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At least 19 recordsLinked to original sources
PubMed1976

Affinity chromatography of tetanus toxin, tetanus toxoid, and botulinum A toxin on synaptosomes, and differentiation of their acceptors.

125I-labelled tetanus toxin and 125I-labelled botulinum A neurotoxin are known to be specifically bound to brain synapotosomes. In order to discriminate between active toxin and inactive admixtures present in the starting material or arising during iodination, synaptosome columns were prepared using bromacetylcellulose and/or kieselgur (Celite) as carriers. Both types of columns absorb the toxins from low ionic strength medium and release them if the pH and ionic strength are raised. Botulinum toxin was eluted with lower ionic strength than tetanus toxin, and could be freed from nontoxic admixtures. Analysis by affinity chromatography disclosed partially toxoided tetanus toxin in both labelled and unlabelled toxin samples. High concentrations of formaldehyde (0.5%) destroyed both toxicity and affinity to the synaptosomes of tetanus toxin. Low concentrations of formaldehyde (0.05%) yielded a derivative of low toxicity which was still however less firmly, bound to synaptosomes. Tetanus and botulinum toxin differ by their acceptors. Whereas unlabelled botulinum toxin is unable to compete with labelled tetanus toxin, unlabelled tetanus toxin slightly competes with botulinum toxin. Both labelled toxins display anomalous binding behavior in that they cannot be displaced completely even with a large excess of unlabelled toxin.

Affinity Labels↗
PubMedDate not supplied

Are DNA-based vaccines useful for protection against secreted bacterial toxins? Tetanus toxin test case.

Polypeptide and DNA vaccine alternatives to the conventional tetanus toxoid were compared. Mouse immunizations with plasmid DNA that encoded the tetanus toxin C fragment polypeptide induced consistently lower antibody responses than direct immunization with the C fragment polypeptide or toxoid, yet provided some degree of protection from a lethal toxin challenge. Cytotoxic T-cell responses dominated DNA immunizations, while specific T-cell proliferation resulted from all vaccines tested. Immune responses to the DNA vaccine exhibited a T-helper type-1 propensity, while polypeptides elicited T-helper type-2 responses. The lower antibody response to the plasmid vaccine was not due to insufficient quantity of C fragment in vivo but was likely the result of a mode of antigen presentation that was less efficient for supporting antibody production. Collectively, these results suggest that polypeptide or toxoid vaccines are preferable to plasmid-based vaccination for control of diseases caused by tetanus toxin.

Amino Acid Sequence↗
PubMed1981

The hydrophobicities of cholera toxin, tetanus toxin and their components.

1. Charge-shift electrophoresis showed that cholera toxin and its subunits have no hydrophobic surfaces. 2. Amino-acid composition and sequence data suggested that the proteins have no masked hydrophobic regions. 3. The A subunit of cholera toxin may interact with polar molecules in the membrane to exert its effect inside the cell. 4. The only hydrophobic part of tetanus toxin was the H-chain.

Amino Acids↗
PubMed2006

Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli.

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.

Animals↗
PubMed1999

Humoral and cellular immune responses in mice immunized with recombinant Mycobacterium bovis Bacillus Calmette-Guérin producing a pertussis toxin-tetanus toxin hybrid protein.

The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60 promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.

Animals↗
PubMed1992

Restoration of exocytosis occurs after inactivation of intracellular tetanus toxin.

Tetanus toxin blocks carbachol-stimulated release of noradrenaline from bovine adrenal chromaffin cells in culture, provided it can gain access to the cells. This can be achieved by electropermeabilization of the plasma membrane or by enriching the membrane with exogenous gangliosides which serve as carriers of the toxin. The inhibition of noradrenaline release persists for at least 6 days, even in the presence of specific anti-tetanus toxin antibodies in the culture medium. However, the block is preventable, for the most part, when antibodies enter chromaffin cells during electropermeabilization, before the uptake of the toxin is facilitated by inserting exogenous gangliosides into the plasma membrane 2 days later. This indicates that the antibodies pass into the cells through the physically induced pores and that these intracellular antibodies neutralize incoming tetanus toxin. If, on the other hand, exocytosis has been inhibited by tetanus toxin, it will recover within 3 days, provided specific anti-tetanus toxin antibodies are introduced into the cells by electropermeabilization. The recovery is not linked to a specific route of entry of the toxin. It is concluded that the restoration of noradrenaline release requires not only the intracellular neutralization of tetanus toxin but also the reconstitution of the as yet unknown target molecule of the toxin.

Adrenal Glands↗
PubMed1996

Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin.

Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani. Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin. A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae. A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed. Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC. The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model. Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC. The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon. Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin. Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin.

Animals↗
PubMed1994

The role of transglutaminase in the mechanism of action of tetanus toxin.

