Journal of Biochemistry Advance Access originally published online on February 21, 2007
Journal of Biochemistry 2007 141(4):593-600; doi:10.1093/jb/mvm064
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© 2007 The Japanese Biochemical Society.
Purification and Substrate Specificity of UDP-D-xylose:ß-D-Glucoside 
-1,3-D-Xylosyltransferase Involved in the Biosynthesis of the Xyl1-3Xyl1-3Glcß1-O-Ser on Epidermal Growth Factor-like Domains
1Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan; 2Department of Chemistry, Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 590-0035, Japan; and 3Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, Hiratsuka, Kanagawa, 259-1292, Japan
*To whom correspondence should be addressed. Tel: +81-6-6850-5380, Fax: +81-6-6850-5383, E-mail: suhase{at}chem.sci.osaka-u.ac.jp
Received January 10, 2007; Accepted February 12, 2007
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Abstract |
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A unique O-glycan structure, Xyl
1-3Xyl1-3Glcß1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-D-xylose:ß-D-glucoside 
1,3-D-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143–146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying ß-glucoside at Ser53. The Km value for this substrate was 34 µM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-D-glucose but does recognize UDP-D-xylose.
Key Words: EGF-like domain, glycosyltransferase, O-glucose, O-glycans, xylose
Abbreviations: EGF, epidermal growth factor; FAB-MS, fast atom bombardment-mass spectrometry; Glc, D-glucose; Xyl, D-xylose; Glcß-EGF, the EGF-like domain of bovine factor IX (45-87) carrying ß-glucoside at Ser53; Glcß-Ser, Glcß1-O-Ser; Glcß-R, 2-[(2-pyridyl)amino]ethyl ß-glucoside
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