Blood-Group ABH-Specific Macroglycolipids of Human Erythrocytes: Isolation in High Yield from a Crude Membrane Glycoprotein Fraction

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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. Serological tests and radioactive labeling 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 .mu.g inhibiting 4 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 1 glycosyl residue per molecule, the average number of sugars in A and B macroglycolipids was 31, and their MW were 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. Macroglycolipids are the predominant ABH-specific component in human erythrocyte membranes; they most likely account for previous observations of ABH activity in membrane glycoprotein fractions.