Specific hydrolysis of intact erythrocyte cell-surface glycosphingolipids by endoglycoceramidase. Lack of modulation of erythrocyte glucose transporter by endogenous glycosphingolipids.
This study represents the specific hydrolysis of cell-surface glycosphingolipids (GSLs) of intact cells by endoglycoceramidase (EGCase; EC.3.2.1.123) which cleaves the linkage between oligosaccharides and ceramides of various GSLs. After a 2-h incubation of horse intact erythrocytes with 20 mU EGCase II in the presence of activator at 37 degrees C, 68% of the N-glycolylneuraminic-acid-containing ganglioside GM3(NeuGc) and 70% of 4-O-acetyl GM3(NeuGc) were found to be hydrolyzed without hemolysis, accompanied by a corresponding increase in ceramide but not sphingosine or N,N-dimethylsphingosine. No hydrolysis was observed for sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, cholesterol or membrane proteins. The decrease in immunoreactivity with GMR8 antibody, specific to NeuGc alpha 2,3Gal- of GM3(NeuGc), corresponded to that of cell-surface GM3(NeuGc) by the enzyme, and almost no immunoreactivity was found when 70% of the GM3(NeuGc) was hydrolyzed. Besides the cell-surface GM3(NeuGc) of horse erythrocytes, Gg3Cer of guinea pig, GM3(NeuAc) and LcCer of human, and bovine and rabbit erythrocyte IV3Gal alpha-nLc4Cer were found to be efficiently hydrolyzed by EGCase II even when present in intact cells, while human erythrocyte Gb4Cer is quite resistant to hydrolysis by the enzyme on the cell surface as well as in detergent micelles. Glucose incorporation via the glucose transporter in erythrocytes was not affected at all by the specific and exhaustive hydrolysis of cell-surface GSLs by EGCase II. This result strongly suggested that glucose transporter function was not directly modulated by endogenous GSLs. In summary, this paper demonstrates that, together with the assistance of activator protein, EGCase II will become a powerful tool for selectively removing sugar chains from cell-surface GSLs without damaging other cell membrane components, and will be useful for describing the biological functions of endogenous GSLs.
Ito
M
M
Laboratory of Glycoconjugate Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Ikegami
Y
Y
Tai
T
T
Yamagata
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002240:Carbohydrate Sequence; D002417:Cattle; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006028:Glycosphingolipids / Q000276:immunology / Q000378:metabolism*; D006168:Guinea Pigs; D006736:Horses; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D006028:Glycosphingolipids; D009004:Monosaccharide Transport Proteins; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase