Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors

Abstract

Five different glycosphingolipid fractions (GL-3, 285 micrograms; GL-5, 1090 micrograms; GL-6, 615 micrograms; GL-7, 555 micrograms; and GL-8, 155 micrograms) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel high-performance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Lec blood-group activities when tested against two different Lec antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: (formula; see text) In contrast to the structurally homogeneous GL-3, minor amounts of 4-O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Lec active structures of GL-3 and GL-6 provide evidence that 3-fucosyl-N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Lec antigenic determinant (as marked in the formula of GL-6).