Characterization of beta-D-xyloside-induced glycosaminoglycans and oligosaccharides in cultured human skin fibroblasts.
Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methylumbelliferyl beta-D-xyloside. Three fluorogenic components were isolated and purified from the culture medium by gel permeation high-performance liquid chromatography. Their structures were then characterized by enzymatic digestion, fast-atom-bombardment mass spectrometry, gas-liquid chromatography, and electrophoresis on cellulose acetate membrane. The results showed that one of the components was a mixture of dermatan sulfate (70%) and chondroitin sulfate (30%), bearing the 4-methylumbelliferone at the reducing termini, and having an average molecular weight of 9,200. The others had the structures galactosyl-galactosyl-xylosyl-4-methylumbelliferone and galactosyl-xylosyl-4-methylumbelliferone, respectively, representing the linkage region between the glycosaminoglycan chains and core protein, except that 4-methylumbelliferone replaced the amino acid. Moreover, it was demonstrated that these oligosaccharides were intermediates of glycosaminoglycan synthesis, not depolymerized products.
Takagaki
K
K
Department of Biochemistry, Hirosaki University School of Medicine, Aomori.
Nakamura
T
T
Kon
A
A
Tamura
S
S
Endo
M
M
D000328:Adult; D002478:Cells, Cultured; D002648:Child; D002851:Chromatography, High Pressure Liquid; D004588:Electrophoresis, Cellulose Acetate; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D012867:Skin / Q000378:metabolism*
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D006923:Hymecromone; C014111:methylumbelliferyl-beta-D-xyloside; D006026:Glycoside Hydrolases