 μM. The specificity was investigated using structurally defined acceptor substrates, which consisted of chemically 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 digestion followed by mercuric acetate treatment. The enzyme utilized a heptasaccharide-serine GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and a pentasaccharide-serine GalNAcβ 4GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser as acceptors. In contrast, neither a trisaccharide-serine Galβ1-3Galβ1-4Xylβ1-O-Ser nor an α-GalNAc-capped pentasaccharide-serine GalNAcα1-4GlcAβ1-3Galβ1-Galβ1-4Xylβ1-O-Ser that is a model compound of the reaction product formed by the action of the α-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190–22195, 1995) was utilized as an acceptor. Besides, all non-sulfated odd-numbered oligosaccharides except for the trisaccharide GalNAcβ1-4GlcAβ1-3GalNAc served as acceptors and the transfer rates roughly increased with increasing chain length. Moreover, 6-O-sulfation of nonreducing terminal GalNAc markedly enhanced GlcA transfer, whereas 4-O-sulfation had little effect on it. These results indicated that at least two glucuronyltransferases are involved in the biosynthesis of CS and that sulfation reactions may play important roles in chain elongation.