Isolation of the Receptors for Wheat Germ Agglutinin and the Ricinus communis Lectins from Human Erythrocytes Using Affinity Chromatography

The glycoproteins of human erythrocyte ghosts were solubilized with 0.5% Triton X-100 in 56 mm sodium borate, pH 8.0. This procedure solubilized 51% of the membrane protein, 81% of the sialic acid, 89% of the receptors for the Agaricus bisporus lectin and 70 to 75% of the wheat germ agglutinin and Ricinus communis lectin receptors. The solubilized glycoproteins were then separated by affinity chromatography on lectin-Sepharose columns. Glycoproteins which adsorbed to the lectin columns were eluted with the appropriate haptene sugar and analyzed for carbohydrate composition, mobility in sodium dodecyl sulfate polyacrylamide gels, and lectin binding ability. Glycoproteins which contained the membrane binding sites for the R. communis and Abrus precatorius lectins adsorbed to ricin and R. communis agglutinin I-Sepharose columns while the glycoproteins containing most of the receptors for the A. bisporus phytohemagglutinin (PHA), Phaseolus vulgaris erythroagglutinating (E)-PHA, Lens culinaris PHA, and wheat germ agglutinin passed through the column. The adsorbed glycoproteins were 1200 times more potent than galactose as haptene inhibitors of R. communis lectin binding to cells. The carbohydrate composition of these glycoproteins was determined to be (in residues relative to N-acetylglucosamine): N-acetylglucosamine (3), N-acetylgalactosamine (0.2), galactose (3.0), mannose (0.7), fucose (0.5), and sialic acid (0.3). When examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the glycoproteins could not be detected with the periodic acid-Schiff stain but were detected as several glycoprotein peaks by direct amino sugar analysis of the gels. When Triton-solubilized material was passed over the wheat germ agglutinin-Sepharose column, only one glycoprotein adsorbed to the column. This glycoprotein was a potent haptene inhibitor of wheat germ agglutinin, A. bisporus PHA, and P. vulgaris E-PHA. On sodium dodecyl sulfate polyacrylamide gels the glycoprotein had a mobility identical with that of the major sialoglycoprotein of the erythrocyte. Its carbohydrate composition was determined to be (in residues relative to N-acetylglucosamine): N-acetylglucosamine (3), N-acetylgalactosamine (7.5), galactose (6.9), mannose (1.5), fucose (0.9), and sialic acid (9.9). This composition is very similar to that of the major sialoglycoprotein. Pronase digestion of this glycoprotein reduced its lectin haptene inhibitory activity by greater than 98%. These data demonstrate that affinity chromatography of human erythrocyte membranes on lectin-Sepharose columns results in the separation of two distinct classes of glycoproteins with different carbohydrate compositions and different lectin binding properties.