Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound β-glucuronosyltransferases from radish primary roots

A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained beta-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [C-14]GlcA from UDP-[C-14]GlcA on to beta-(1 -> 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50-150 pmol min(-1) (mg protein)(-1). The enzyme specimen also catalyzed the transfer of [C-14]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [C-14]GlcA occurred preferentially on to consecutive (1 -> 3)-linked beta-Gal chains as well as single branched beta-(1 -> 6)-Gal residues through beta-(1 -> 6) linkages, producing branched acidic side chains. The enzymes also transferred [C-14]GlcA residues on to several oligosaccharides, such as beta-(1 -> 6)- and beta-(1 -> 3)-galactotrioses. A trisaccharide, alpha-l-Araf-(1 -> 3)-beta-Gal-(1 -> 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, alpha-l-Araf-(1 -> 3)[beta-GlcA-(1 -> 6)]-beta-Gal-(1 -> 6)-Gal. We report the first in vitro assay system for beta-GlcATs involved in the AG synthesis as a step toward full characterization and cloning.