Characterization and biological significance of sialyl alpha 2-3galactosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone) synthesized in cultured human skin fibroblasts.

K Takagaki, T Nakamura, S Shibata, T Higuchi, M Endo

Index: J. Biochem. 119(4) , 697-702, (1996)

Full Text: HTML

Abstract

Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methyl-umbelliferyl-beta-D-xyloside (Xyl-MU), then the cultured medium was recovered, concentrated with a lyophilizer, and dialyzed against distilled water. The structures of the Xyl-MU derivatives purified from the dialyzable fraction were investigated. In addition to established glycosaminoglycans-MU (GAGs-MU), Gal-Gal-Xyl-MU, Gal-Xyl-MU, sulphate-GlcA-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU, which were induced by Xyl-MU, an oligosaccharide having fluorescence was purified using a combination of gel filtration, ion-exchange chromatography and high-performance liquid chromatography, then subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation, 1H-NMR, and ion-spray mass spectrometric analysis. From the data obtained, the oligosaccharide was considered to have the structure SA alpha 2-3Gal beta 1-4Xyl beta 1-MU. The amount of MU-oligosaccharide in the cell culture increased with time and was dependent on the amount of Xyl-MU added. Its production was also different from that of Gal-Gal-Xyl-MU and Gal-Xyl-MU, which are biosynthetic intermediates of GAG-MU. Addition of CDP, an inhibitor of sialytransferase, to the cell culture medium increased the secretion of GAG-MU. These results suggest that SA-Gal-Xyl-MU production may be related to the regulation of GAG-MU biosynthesis.