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.
Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 338-8570, Japan.
Maya
M
Endo
Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 338-8570, Japan.
Toshihisa
T
Kotake
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo, 186-8650, Japan.
Yoko
Y
Watanabe
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo, 186-8650, Japan.
Kazumasa
K
Kimura
Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 338-8570, Japan. tsumura@mail.saitama-u.ac.jp.
Yoichi
Y
Tsumuraya
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000096:biosynthesis / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009088:Mucoproteins / Q000737:chemistry* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D031224:Raphanus / Q000378:metabolism*; D013379:Substrate Specificity
D002241:Carbohydrates; D009088:Mucoproteins; D010940:Plant Proteins; C006619:arabinogalactan proteins; D014453:Glucuronosyltransferase
Cell wall; Glucuronic acid; Glycosyltransferase; Raphanus