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^a 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-Sd^a antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Gk-Gal-GlcNAcGalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05,. Its chromatographic mobility was between that of G_M1 and G_D3. After treatrnent with β-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV³NeuAcnLc4OseCer), 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 (nLc₄OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAcβ(1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAcβ(1-4)[NeuAcα(2-3)]Galβ(1-4)GlcNAcβ(1-3)Galβ(1- 4)GlcCer. ¹H NMR analysis also supports the conclusion that the terminal GalNAc is linked β(1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination. In addition 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^a antigen remains to be identified.