ganglio-n-triaosylceramide and Leukemia-L5178

To characterize the process by which glycolipids are shed from cell membranes, the cellular and supernatant glycolipids were compared from a variant of the mouse lymphoma L5178Y which had been selected for strong expression of the neutral glycolipid gangliotriaosylceramide (GgOse3Cer). This glycolipid was present in three forms which differed in their fatty acid composition. Whereas the major cell-associated form of GgOse3Cer contained C24 fatty acids, the predominant form shed into the culture supernatant contained C16 fatty acids. Ultracentrifugation of the culture medium yielded a pellet with a GgOse3Cer profile similar to that of the cells and a supernatant enriched in the C16 fatty acid form. Gel filtration of the culture medium revealed two GgOse3Cer-containing pools. The first was excluded from Sepharose CL-2B and had a GgOse3Cer profile similar to that of the cells, while the second migrated with proteins in the range of 25,000-500,000 daltons and was enriched in the C16 fatty acid form. These results suggest two forms in which glycolipids are released from cell membranes. The first is in a large complex, possibly a membrane vesicle, which retains the glycolipid profile of the membrane of intact cells while the second form appears to result from the preferential release of particular glycolipid components.

Topics: Animals; Chromatography, Gel; Fatty Acids; Gangliosides; Glycosphingolipids; Leukemia L5178; Leukemia, Experimental; Membrane Lipids; Mice; Ultracentrifugation

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.

Topics: Animals; Antibodies, Monoclonal; Chromatography, Thin Layer; Cytotoxicity, Immunologic; Epitopes; Gangliosides; Glycolipids; Glycosphingolipids; Leukemia L5178; Leukemia, Experimental; Mice; Neuraminidase; Sialic Acids

Gangliotriaosylceramide (Gg3Cer) was previously described as a tumor-associated antigen in murine L5178Y lymphoma [Young, W. W., Jr., and Hakomori, S., Science (Wash. D.C.), 211: 487-489, 1981]. This paper describes the major factors affecting the expression of Gg3Cer at the surface of various clones of L5178Y lymphoma. Of 26 sublines that were recloned, six cell lines showing different degrees of Gg3Cer expression at the cell surface were used for analysis of the glycolipid composition as related to its cell surface antigenicity. Three remarkable correlations between glycolipid composition and the antigenicity of Gg3Cer have been found: (a) high-expressor sublines were characterized by a large proportion of a unique molecular species of Gg3Cer having alpha-hydroxypalmitic acid in its ceramide moiety in striking contrast to low expressors which did not contain this molecular species; (b) low expressors contained a large quantity of ganglio-N-tetraosylceramide (Gg4Cer) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1 Cer (GM1b) gangliosides, whereas these glycolipids were almost absent in high-expressor clones; and (c) nonexpressors, which were converted from the high expressors in vivo through immunotherapy with the monoclonal antibodies to Gg3Cer, contained a large quantity of ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. The nonexpressors should have an induced enzyme system to metabolize Gg3Cer to ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. Three factors, i.e., ceramide composition, coexisting glycolipids, and an antibody-dependent glycolipid change, are therefore important in determination of glycolipid antigenicity and antigen modulation by antibodies. The ceramide composition may affect glycolipid organization in membranes, and the coexisting glycolipid having a longer carbohydrate chain may mask the accessibility of antibody to the antigenic glycolipid. The antigenic modulation by the action of the antibody in vivo may be based on activation of a new glycosyltransferase.

Topics: Animals; Antigens, Neoplasm; Ceramides; Fatty Acids; G(M1) Ganglioside; Gangliosides; Glycolipids; Glycosphingolipids; Leukemia L5178; Leukemia, Experimental; Mice

NK cells lyse an uncommonly wide range of cell types, implying either that they (the NK cells) have clonally distributed receptors each of which is capable of interacting with a very limited number of cell types or, alternatively, that susceptible target cells share a common characteristic. A number of experimental approaches have suggested that the cytotoxic 'specificity' of NK cells is not clonally distributed. Thus, clones of NK cells, established from mouse spleen cell suspensions, showed no greater restriction in the spectrum of target cells which they could lyse, than did the parent spleen cell populations from which they were derived. It seems likely therefore that the wide range of target cell types that can be lysed by NK cells share common cell surface characteristics which render them susceptible. As lysis results from membrane-membrane interactions, it seemed logical that a search for 'hallmarks' of NK susceptibility should begin with a detailed examination of the plasma membrane of susceptible cells. Analysis of one pair of lymphoma cell variants, selected on account of their markedly different susceptibility to NK cells, suggests that cell surface glycoconjugates may be of significance in determining those effector cell-target cell interactions that lead to lysis. This review outlines attempts to characterize such glycoconjugates.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Carbohydrates; Cell Adhesion; Cell Membrane; Cells, Cultured; Clone Cells; Cytotoxicity, Immunologic; Gangliosides; Glycolipids; Glycosphingolipids; Humans; Killer Cells, Natural; Leukemia L5178; Mice; Rabbits; Rats