Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.
We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.
Minamida
S
S
Department of Chemistry, Osaka University College of Science.
Aoki
K
K
Natsuka
S
S
Omichi
K
K
Fukase
K
K
Kusumoto
S
S
Hase
S
S
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D010430:Pentosyltransferases / Q000032:analysis*; D012995:Solubility; D014407:Tumor Cells, Cultured; D000097608:UDP Xylose-Protein Xylosyltransferase
D002413:Cations, Divalent; D004492:Edetic Acid; D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase