Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides

Abstract

Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration. In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm. A linear increase in difference absorption was observed at these wavelengths vs. oligosaccharide concentration; a saturation effect occurred when the molar ratio of oligosaccharide to cholera toxin was higher than 5. The features of the spectra indicated that the binding with the oligosaccharide affected the environment of tryptophan and tyrosine residues of protomer B. In good agreement with the above results, circular dichroic spectra indicated also a local effect of the binding, mostly restricted to protomer B, while the residues of protomer A remained largely unperturbed. Difference absorption spectra were also measured for cholera toxin in the presence of ganglioside and detergent micelles. The employed gangliosides GD1a and GT1b, unable to bind cholera toxin, interact with the protein by way of contaminating traces of GM1. The preparations of GD1a and GT1b contained 0.8-1.0% (w/w) and 0.4-0.5% (w/w) of GM1, respectively. The results obtained with ganglioside GD1a and GT1b in contrast with the observations made with the oligosaccharide of GM1 indicated a major conformational change of the toxin structure. Upon comparison of the conformational change induced by ganglioside micelles with that induced by sodium dodecyl sulfate it may be suggested that the ganglioside micelle, behaving as a detergent, alters the structure of the toxin such as to induce the penetration of protomer A into the lipid milieu.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, stability, and receptor interaction of cholera toxin as studied by Fourier-transform infrared spectroscopy

“…A quantitative analysis of the spectra allowed us to estimate that no greater than 3-4% change in @-sheet or a-helix content could Occur in the presence of the receptor. Previous fluorescence and absorption spectroscopy titration experiments with oligo-GMl (Fishman et al, 1978;Tomasi et al, 1984), as well as the estimated toxinreceptor binding constants (Sattler et al, 1977(Sattler et al, , 1978Fishman et al, 1978), strongly indicate that under the experimental conditions of this study (receptor to B subunit monomer molar ratio of 2: 1) virtually all the protein present in solution is associated with the receptor. The absence of unbound protein in our samples of subunit B mixtures with ganglioside GMl or oligo-GM, is further corroborated by the studies of temperature dependence of infrared spectra (Figures 2 and 3), which demonstrate a complete elimination of a thermotropic transition characteristic of the free protein.…”

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

“…The calorimetric results presented in this paper revealed that the major effect of ganglioside is on the cooperative interactions between the B subunits and demonstrate further that there is little, if any, direct interaction between the A subunit and ganglioside GMl. This conformational difference probably does not represent a gross structural rearrangement (Dwyer & Bloomfield, 1982) but rather local amino acid side chain reorientation due to ganglioside GMi association (Tomasi et al, 1984). The binding site for the ganglioside GMl has been postulated as being at the interface between B subunit monomers (DeWolf et al, 1981), thus providing a structural basis for the observed enhancement in intersubunit interactions.…”

“…By contrast, incubation of intact toxin with ganglioside GMl results in only a slight increase in the transition temperature for the A subunit transition. The results from several spectroscopic investigations (Fishman et al, 1978;Sillerud et al, 1981;Tomasi et al, 1984) have suggested the existence of a conformational change in the B subunit upon binding to ganglioside GMl. The calorimetric results presented in this paper revealed that the major effect of ganglioside is on the cooperative interactions between the B subunits and demonstrate further that there is little, if any, direct interaction between the A subunit and ganglioside GMl.…”

pH-induced transitions in cholera toxin conformation: a fluorescence study

“…This would result in the dissociation of CT into CT A and CT B and subsequent penetration of the A component into the cell membrane. The initial highly specific interaction of CT with the oligosaccharide moiety of GM, (oligo-GM,) appears to occur without any substantial conformational change (De Wolf et al, 1981b;Tomasi et al, 1984). It was shown (De Wolf et al, 1981a,b;Sillerud et al, 1981;Tomasi et al, 1984;Ludwig et al, 1985) that the lone Trp-88 residue of each /3-polypeptide chain is an important determinant in this interaction.…”

“…The initial highly specific interaction of CT with the oligosaccharide moiety of GM, (oligo-GM,) appears to occur without any substantial conformational change (De Wolf et al, 1981b;Tomasi et al, 1984). It was shown (De Wolf et al, 1981a,b;Sillerud et al, 1981;Tomasi et al, 1984;Ludwig et al, 1985) that the lone Trp-88 residue of each /3-polypeptide chain is an important determinant in this interaction. In a previous paper (De Wolf et al, 1985), the pH dependence of the fluorescence characteristics of CT that are related to this lone Trp-88 were examined.…”