Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

Chloroquine inhibition of cholera toxin

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.     

Vibrio cholerae: cholera toxin

The bacterial protein toxin of Vibrio cholerae, cholera toxin, is a major agent involved in severe diarrhoeal disease. Cholera toxin is a member of the AB toxin family and is composed of a catalytically active heterodimeric A-subunit linked with a homopentameric B-subunit. Upon binding to its receptor, GM0(1), cholera toxin is internalized and transported in a retrograde manner through the Golgi to the ER, where it is retrotranslocated to the cytosol. Here, cholera toxin reaches its intracellular target, the basolaterally located adenylate cyclase which becomes constitutively activated after toxin-induced mono-ADP-ribosylation of the regulating G(S)-protein. Elevated intracellular cAMP levels provoke loss of water and electrolytes which is manifested as the typical diarrhoea. The cholera toxin B-subunit displays the capacity to fortify immune responses to certain antigens, to act as a carrier and to be competent in inducing immunological tolerance. These unique features make cholera toxin a promising tool for immunologists.     

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1

BT23, a Vibrio cholerae O1 El Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.     

Primary structure of cholera toxin B-subunit

The primary structure of cholera toxin B-subunit, responsible for the binding of the toxin to cell surfaces, has been elucidated. The polypeptide contains 103 amino acid residues and one intra-chain disulfide bridge between Cys 9 and Cys 86. The molecular weight is calculated to be 11,637, 15–20% higher than the values estimated by physicochemical methods. This value is consistent with a structure containing five moles of B-subunits per mole of cholera toxin.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Cholera toxin as Vibrio cholera superantigen

Experimental data confirming our earlier suggestion, that cholerae toxin (CT) possesses superantigen (SA) properties are presented. When used in very small doses, CT has been found to induce polyclonal activation of T lymphocytes, essentially exceeding that observed in classical T mitogens characteristic of SA. CT, in contrast to mitogens and similarly to other SA, is shown to display this activity only in the presence of antigen-presenting cells. Experiments with the use of monoclonal antibodies to the variable region of the beta-chain of the T-cell receptor (V beta TCR) have demonstrated that CT, similarly to other SA, are capable of inducing expression of certain types of V beta TCR and causing polyclonal activation of T lymphocytes carrying these types of V beta TCR. The presence of these properties gives grounds for regarding CT as SA. The SA activity of CT has been found to be linked with its subunit A.     

ADP-ribosylation of bovine S-antigen by cholera toxin

The S-antigen (alias 48K protein or arrestin) of bovine rod photoreceptors contains two stretches of amino acid sequence homologous to the ADP-ribosylation sites of the alpha subunit of transducin (Ta). We have found that cholera toxin transfers the ADP-ribosyl group from NAD to purified bovine S-antigen as well as to S-antigen in rod outer segment membranes, while Bordetella pertussis toxin is unable to catalyze the transfer reaction efficiently. Under the same conditions, both toxins catalyzed ADP-ribosylation of Ta in rod outer segments. The ADP-ribosylation of S-antigen by cholera toxin indicates that S-antigen not only exhibits sequence homology with the ADP-ribosylation sites of Ta, but it must also resemble Ta in the tertiary structure of the domain which determines the susceptibility of S-antigen to the catalytic action of cholera toxin. These results suggest that S-antigen may function as a competitor of Ta in some stage of the cGMP cascade of visual transduction.     

Photolabelling of cholera toxin by NAD+.

When cholera toxin is incubated under u.v. light with NAD+ labelled in either the adenine or the nicotinamide moiety, radioactivity becomes covalently bound to the protein. The reaction is specific for cholera toxin, and is inhibited by excess unlabelled NAD+ or NAD analogues. Only the active A 1 chain of the toxin is labelled. The u.v.-absorption spectrum of the product is very similar to that of NAD+, and shows the same reaction with cyanide. The nature of the product is therefore different from that found when diphtheria toxin is photolabelled [Carroll & Collier (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3307-3311] in that the yield is lower, but both moieties of the NAD molecule become bound.     

Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer

Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.     

Immunomodulatory effects of cholera toxin in mice

Immunomodulatory effects of cholera toxin (CT) were investigated in a murine model using various immunological parameters. C3H/HeN mice were injected with 2 micrograms of CT at various intervals (from 6 h to 21 days) before the immunological assays. Thymocytes were markedly decreased in their absolute number, and the phenotypes in such cells were clearly shifted from Thy1.2high+ PNAhigh+ to Thy1.2low+ PNAlow+ 2-4 days after the CT treatment. Spleen T cells were relatively increased, while surface IgM positive B cells were rather decreased. Natural killer activity and in vivo and in vitro cytotoxic T lymphocyte activity were markedly suppressed during the early stages after the CT treatment but recovered completely within 21 days. Mixed lymphocyte reaction was profoundly suppressed at least for the 1st week after the CT treatment. Furthermore, EL-4 tumor of C57BL/6 origin grew progressively and killed the recipient C3H mice when such tumor cells were inoculated 6 h after the CT treatment. On the contrary, a marked augmentation of direct (IgM) and indirect (IgG) plaque-forming cell responses to sheep red blood cells was seen after CT treatment. Delayed footpad reaction to SRBC was also augmented after CT treatment. As the mechanisms, both direct augmentation of CD4+ T cells and direct suppression of CD8+ T cells appeared to occur at a time due to the CT treatment. An indirect effect of CT through the release of the endogenous steroids was dismissed in the present study. Taken together, CT appears to have differential immunomodulatory effects on various immune effector cells through various mechanisms.     

Quantification of gangliotetraose gangliosides with cholera toxin

A procedure is described for assay of GM1 and other gangliotetraose-type gangliosides at the picomole level. The gangliosides are absorbed onto polystyrene microwells and treated with neuraminidase and then with cholera toxin B subunits conjugated to horseradish peroxidase. Color is developed and quantified spectrophotometrically. Omission of neuraminidase gives a measure of GM1 alone. Linearity was obtained between 0.5 and 3 pmol. This procedure was applied to ganglioside mixtures isolated fron neuro-2A neuroblastoma and PC12 pheochromocytoma cells. For the latter, an additional step involving reaction with fucosidase increased the yield of GM1 due to the presence of fucosylated gangliosides. Application of the same reagents as a TLC overlay procedure to the gangliosides from neuro-2A cells revealed the presence of GD1a, GD1b, and GT1b in addition to GM1, thus confirming the presence of a family of gangliotetraose gangliosides.     

The arrangement of subunits in cholera toxin.

Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion.     

Binding of NAD+ by cholera toxin.

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.     

Interaction of cholera toxin B-subunit with human T-lymphocytes

In this work, ¹²⁵I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (K _d = 3.3 nM) was determined. The binding of the ¹²⁵I-labeled CT-B was inhibited by unlabeled interferon-α₂ (IFN-α₂), thymosin-α1 (TM-α₁), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α₁ and 131-135 of IFN-α₂ (K _i 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (K _i > 1 μM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.