The Role of the Reactive Disulfide Bond in the Interaction of Cholera‐Toxin Functional Regions

Abstract

The chemical reactivity of disulfide bonds towards reducing agents, in the absence of denaturing conditions, in cholera toxin has been studied. Treatment of the toxin with dithiothreitol or other mercaptans gave selective reduction of one of the six disulfide bonds of the protein. This reactive disulfide links two distinct functional regions of the toxin, fragment CI, which activates adenylate cyclase, and fragment y/35, which recognizes the cell surface receptors. Upon reduction, the two fragments remain bound together and the secondary structure of the protein is retained. The two functional regions have been separated and purified only by methods based on charge differences. When mixed together, purified a and purified y/35 fragments spontaneously and rapidly re-form the disulfide bond. However, reduction of the disulfide bond is an absolute requirement for freeing the catalytic site of the CI functional region. Thus, while other non-covalent binding regions are involved in maintaining cholera toxin molecular structure, the reactive disulfide bond may play a role in the mechanism of cell intoxication.Cholera toxin is a protein extremely potent in stimulating the membrane-bound enzyme, adenylate cyclase, in a variety of mammalian cells [1,2]. It has been shown that the toxin molecule is composed of two protomeric species A and B [3-81. Protonier A contains two non-identical polypeptide chains CI and y, linked through a single disulfide bond. The remainder of the molecule consists of five identical polypeptide chains [9], each containing a single intra-chain disulfide bond, forming a stable aggregate, protomer B.Thus, the toxin has a molecular formula ay/35. The A protomer is required for the activation of adenylate cyclase [lo], while protomer B is responsible for the binding of the toxin to the cell [ll,12], interacting specifically with the hydrophilic moiety of ganglioside GMI [13].Protomer A and B are very tightly bound as shown by their slow dissociation in sodium dodecyl sulfate or urea solutions. Dissociation conditions in acid pH, with or without unfolding agents, separate cholera toxin into two fractions, protomer A and the disaggregated /3 polypeptide chains [3,6,7]. Although the lipid environment of the cell membrane might ayl-(N-acetylneuraminyl)-galactosylglucosylceramide.mimic the properties of detergents, no direct evidence of the dissociation in vivo of protomers A and B has been described. It seems more probable, as suggested by Gill [8], that one of the commited steps in the intoxication process is the cleavage of one of the six disulfide bonds of cholera toxin. Exposure of native toxin to dithiothreitol, in the absence of denaturing agents, splits selectively the reactive disulfide bond joining the CI and y polypeptide chains. This partial reduction results in the formation of two fragments, possessing functional properties, the a chain and a complex of the y polypeptide chain and protomer B, y/35 [3 -8,141. The a fragment stimulates adenylate cyclase in cell-free systems in an NAD-dependen...

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

“…For isolation of EVs, subconfluent monolayers of CHO and Me665 cells in exponential growth were incubated in DMEM supplemented with 0.3 % FBS with or without 12 nM CT, purified from culture filtrates of Vibrio Cholerae 569 B, serotype Inaba as described by [40]. After 2 h of CT treatment, control and treated cells were washed in PBS and further cultured in fresh DMEM medium for 24 h before collection of medium for EVs isolation.…”

Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS

“…In comparison to published on-plate digestion of proteins, 57% coverage is in the range that is considered acceptable [53,54]. Sequences 1-22 and 82-103 are connected through intra-chain disulfide bonds [55] and were not detected; it is possible that disulfide bridge reduction may be necessary to achieve full coverage. In comparison, van Baar et al [39] used liquid chromatography-electrospray MS to characterize cholera toxin.…”

Toxin Instability and Its Role in Toxin Translocation from the Endoplasmic Reticulum to the Cytosol

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