Purification and substrate specificity of UDP-D-xylose:beta-D-glucoside alpha-1,3-D-xylosyltransferase involved in the biosynthesis of the Xyl alpha1-3Xyl alpha1-3Glc beta1-O-Ser on epidermal growth factor-like domains.
A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-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:beta-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 beta-glucoside at Ser53. The Km value for this substrate was 34 muM. 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.
Ishimizu
Takeshi
T
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan.
Sano
Kyoko
K
Uchida
Takashi
T
Teshima
Hiroshi
H
Omichi
Kaoru
K
Hojo
Hironobu
H
Nakahara
Yoshiaki
Y
Hase
Sumihiro
S
D000818:Animals; D002417:Cattle; D004815:Epidermal Growth Factor; D008099:Liver / Q000201:enzymology; D009844:Oligosaccharides / Q000378:metabolism*; D010430:Pentosyltransferases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism
D009844:Oligosaccharides; D014540:Uridine Diphosphate Xylose; D004815:Epidermal Growth Factor; D010430:Pentosyltransferases; C105769:UDP-D-xylose-beta-D-glucoside alpha-1,3-xylosyltransferase; D014532:Uridine Diphosphate Glucose