Characterization and biological significance of sialyl alpha 2-3galactosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone) synthesized in cultured human skin fibroblasts.
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.
Takagaki
K
K
Department of Biochemistry, Hirosaki University School of Medicine.
Nakamura
T
T
Shibata
S
S
Higuchi
T
T
Endo
M
M
D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D003565:Cytidine Diphosphate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006026:Glycoside Hydrolases; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors; D012867:Skin / Q000166:cytology
D002241:Carbohydrates; D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D009844:Oligosaccharides; C101395:sialyl-2-3-galactosyl-1-4-xylosyl-1-(4-methylumbelliferone); D006923:Hymecromone; D003565:Cytidine Diphosphate; C014111:methylumbelliferyl-beta-D-xyloside; D012799:Sialyltransferases; D006026:Glycoside Hydrolases