External Labeling of Cell Surface Galactose and Galactosamine in Glycolipid and Glycoprotein of Human Erythrocytes

Abstract

Abstract Treatment of erythrocytes with galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride (NaB3H4) at pH 7.4 allowed the labeling of galactosyl and N-acetylgalactosaminyl residues on external surfaces of cells with tritium (3H). Labeling patterns and specific activities of galactose and galactosamine in glycolipids and glycoproteins were determined after separation with gel electrophoresis and thin layer chromatography. The labeling patterns of normal adult cells differed greatly from fetal cells, and were significantly altered when cell surfaces were modified by proteases and neuraminidase. The results of analysis indicated that (a) the carbohydrate moieties of two glycolipids (globoside and ceramide trihexoside) and at least three glycoproteins (molecular weight 9.5, 8.2, and 6.4 x 104) were exposed to the external environment, but not ceramide dihexoside, ceramide monohexoside, or other glycoproteins with higher molecular weights; (b) the specific activities of galactosamine in glycolipid and of galactose in glycoprotein increased after protease treatment, although total activity of glycoprotein did not change; (c) labeling of glycoprotein was greatly enhanced by neuraminidase treatment, while that of glycolipid was enhanced to a lesser degree; (d) "the relative exposures" of glycoprotein and glycolipid differed greatly between normal and fetal erythrocyte surfaces. Glycoproteins of fetal cells had a very low label as compared to glycolipid.