Tetanus toxin is a zinc-dependent metalloendoprotease that cleaves synaptobrevin, a polypeptide found in the membranes of synaptic vesicles. This action is thought to account for toxin-induced blockade of transmitter release. However, Facchiano and Luini (Fachiano, F., and Luini, A. (1992) J. Biol Chem. 267, 13267-13271) have proposed that tetanus toxin can stimulate transglutaminase, and Facchiano et al. (Facchiano, F., Benfenati, F., Valtorta, F., and Luini, A. (1993) J. Biol Chem. 268, 4588-4591) have further proposed that the stimulated enzyme produces cross-linking of synapsin. These actions might also account for toxin-induced blockade of exocytosis. Therefore, a series of experiments were performed to evaluate the possibility that tetanus toxin exerts its effects via transglutaminase. The results indicated that clostridial neurotoxins were poor substrates for the cross-linking effects of transglutaminase, and transglutaminase was a poor substrate for the proteolytic actions of tetanus toxin. In addition, at concentrations relevant to blockade of exocytosis, clostridial neurotoxins did not act on intact cells to stimulate transglutaminase, nor did they act on the isolated enzyme to stimulate cross-linking of putrescine and dimethylcasein. When used as competitive inhibitors of endogenous transglutaminase substrates, glycine methyl ester and monodansylcadaverine did not block toxin action. Furthermore, concentrations of calcium that were too low to support transglutaminase activity did not prevent toxin action. The data suggest that stimulation of transglutaminase is not the principal mechanism by which tetanus toxin blocks exocytosis in nerve cells.

Amino Acid Sequence↗
PubMed1990

An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin.

Tetanus toxin is composed of a heavy chain (100 kDa) and a light chain (50 kDa) held together by a single interchain disulfide bridge. An additional intrachain disulfide is present in the carboxy-terminal part of the heavy chain. Reduction of the two disulfide bonds in tetanus toxin with both chemical and proteinaceous reducing agents was studied. Dithiothreitol and 2-mercaptoethanol cleaved both the inter- and intrachain disulfide bridges of the toxin, while glutathione and cysteine were ineffective. Specific reduction of the single interchain disulfide link was achieved with the thioredoxin-thioredoxin reductase system, thus indicating that this bond is exposed at the protein surface. Also, dead or permeabilized cells were able to reduce the toxin. Such reduced toxin bound to neuronal membranes as well as the native toxin but was not neurotoxic. These findings open the possibility that reduction by cytoplasmic agents released by dead cells contributes to detoxification of tetanus toxin. Moreover, together with the notion that the light chain is the active form of the toxin in the cytoplasm, these results suggest that the interchain disulfide bond of tetanus toxin plays a role in nerve cell penetration.

Animals↗
PubMed1984

Analysis of the immune response to papain digestion products of tetanus toxin.

Tetanus toxin was degraded by papain and the previously characterized Fragment B and Fragment C were isolated. The induction of protective immunity against tetanus toxin was subsequently investigated in two animal models using Fragment B and Fragment C, as well as conventional tetanus toxoid for comparison. In guinea pigs, Fragment B showed an immunizing potency similar to that recorded for an equal amount of tetanus toxoid, whereas Fragment C was considerably less efficient, on an equal weight basis. In contrast, mice immunized with Fragment C reached a protection level similar to that achieved after immunization with the complete tetanus toxoid antigen. In both animal models, Fragments B and C acted in a synergistic fashion when injected together. Analysis of sera from guinea pigs using ELISA, after administration of a low dose of tetanus immunogen corroborated these results. The data indicate that antigenic determinants in distinct regions of the toxin molecule independently may induce antibodies neutralizing the lethal action of tetanus toxin. The immune response to the complete toxoid antigen may be subject to influence by intramolecular competition between antigenic determinants. The immunogenicity of the fragments as measured by the protection rate after active immunization may depend upon the animal species used.

Animals↗
PubMed2000

Identification of novel small molecule ligands that bind to tetanus toxin.

Tetanus toxin belongs to a family of clostridial protein neurotoxins for which there are no known antidotes. Another closely related member of this family, botulinum toxin, is being used with increasing frequency by physicians to treat severe muscle disorders. Botulinum toxin has also been produced in large quantities by terrorists for use as a biological weapon. To identify small molecule ligands that might bind to the targeting domain of tetanus and botulinum toxins and to facilitate the design of inhibitors and new reagents for their detection, molecular docking calculations were used to screen a large database of compounds for their potential to bind to the C fragment of tetanus toxin. Eleven of the predicted ligands were assayed by electrospray ionization mass spectrometry (ESI-MS) for binding to the tetanus toxin C fragment, and five ligands (45%) were found to bind to the protein. One of these compounds, doxorubicin, was observed to have strong hydrophobic interactions with the C fragment. To check the ligands for their ability to compete with ganglioside binding, each was also tested using a GT1b liposome assay. Doxorubicin was the only ligand found to competitively bind the tetanus toxin C fragment with an appreciable binding constant (9.4 microM).

