Studies of the Subunit Structure of Choleragen

Abstract

The two choleragen protein constituents were isolated and characterized. Protein I has a molecular weight of approximately 54 000. It consists of subunits of approximate molecular weight 10 000. Protein I1 with molecular weight of approximately 32000 is cleaved by 2-mercaptoethanol into two fragments, protein 11, (N-terminal Asx, M , = 25000) and protein 11, (N-terminal Ser, M , = 7000).Proteins 111 and 112 could be recombined by oxidation to yield protein 11. Upon treatment of choleragen with 2-mercaptoethanol protein 11, precipitates quantitatively. The remaining protein consisting of proteins I and II,, was quantitatively precipitated by ganglioside GGtetl. Of the separated choleragen subunit proteins, only protein I and not protein I1 complexed specifically with ganglioside GGtetl. The isolated proteins I and I1 were considerably less toxic in the skin test but almost full toxicity was recovered after mixing the two proteins I and 11. Antisera against protein I and protein 11 revealed no immuno-cross reactivity between the two proteins. Both antisera inhibited the biological effects of choleragen in the skin and ileal loop tests. A molecular model for the constitution of choleragen is proposed.The pathogenic effects of cholera bacteria in vivo are caused by the action of a secreted toxin upon the mucosal cells. This cholera exo-entero-toxin, choleragen, induces major irreversible membrane permeability changes including the massive outflow of fluid into the intestine. Choleragen also acts on numerous other cell types by way of stimulation of adenylate cyclase (for review see [3]).The work of van Heyningen and collaborators first identified ganglioside to be the cell receptor for choleragen [l 1. These authors discovered that choleragen complexes very specifically with the major monosialo-ganglioside of brain, ganglioside GGtetl .In an earlier investigation it was shown that both the lipophilic and the hydrophilic moiety of the glycolipid contribute to the strength of the binding [Z]. It was also reported that the precipitation of choleragen with ganglioside GGtetl leads to the formation of an apparently high-molecular-weight aggregate of one of the two protein subunits of the toxin. Theother protein subunit, however, showed a less tight binding to the toxin-ganglioside complex and could be readily dissociated by detergents.The ganglioside complexing ability does not appear to be directly related to the toxic activity of the choleragen in vivo. This is shown by the fact that ganglioside is also bound to choleragenoid, which itself is nontoxic and consists only of one type protein of the original choleragen. Furthermore, gangliosidoides i.e. analogues of the ganglioside GGtetl, display comparable binding properties to choleragen, but in contrast to the natural ganglioside still do not inhibit its toxicity in vivo.In order to study the role of the subunits of choleragen in the binding to ganglioside GGtJ as well as choleragenicity, the two proteins were separated and isolated in pure form.The two choleragen prot...

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

“…1, was an association of the p and y chains attached to form the y p~ complex, while the faster-migrating fragment was identical to the CI chain, as already reported [14]. Amino acid analyses of the fragments, isolated by preparative disc gel electrophoresis, confirmed such a polypeptide chain composition, and were in agreement with data from other laboratories [4,7]. Further evidence on the identity of the fragments was obtained by studies on their biological properties.…”

Mechanism of action of choleragen

“…The A subunit consists of two peptide chains, A,(20,000-25,000 daltons) and A2 (2,500-9,700 daltons), linked through a single disulfide bond [25][26][27][29][30][31][32][33][34]. The complete amino acid sequence of the B subunit and parts of the A i and A2 peptideshave recently been determined [30,32,36--38,41,42].…”

Protein secretion and the pathogenesis of bacterial infections

“…Secreted CtxB 5 binds to G m1 ganglioside , a sphingoglycolipid, on the surface of human intestinal cells (Cuatrecasas 1973;Holmgren et al 1973;King and Van Heyningen 1973), causing toxin uptake and reduction of CtxA by cytosolic thioredoxin. The reduced CtxA fragment is released from the membranebound receptor complex (Sattler and Wiegandt 1975; To- Figure 6. Type II secretion across the outer membrane.…”