BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

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Ganglioside G(M1) is the cell surface receptor for cholera toxin (CT). The oligosaccharide chain (G(M1)OS) is recognized by the B or binding subunits of CT whereas the A subunit of the toxin activates adenylyl cyclase by ADP-ribosylation of the stimulatory G protein (Gs) of the cyclase system. The pathway by which the A subunit gains access to Gs has not yet been established. We previously had shown that neoganglioproteins generated by covalently attaching G(M1)OS to cell surface proteins are nonfunction receptors for CT. Thus, CT binds to rat glioma C6 modified with G(M1)OS but is unable to stimulate adenylyl cyclase. We recently found that in the presence of chloroquine, these neoganglioproteins became functional receptors for CT. To further explore the role of neoganglioproteins as toxin receptors,we covalently attached G(M1)OS to human transferrin. The derivative bound with high affinity to transferrin receptors on HeLa cells and supported the binding of CT. The toxin, however, was unable to activate adenylyl cyclase in contrast to G(M1)-treated HeLa cells. Chloroquine treatment allowed CTL activate adenylyl cyclase in HeLa cells treated with G(M1)-transferrin. By contrast, pretreatment of either control or G(M1)-treated HeLa or C6 cells with chloroquine had no effect on toxin stimulation of adenylyl cyclase. These results indicate that CT bound to G(M1) gains access to adenylyl cyclase through a different pathway than when bound to neoganglioproteins. E. coli heat-labile enterotoxin (LT) is structurally similar to CT, binds to GM, and activates adenylyl cyclase. A human intestinal cell line was found to have additional binding sites for LT on the brush border membranes. These sites were identified as galactoproteins. We are now determining whether these binding sites can serve as functional receptors for LT and allow LT to enter the cells through a different pathway than CT.