Characterization of tumor-associated ganglio-N-triaosylceramide in mouse lymphoma and the dependency of its exposure and antigenicity on the sialosyl residues of a second glycoconjugate.

Ganglio-N-triaosylceramide (GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer), a tumor-associated marker for L5178 cells, was previously reported to separate on thin layer chromatography into three distinct bands (bands a, b, and c). The present paper describes the characterization of these bands and the factor that determines the degree of glycolipid exposure at the cell surface and its antigenicity. 1) The resolution of ganglio-N-triaosylceramide into three bands was found to be due to molecules having different fatty acid compositions. Band a contained nervonic (C24:1) and lignoceric (C24:0) acids, band b contained palmitic acid (C16:0), and band c contained alpha-hydroxypalmitic acid. 2) Surface labeling of L5178c127 cells with galactose oxidase/sodium borotritide, followed by fluorography of the isolated glycolipids, revealed that all three bands were exposed on the surface of the cell. However, treatment of cells with sialidase before treatment with galactose oxidase resulted in a 10-fold increase of label incorporated into ganglio-N-triaosylceramide. Since no sialylated form of ganglio-N-triaosylceramide was detected on these cells, and no change in the chemical amount of this glycolipid could be detected, the increase of label into this molecule was due to the exposure by sialidase of a normally cryptic glycolipid. The exposure of ganglio-N-triaosylceramide after sialidase treatment was also reflected by the increased sensitivity of these cells to monoclonal antibodies to the glycolipid and complement after enzyme treatment. Thus, the results provide clear evidence that crypticity, as well as antigenicity, of a membrane glycolipid is determined by the degree of sialylation in a second membrane glycoconjugate.