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

• Published in: Planta Vol. 238; no. 6; p. 1157
• Main Authors: Endo, Maya, Kotake, Toshihisa, Watanabe, Yoko, Kimura, Kazumasa, Tsumuraya, Yoichi
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer Nature B.V 01-12-2013
• Subjects: Biosynthesis; Cloning; Roots
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-013-1959-0

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 [^sup 14^C]GlcA from UDP-[^sup 14^C]GlcA on to [beta]-(1 [arrow right] 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50-150 pmol min^sup -1^ (mg protein)^sup -1^. The enzyme specimen also catalyzed the transfer of [^sup 14^C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [^sup 14^C]GlcA occurred preferentially on to consecutive (1 [arrow right] 3)-linked [beta]-Gal chains as well as single branched [beta]-(1 [arrow right] 6)-Gal residues through [beta]-(1 [arrow right] 6) linkages, producing branched acidic side chains. The enzymes also transferred [^sup 14^C]GlcA residues on to several oligosaccharides, such as [beta]-(1 [arrow right] 6)- and [beta]-(1 [arrow right] 3)-galactotrioses. A trisaccharide, [alpha]-l-Araf-(1 [arrow right] 3)-[beta]-Gal-(1 [arrow right] 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, [alpha]-l-Araf-(1 [arrow right] 3)[[beta]-GlcA-(1 [arrow right] 6)]-[beta]-Gal-(1 [arrow right] 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.[PUBLICATION ABSTRACT]