Structure of forssman hapten glycosphingolipid from sheep erythrocytes

Abstract

The structure of Forssman glycosphingolipid hapten of sheep erythrocytes was worked out as follows. Earlier composition data showing it to be a pentahexosyl ceramid containing 2 mole of N-acetylgalatosamine, 2 mole of galactose and 1 mole of glucose per mole of hapten were confirmed. Positions of linkages between monosaccharides were determined by methylation-gas chromatographic-mass spectrometric analysis. Results of this study were confirmed by periodate oxidation which also established the sequence of monosaccharide residues. Double-labeling of Forssman hapten with 3H and 14C followed by selective enzymatic hydrolysis determined anomeric configurations of the acetal linkages. These results were verified by chromium trioxide oxidation of peracetylated hapten. The propose structure was supported by sequential enzymatic hydrolysis and assay of the products by inhibition of complement-medited hemolysis. It agrees with the carbohydrate sequences reported by Siddiqui and Hakomori [ J. biol. Chem. 246, 5766 (1971)] for horse kidney Forssman hapten and by Sung et al. [ J. biol. Chem. 248, 6528 (1973)] for canine kidney hapten. Gas chromatographic analysis revealed that sphinganine and lignoceric, nervonic and behenic acids were components. A limited interchangeability of fatty acid moieties is suggested. The structure proposed for Forssman hapten from sheep erythrocytes is N-acetylgalactosaminosyl-( α1 → 3)-N-acetylgalactosaminosyl-( β1 →3)-galactosyl-( α1 → 4)-galactosyl -( gb1 → 4)-glucosyl-( β1 → 1)-O-(acyl-N-sphinganine).