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

Mj, De Wolf; Ga, Van Dessel; Ar, Lagrou; Hj, Hilderson; Ws, Dierick

doi:10.1021/bi00387a010

Cited by 31 publications

(21 citation statements)

References 35 publications

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“…A non-native pentamer had already been reported for the B subunits of various AB 5 toxins [12], [13], [18]. The non-native pentameric conformation was adopted after treatment of the native pentamer at pH 5.0 and did not result from disassembly/reassembly reactions.…”

Section: Discussionmentioning

confidence: 86%

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Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

et al. 2010

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The cholera toxin B pentamer (CtxB5), which belongs to the AB5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB5 and the related heat labile enterotoxin LTB5 have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.

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Section: Discussionmentioning

confidence: 86%

“…Among AB 5 members, the in vitro disassemblies of the cholera toxin B subunit (CtxB 5 ) and of the human heat-labile enterotoxin B subunit (LTB 5 ) are well documented [11], [12], [13], [14], [15], [16], [17]. The two toxins in vitro assemblies have also been reported [18], [19], [20], [21], [22].…”

Section: Introductionmentioning

confidence: 99%

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

et al. 2010

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“…The structural properties of subunits B and A are generally consistent with the view (Goins & Freire, 1988) that the main role of the B subunit is to provide a water-soluble carrier to the A subunit and to place it in close proximity of the membrane surface. The actual mechanism of membrane translocation of the toxic A subunit is likely to take advantage of the intrinsic properties of this subunit, such as its hydrophobicity (Moss et al, 1977b;Goins & Freire, 1985;De Wolf et al, 1987) and a relatively loose folding. G,,, 37758-47-7. containing domains usually also have two Asp/Glu N-terminal to the first Cys residue (Figure 1).…”

Section: Discussionmentioning

confidence: 99%

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

1990

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The structure and thermal stability of isolated B and A subunits of cholera toxin, as well as the interaction of the B subunit with a ganglioside GM1 receptor, were studied by Fourier-transform infrared spectroscopy. The B subunit of the toxin is highly folded; its secondary structure consists predominantly of beta-sheets. The temperature dependence of the infrared spectrum indicates that the B subunit undergoes thermal unfolding in the temperature range between approximately 66 and 78 degrees C. Binding to the ganglioside GM1 receptor or to its oligosaccharide moiety results in only marginal, if any, change in the secondary structure of the B subunit; however, the receptor-associated subunit does show a markedly increased thermal stability. The secondary structure of the enzymatically active A subunit is less ordered and much less stable than that of the B subunit. The relatively loose folding of the A subunit is likely to be of importance for the effective membrane translocation of this subunit.

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“…Proteolytic processing of CT by a metalloendoprotease, on the other hand, may offer an explanation for the observation that a serine protease-resistant CT mutant (CTR 192H) where Arg-192 has been replaced by a His residue, inactivating the nicking site connecting the A 1 and A 2 polypeptides of CT-A, was still able to elicit a secretory response in polarized T84 cells (35). We assume that the holotoxin-GM 1 complex is transported to the ER, which would fit with the marked stability of this complex (36,37). Proteolytic FIG.…”

Section: Is the Inhibitory Effect Of Cbz-gly-phe-nh 2 -Mediated By A mentioning

confidence: 95%

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

Wolf

2000

Journal of Biological Chemistry

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Prior exposure (15 min at 37°C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mM N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH 2 ), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH 2 , an inactive analogue of Cbz-Gly-Phe-NH 2 . The effect was reversible and dose-(IC 50 as low as 0.2 mM depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH 2 during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A 1 peptide of CT and NAD ؉ activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH 2 -treated cells or when Cbz-Gly-Phe-NH 2 was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH 2 and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFAresistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH 2 on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca 2؉ homeostasis by Cbz-Gly-Phe-NH 2 is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of 125 I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH 2 did not completely suppress the formation of the catalytically active A 1 fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH 2 cannot be excluded.Cholera toxin (CT), 1 the enterotoxin secreted by Vibrio cholerae classical as well as El Tor biotypes, is the major causative agent of the acute diarrheal disease of humans. CT and the Escherichia coli heat-labile enterotoxin (LT), are structurally and immunologically highly homologous, belonging to the same enterotoxin family (1, 2). Both are oligomeric proteins of the A-B type. CT is composed of one A or activating subunit (CT-A M r 27,400), which consists of two distinct polypeptide chains CT-A 1 (M r 22,000) and CT-A 2 (M r 5,400), linked by a single disulfide bridge and five identical B subunits (M r 11,600) arranged in a ring-like configuration (CT-B).The subunits are arranged such that CT-A occupies the central channel of the CT-B pentamer extending well above the plane of the pentameric ring (3, 4). The CT-A 2 peptide goes through the pore in the doughnut-like structure of the CT-B pentamer and protrudes on the side that binds cell surface receptors with its COOH-termi...

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

Cited by 31 publications

References 35 publications

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

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

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

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