NEOGLYCOLIPID ANALOGS OF GANGLIOSIDE-GM1 AS FUNCTIONAL RECEPTORS OF CHOLERA-TOXIN

We synthesized several lipid analogues of ganglioside G(M1) by attaching its oligosaccharide moiety (G(M1)OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated G(M1)-deficient rat glioma C6 cells with each of the derivatives as well as native G(M1) and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of I-125-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C-10 derivative was ineffective, C-12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native G(M1), whereas the phospholipid derivatives were less effective. The distance between G(M1)OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of G(M1)OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching G(M1)OS to PE by reductive amination. Furthermore, insertion of a C-8 spacer in the former neoglycolipid rendered it even less effective. Finally, cells treated with neoglycolipids based on phospholipids exhibited a longer lag period before a response to cholera toxin was observed compared to cells treated with G(M1) or neoglycolipids based on stearylamine or cholesterol. Our results indicate that although G(M1)OS provides the recognition site for the binding of cholera toxin, the nature of the lipid moiety plays an important role in the action of the toxin.