Calorimetric and fluorescent characterization of the interactions of cholera toxin with its cell surface receptor, ganglioside GM₁₊

The physical nature of the sequence of events associated with the binding and insertion of cholera toxin into membranes was studied using high sensitivity differential scanning calorimetry (DSC), fluorescence spectroscopy, isothermal reaction calorimetry and differential solubility thermal gel analysis. This sequence of events consists of 1) association of toxin to membranes containing ganglioside G_M1, the toxin cell surface receptor and 2) release and penetration of the toxin bioactive (A) subunit into receptor-containing membranes.

The effect of ganglioside G_M1 association on toxin structure and function was studied by monitoring toxin thermal stability using DSC and differential solubility thermal gel analysis. The results of this investigation showed 1) stabilization of the B subunit following ganglioside binding 2) enhancement of the cooperative interaction between B monomers in the B pentamer and 3) absence of an exothermic heat effect due to A subunit precipitation.

The interactions of toxin with phospholipid-G_M1 bilayers indicate the toxin associates to both phases of the bilayer. Also toxin association results in only a small decrease in the ganglioside lateral mobility. DSC studies of intact toxin or its isolated subunits with phospholipid-G_M1 bilayers showed that the B subunit associated to and remained on the bilayer surface. In addition, these calorimetric experiments indicated that the toxin insertional mechanism was by a direct penetration of the A subunit into the lipid m bilayer matrix. The insertional event was facilitated under conditions of lateral phase separation induced by calcium. These results are in agreement with toxin thermal denaturation studies using thermal gel analysis and DSC that there is a hydrophobic interaction between the A subunit and the lipophilic matrix of ganglioside_M1 micelles and bilayers containing ganglioside_M1.

This study represents the first time that the interactions of a ligand-receptor system have been investigated at this detailed molecular level.