Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sd' blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc- GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GMl and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside ( IV3NeuAcnLc40seCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc40seCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad gangli&ide with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta( 1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta( 1-4) [NeuAc alpha(2-3)]Gal beta3( 1-4)GlcNAc beta( 1-3)Gal beta( 1-4)GlcCer. 'H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta(1-4) to GAL. High-performance thin-layer chromatographic ganglioside patterns froni three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen exptession on the erythrocytes, as measured by hemagglutination. In additibn to the major Cad ganglioside, a minor, slower moving component reactive with H. pomatia lectin WAS detected in all three Cad samples. No H. pomatia reactive bands were detected in gangliosides isolated from Sd(a+) cells, and the red cell component carrying the Sd' antigen remains to be identified