Recognition by a natural cytotoxic antibody of lactoneotetraglycosyl ceramide as an antigenic molecule in a syngeneic rat fibrosarcoma.

Conventionally bred rats possess in their sera an NA which has a cytotoxic effect on a tumor cell line (KMT-17) derived from a fibrosarcoma induced by 3-methylcholanthrene and which can be absorbed by normal rat tissues. We have succeeded in purifying a glycosphingolipid as an antigen reactive to NA. GSLs isolated from the tumor cells were separated into neutral and acidic fractions. The former fraction was judged to be antigenic as detected by its capacity to absorb NA. The antigenic fraction was further separated into 7 fractions (Frs. A to G) by silicic acid chromatography. The antigenic activity was detected in only Frs.D and E, although Fr.D was a more potent antigen than Fr.E. Chemical and immunochemical analyses showed that both fractions are composed of lactoneotetraglycosyl ceramide (paragloboside), Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)ceramide, and that the more active GSL, Fr.D, contains larger amounts of long fatty-acid chains. Inhibition studies using disaccharides and monosaccharides indicated that a N-acetyllactosamine moiety, Gal(beta 1-4)GlcNAc, of the GSL is a specific site of NA. These results suggest that paragloboside is an NA antigen and that a sugar chain portion of this GSL is required for defining specificity while the ceramide portion plays a role in potentiating the antigenicity of this GSL antigen for NA.