Blood-group ABH-specific macroglycolipids of human erythrocytes: isolation in high yield from a crude membrane glycoprotein fraction

Abstract

Highly glycosylated, water-soluble ABH-specific sphingolipids, designated macroglycolipids, were isolated in high yield, up to 5 mg per unit of blood, from the crude human-erythrocyte-membrane glycoprotein fraction which is obtained by extraction of the membranes with chloroform/methanol/water. Both serological tests and radioactive labelling experiments indicated that these substances, rather than the glycoproteins, are the principal ABH-components in this fraction. The activities of A-specific, B-specific and H-specific macroglycolipids were very high, approximately 0.1 microgram inhibiting four hemagglutinating doses of the respective agglutinating reagents, and were thus comparable to those of secreted blood-group ABH-specific glycoproteins. The substances were stable to mild alkaline conditions. They contained fucose, galactose, glucosamine, glucose, sialic acid, sphingosine and fatty acids; blood-group-A-specific substances contained, in addition, galactosamine. No amino acids were detected. Assuming one glycosyl residue per molecule, the average number of sugars in A and B macroglycolipids was 31, and their molecular weights approximately 6100. The presence of beta-D-galactosidase-labile and sialic acid residues indicated that these substances contain nonreducing termini additional to the ABH immunodeterminants. In the B macroglycolipid, the ratio between nonreducing terminal alpha-D-galactopyranosyl and beta-D-galactopyranosyl residues was 1.7:1.0. The macroglycolipids formed clear aqueous solutions at concentrations as high as 30 mg/ml, were insoluble in 60--70% aqueous ethanol, and did not migrate on thin-layer chromatography unless they were acetylated. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed the macroglycolipids to be a heterogeneous mixture migrating throughout most of the region in which the periodic acid/Schiff-positive membrane glycoproteins are found. On the basis of the evidence presented, it is concluded that macroglycolipids are the predominant ABH-specific component in human erythrocyte membranes, and that they most likely account for previous observations of ABH activity in membrane glycoprotein fractions.