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 β-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [(14)C]GlcA from UDP-[(14)C]GlcA on to β-(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 [(14)C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [(14)C]GlcA occurred preferentially on to consecutive (1 → 3)-linked β-Gal chains as well as single branched β-(1 → 6)-Gal residues through β-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [(14)C]GlcA residues on to several oligosaccharides, such as β-(1 → 6)- and β-(1 → 3)-galactotrioses. A trisaccharide, α-l-Araf-(1 → 3)-β-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-l-Araf-(1 → 3)[β-GlcA-(1 → 6)]-β-Gal-(1 → 6)-Gal. We report the first in vitro assay system for β-GlcATs involved in the AG synthesis as a step toward full characterization and cloning. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00425-013-1959-0) contains supplementary material, which is available to authorized users.