Immunochemistry of Ii-Active Glycosphingolipids of Erythrocytes

Abstract

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosy1)ceramides. In contrast to poly(glycosy1)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosy1)ceramides.Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transferase of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAcl + 6Gal structures.

Both polyglycosylceramides and polyglycosylpeptides are unbranched in i erythrocytes

“…They were isolated from pronase digests of erythrocyte membranes and variously designated polyglycosylpeptides [6] or erythroglycan [ 71. Polyglycosylpeptides are probably derived from band 3 and band 4.5 glycoproteins [4]. We had established that polyglycosylceramides are largely unbranched in erythrocytes of a rare i phenotype [8]. We now show that the impaired branching is also a feature of i erythrocyte polyglycosylpeptides.…”

“…Control I erythrocytes were obtained from a type B blood donor. Polyglycosylceramides were isolated as in [8] while polyglycosylpeptides as in [7]. The starting material was erythrocyte stroma prepared as in [8].…”

“…The isolated materials were analysed for carbohydrate constituents as in [8]. Sphingosine was deter-* To whom correspondence should be addressed ElsevieriNorth-HoIland Biomedical Press mined as in [lo].…”

“…The number of branching points was estimated on the basis of 2,4-di_O-methylgalactose contents as determined in the methylated and acetolysed samples [8]. Hemagglutination inhibition assays were done as in [8] using blood group 0 erythrocytes as indicator cells. Carrier auxilliary lipid was not added.…”

“…Carrier auxilliary lipid was not added. Some of the anti-1 and anti-i serums employed in this study were used in [8] for measurements of Ii activities of polyglycosylceramides isolated from cord, adult and i erythrocytes.…”