INTERACTION OF A CHOLERA-TOXIN DERIVATIVE CONTAINING A REDUCED NUMBER OF RECEPTOR-BINDING SITES WITH INTACT-CELLS IN CULTURE

Hybrid CTB (hCTB), having only one or two functional binding sites, has been constructed from two chemically inactivated derivatives of CTB. One inactive derivative consisted of CTB formylated in the lone Trp-88 of each beta-chain (fCTB), whereas the other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site (sssCTB). hCTB, fCTB and sssCTB were able to reassociate with CTA and form the corresponding holotoxins hCT, fCT and sssCT as measured by gel filtration chromatography. In contrast to fCT and sssCT, hCT could increase the cAMP content of intact Vero cells in a time- and dose-dependent way: concentrations as low as a few nanograms of hCT per milliliter caused a significant increase in the intracellular cAMP level. The maximal cAMP level induced by hCT (1 mu g/ml) was, however, more than 2-fold lower than that elicited by its native counterpart. At saturating ligand concentrations and at 37 degrees C, the lag periods and rates of CT and hCT induced cAMP accumulation were essentially the same. Treatment of Vero and HeLa cells with GM(1) did not affect their difference in response to CT and hCT. When Vero cells treated with hCT were incubated for longer periods of time, a further slow accumulation of cAMP occurred until after about 20 h cAMP levels of cells exposed to CT or hCT were essentially the same. In contrast to Vero and HeLa cells, human skin fibroblasts exhibited an almost identical response to CT as well as to hCT. Acidotropic agents such as chloroquine and monensin affected the CT and hCT induced increase in cAMP content of Vero cells, fibroblasts and GM(1) treated Hela cells in a similar way. The results are consistent with the view that CT receptor recognition domains are shared between adjacent beta-chains, that pentavalent binding appears not to be essential for cytotoxicity and that in the cell types studied intracellular processing of CT, hCT is involved.