Binding Sites↗
PubMed1985

Neuropharmacological characterization of fragment B from tetanus toxin.

Tetanus toxin and Fragment B from tetanus toxin were assayed for activity on the mouse phrenic nerve-hemidiaphragm preparation. Both molecules produced blockade of neuromuscular transmission, but the parent molecule was at least two orders of magnitude more potent than the fragment. Experiments were done to determine whether the toxicity attributed to Fragment B was authentic or due to contamination with the parent molecule. Analysis of the fragment by high-performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed trace contamination. Removal of the major contaminant (1-2%) did not abolish toxicity of the material. However, in pharmacological experiments with native toxin and its fragment, the latter behaved indistinguishably from the former. At equiactive concentrations, both were antagonized by Fragment C and both were antagonized by lysosomotropic agents (ammonium chloride and methylamine hydrochloride). In addition, monoclonal antibodies directed against epitopes in Fragment C neutralized both native toxin and the material presumed to be Fragment B. The studies with antagonists and antibodies suggest that the toxicity apparently associated with Fragment B was in fact due to trace contamination with the parent molecule. In experiments on planar lipid bilayers, Fragment B formed pH-dependent channels. This activity was not abolished by monoclonal antibodies directed against epitopes in Fragment C. The data indicate that Fragment B retains the ability to form channels in membranes, but in the absence of Fragment C it retains little ability to paralyze neuromuscular transmission.

Animals↗
PubMed1988

Motoneuron uptake from the circulation of the binding fragment of tetanus toxin.

Tetanus toxin enters the central nervous system from the systemic circulation after it is internalized by motoneuron terminals at the neuromuscular junction. We have demonstrated that the atoxic binding fragment (C-fragment) of tetanus toxin is internalized preferentially by motoneurons. We examined the distribution of C-fragment after intravenous injection in the nervous systems of mice by immunohistochemical methods. All animals remained asymptomatic until killed one to two days after injection. C-fragment was found only within neurons with processes outside the blood-brain barrier. Large motoneurons of the spinal cord showed the greatest accumulation of C-fragment. Motoneurons of brain-stem nuclei (particularly facial and trigeminal), also showed substantial label of C-fragment. Small amounts of C-fragment were detected in dorsal root ganglion cells. Affinity of a systemically distributed substance for synaptic components, as well as an inability to cross the blood-brain barrier, may lead to its preferential localization in motoneurons.

Animals↗
PubMed1993

The transglutaminase hypothesis for the action of tetanus toxin.

Tetanus toxin potently and almost irreversibly inhibits the release of neurotransmitters from nerve terminals. The toxin binds to and activates transglutaminase, a Ca(2+)-dependent enzyme that can form stable crosslinks between substrate proteins. Transglutaminase is present in nerve terminals and recognizes synapsin I, an abundant synaptic vesicle phosphoprotein involved in neurotransmission, as an excellent substrate. The neuroparalytic action of tetanus toxin might be due, at least in part, to the stimulation of synaptic transglutaminase and the consequent crosslinking of synapsin I.

Animals↗
PubMed1992

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

Amino Acid Sequence↗
PubMed1993

Disulfide linkage between C3b and tetanus toxin on tetanus toxin-specific EBV-transformed B cells.

EBV-transformed B cells specific for tetanus toxin were found to bind C3b in excess over the expected figures based on the number of complement receptors CR1. This was confirmed by analysis of cell extracts by SDS-PAGE giving evidence for C3b-membrane protein complexes that were disrupted upon reduction. Alkylation of C3b-free cysteine abolished formation of these complexes and only a noncovalent binding of C3b to CR1 was observed, which could be inhibited by mAb to CR1. When C3b was incubated with the same cells coated with tetanus toxin bound to their specific membrane Ig, preferential formation of disulfide-bonded complexes between tetanus toxin and C3b was observed. These observations correspond to a novel capacity of C3b to interact covalently through its cysteine 1010 with free SH groups of protein acceptors. One hypothesis is that the disulfide bond formation is catalyzed by a thioredoxin-like protein secreted and expressed on the membrane of EBV-transformed B cells. In the context of Ag processing and presentation by B cells, disulfide binding of chaperone C3b to Ag is likely to persist during transcytosis and to play a significant role in the modulation of the processing.

B-Lymphocytes↗
PubMed1984

Botulinum toxin and tetanus toxin recognize similar membrane determinants.

The binding fragment of tetanus toxin (50,000 dalton carboxy-terminus of heavy chain) does not block neuromuscular transmission, but it does antagonize the ability of native tetanus toxin to block neuromuscular transmission. The binding fragment of tetanus toxin also antagonizes certain botulinum toxins, including types C and E. Antagonism is at the cell surface, suggesting that the various molecules compete for a similar membrane binding site. The data indicate that the binding site is specific for botulinum toxin rather than tetanus toxin.

Animals↗