A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

Abstract

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4+ T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts

“…It fails to induce a potent anti-B-subunit response in mice, and it also fails to serve as mucosal adjuvant. In the same manner, CTB (H57A) binds GM1 but lacks immunomodulatory or toxic activity (39). In the present study, we demonstrate that GM1 ligation alone, via rabbit polyclonal Abs anti-GM1, is not able to inhibit CD4 ϩ T lymphocyte activation and proliferation.…”

“…We demonstrate that GM1 ligation by Abs does not lead to SMase activation and SM hydrolysis (data not shown). Aman et al (39) hypothesize that CT may require interaction, not only with GM1, but also with another molecule to exert its biological activity. For Aman et al (39), it is conceivable that rCTB binding to GM1 in lipid rafts would position it to interact with signaling molecules that participate in toxin-mediated immune cell modulation.…”

“…Aman et al (39) hypothesize that CT may require interaction, not only with GM1, but also with another molecule to exert its biological activity. For Aman et al (39), it is conceivable that rCTB binding to GM1 in lipid rafts would position it to interact with signaling molecules that participate in toxin-mediated immune cell modulation. In respect with our experimental data, it is quite conceivable that the intermediary molecule involved in the toxin action would be a raft-resident neutral SMase.…”

Ganglioside GD1a Is an Essential Coreceptor for Toll-like Receptor 2 Signaling in Response to the B subunit of Type IIb Enterotoxin

“…56). The ganglioside binding activity of the B pentamers is important but not sufficient for their immunomodulatory effects, since certain point mutations that do not preclude ganglioside binding result in defective immunomodulatory action (57,58). These findings suggest that additional receptors may be required for optimal immunomodulatory signaling by enterotoxin B pentamers.…”

Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit

“…It was initially thought that the adjuvant properties of EtxB were dependent on receptor binding activity as a nonbinding point mutant of EtxB (G33D), lost all immunomodulatory activity. However, the CtxB mutant, CtxB (H57A) was shown to retain the ability to bind to G M1 but was unable to act as an immunomodulator (Truitt et al, 1998;Aman et al, 2001;Fraser et al, 2003). It is now theorized that structural alterations in CtxB (H57A), while not preventing binding to G M1 , may preclude interactions with additional co-receptors required for signaling (Fraser et al, 2003).…”