Exposure of the major human red-cell glycolipid, globoside, to galactose oxidase

Abstract

Plasma membrane glycolipids are localized at the outer leaflet of the lipid bilayer, and their carbohydrate portions are exposed to the environment. The efficiency of exposure has, however, not been known. We have been able to determine the availability of the major red cell glycolipid, gIoboside, to externally added galactose oxidase. Red cells were extensively treated with the enzyme and the oxidized cells reduced with NaBD4. After isolation the extent of exposed globoside was estimated by mass spectrometry. The results show that the exposure of globoside varies in red cells of different individuals from 37 -66%. The fatty acid composition of externally available globoside was the same as that of non-oxidized globoside. The exposure was not influenced by protease treatment of intact cells and no correlation was found with different ABO blood groups.Glycolipids are ubiquitous mammalian cell-surface components. Their carbohydrate head-groups can act as receptors for antibodies, lectins, bacterial toxins, and microbes [l]. They may also pIay a role in cell-cell interactions.Several studies have demonstrated that glycolipids in plasma membranes are localized at the outer cell surface and no glycolipids are found at the cytoplasmic aspect [2, 31. There is, however, evidence that glycolipids are only partially exposed to the environment [4]. The crypticity may vary during the cell cycle and by transformation [5]. Essentially nothing is, however, known about the efficiency of exposure [l].It would obviously be important to determine the degree of exposure and its variation under different conditions. We have therefore treated human erythrocytes with galactose oxidase until all accessible glycolipid was oxidized, and thereafter reduced the cells with NaBD4. The glycolipids were isolated and the ratio of surface lipid to inaccessible lipid determined by mass spectrometry. By using this technique we show that only part of the major glycolipid, globoside, is exposed in human erythrocyte membranes, and that the degree of exposure differs in different individuals. MATERIALS AND METHODS CellsBlood samples of known ABO blood groups were taken into heparinized tubes, and the erythrocytes were washed by centrifugation four times with 0.15 M NaCl-0.01 M sodium phosphate, pH 7.4 (NaC1/PO4). All labeling experiments were done using freshly drawn cells. EnzymesIn the early experiments galactose oxidase (Dactylium dendroides) was obtained from Kabi Ltd (Stockholm, Enzymes. Galactose oxidase (EC 1 .I -3.9); horseradish peroxidase (EC 1.11.1.7); catalase (EC 1.11.1.6); trypsin (EC 3.4.21.4); Streptomyces griseus protease (EC 3.4.24.4); Vibrio cholerae neuraminidase (EC 3.2.1.18). Sweden), later galactose oxidase (type V) from Sigma Chemical Co. (St. Louis, MO) was used. The enzyme was heated for 30 min at 56°C before use to destroy possibly contaminating proteases [3]. No difference was found between the preparations. Horseradish peroxidase (type II), bovine liver catalase, trypsin from bovine pancreas (type XI) and Streptomyces gris...

Exposure of major neutral glycolipids in red cells to galactose oxidase

“…Normal human red cells were treated as previously described [6]. Heparinized porcine blood was obtained from a local slaughterhouse.…”

“…Labeling of erythrocytes and treatments of cells with pronase and trypsin were done as previously described [6]. When neuraminidase treatment was used in the galactose oxidasel NaB2H4 labeling, the cells were incubated with 50 pl neuraminidase/ml packed cells simultaneously with galactose oxidase for 8 h. Erythrocyte membranes were prepared by lysing the cells in 5 mM Tris buffer, pH 8.0, followed by centrifugation and washings at 4 "C [9].…”

“…We have recently developed a method by which the exposure of membrane-bound glycolipids to galactose oxidase can be quantitatively determined [6]. The cells are first extensively oxidized with the enzyme, and the aldehyde groups in terminal galactose and N-acetylgalactosamine residues are reduced with NaB2H4.…”

“…Sialylated glycoconjugates have been shown to have a masking effect in some cases [3, 41. Moreover, suppression of antigenicity by sialic acids seems to be a general feature of molecules and cells [5].We have recently developed a method by which the exposure of membrane-bound glycolipids to galactose oxidase can be quantitatively determined [6]. The cells are first extensively oxidized with the enzyme, and the aldehyde groups in terminal galactose and N-acetylgalactosamine residues are reduced with NaB2H4.…”

Specific hydrolysis of intact erythrocyte cell‐surface glycosphingolipids by endoglycoceramidase

“…Lampio et al examined the action of galactose oxidase on both galactose and N-acetylgalactosamine residues of erythrocyte cell-surface GSLs [30]. They found that 60-70% of human and porcine erythrocyte Gb,Cer were oxidized by the enzyme, while only 4-12% of guinea pig erythrocyte Gg,Cer was accessible.…”