Detection of UDP-D-Xylose: -D-Xyloside ->3Xylosyltransferase Activity in Human Hepatoma Cell Line HepG2

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Abstract
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 (Xylα1-3Glc) and a trisaccharide (Xylα1-3Xylα1-3Glc) 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 a1→xylosyltransferase for their biosynthesis. We report here on the detection of a1→3xylosyltransferase activity which catalyzes the transfer of xylose to Xylα1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xylα1-3Glc 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 Xylαl-3Xylα1-3Glc by Smith degradation, mass spectrometry, and α- and β-xylosidase digestions. The apparent Km value for pyridylaminated Xylα1-3Glc 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 Xylα1-3Glc, forming Xylα1-3Xylα1-3Glc in human cells.

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