Specific hydrolysis of intact erythrocyte cell‐surface glycosphingolipids by endoglycoceramidase

Abstract

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 I1 in the presence of activator at 37"C, 68% of the N-glycolylneuraminic-acidcontaining ganglioside GM3(NeuGc) and 70% of 4-0-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 NeuGca2,3Gal-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, Gg,Cer of guinea pig, GM3(NeuAc) and LcCer of human, and bovine and rabbit erythrocyte IV3Galu-nLc,Cer were found to be efficiently hydrolyzed by EGCase I1 even when present in intact cells, while human erythrocyte Gb,Cer 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 11. 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 I1 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.Glycosphingolipids (GSLs) are recognized as characteristic constituents of the plasma membrane of vertebrates. Since GSL sugar chains are oriented towards the external environment and exhibit structural heterogeneity, GSLs seem to be well suited for taking part in various biological events on the cell surface [l]. During the past decade, the importance of GSLs in cell activities has been increasingly recognized following the demonstration of profound changes in the quantity as well as quality of GSLs accompanying cellular differentiation, oncogenic transformation and the cell cycle [ 11. Furthermore, exogenously added GSLs, especially sialic-

Qualitative and Quantitative Cellular Glycomics of Glycosphingolipids Based on Rhodococcal Endoglycosylceramidase-assisted Glycan Cleavage, Glycoblotting-assisted Sample Preparation, and Matrix-assisted Laser Desorption Ionization Tandem Time-of-flight Mass Spectrometry Analysis

“…This criterion does not necessarily indicate that the release of glycans is quantitative. However, considering our previous observation that ϳ70% of cell surface GM3 on intact erythrocytes was hydrolyzed by EGCase II (20 milliunits) in the absence of detergent (30), fairly good releasing efficiency, if not quantitative, can be expected under the conditions of this study where detergent was present (a preferred condition for the hydrolysis by EGCases).…”

Homeostasis of Cell-surface Glycosphingolipid Content in B16 Melanoma Cells

“…By using EGCase with the assistance of its protein activator (7,8), cell-surface GSLs of erythrocytes were found to be hydrolyzed specifically without damaging other cell membrane components (9,10). We observed that the decrease of the GM 3 (NeuAc) content of B16 melanoma cells during EGCase treatment was much slower than GM 3 (NeuGc) of equine erythrocytes, although the initial velocity of EGCase toward GM 3 (NeuAc) is exactly the same as that toward GM 3 (NeuGc) (10).…”

Regulation of Intracellular Ceramide Content in B16 Melanoma Cells

“…that hydrolyzes the glycosidic linkage of ceramide and sugar chains of various GSLs (8). The cell surface GSLs of various erythrocytes (9) and cultured mammalian cells (10) were hydrolyzed by the purified rhodococcal endoglycoceramidase (11) with the assistance of its protein activator (12). We found that treatment of B16 melanoma cells with a microbial endoglycoceramidase activated GSL synthesis via transient up-regulation of UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1, glucosylceramide synthase; EC2.4.1.80) (13).…”