Liberation of oligosaccharides from glycosphingolipids on PC12 cell surface with endoglycoceramidase.
Endoglycoceramidase (EGCase) cleaves the linkage between the oligosaccharide and ceramide of glycosphingolipids (Ito, M., and Yamagata, T., J. Biol. Chem. 261, 14278-14282, ibid, 264, in press). Intact cells of rat pheochromocytoma line PC12 were treated with the highly purified EGCase I and the oligosaccharides released were analyzed by HPLC. Cleavage of the oligosaccharides with the enzyme reached a plateau as the amount of the enzyme was increased. At maximum, 42% of the oligosaccharides from globoside, 40% from GalGb3Cer, and 60% from Gb3Cer were liberated during 1h of incubation without impairing the viability of cells. The only partial liberation indicates that not all oligosaccharides of cell surface glycosphingolipids are accessible to the enzyme. The use of EGCase offers an important new method to access the functions of glycosphingolipids on cell surface in situ.
Laboratory of Glycoconjugate Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
M L
ML
Rasilo
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M
Ito
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T
Yamagata
D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism*; D066298:In Vitro Techniques; D009844:Oligosaccharides / Q000378:metabolism*; D010673:Pheochromocytoma / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D006028:Glycosphingolipids; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase