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PubMed1994

Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin.

Tetanus toxin (TeTx) and the various forms of botulinal neurotoxins (BoNT/A to BoNT/G) potently inhibit neurotransmission by means of their L chains which selectively proteolyze synaptic proteins such as synaptobrevin (TeTx, BoNT/B, BoNT/F), SNAP-25 (BoNT/A), and syntaxin (BoNT/C1). Here we show that BoNT/D cleaves rat synaptobrevin 1 and 2 in toxified synaptosomes and in isolated vesicles. In contrast, synaptobrevin 1, as generated by in vitro translation, is only a poor substrate for BoNT/D, whereas this species is cleaved by BoNT/F with similar potency. Cleavage by BoNT/D occurs at the peptide bond Lys59-Leu60 which is adjacent to the BoNT/F cleavage site (Gln58-Lys59) and again differs from the site hydrolyzed by TeTx and BoNT/B (Gln76-Phe77). Cellubrevin, a recently discovered isoform expressed outside the nervous system, is efficiently cleaved by all three toxins examined. For further characterization of the substrate requirements of BoNT/D, we tested amino- and carboxyl-terminal deletion mutants of synaptobrevin 2 as well as synthetic peptides. Shorter peptides containing up to 15 amino acids on either side of the cleavage site were not cleaved, and a peptide extending from Arg47 to Thr116 was a poor substrate for all three toxins tested. However, cleavability was restored when the peptide is further extended at the NH2 terminus (Thr27-Thr116) demonstrating that NH2 terminally located sequences of synaptobrevin which are distal from the respective cleavage sites are required for proteolysis. To further examine the isoform specificity, several mutants of rat synaptobrevin 2 were generated in which individual amino acids were replaced with those found in rat synaptobrevin 1. We show that a Met46 to Ile46 substitution drastically diminishes cleavability by BoNT/D and that the presence of Val76 instead of Gln76 dictates the reduced cleavability of synaptobrevin isoforms by TeTx.

Amino Acid Sequence↗
PubMed1980

[Contractile proteins of nerve endings as a possible target of the presynaptic action of tetanus toxin].

Tetanus toxin (TT) in a dose of 0.25--250 DLM/ml inhibits the superprecipitation reaction of actomyosin-like protein (AMLP) of the bovine brain in response to Mg-ATP addition. Gangliosides of the bovine brain exert a similar action. Combined action of gangliosides (0.25 g/ml) and TT (0.25 DLM/ml) results in additivity of their effects. TT in a dose of 1600 DLM/ml blocks the formation of a complex of isolated synaptic vesicles (SV) of the rat brain. This effect was assessed from light scattering in response to MgCl2 and ATP addition. Under these conditions TT also inhibits release of the endogenous mediator noradrenaline from SV of bovine hypothalamus, with antitetanus serum reversing the effect of toxin. It is postulated that ALP associated with the synaptic membrane may be a potential TT target in the nerve endings.

Adenosine Triphosphate↗
PubMed2001

The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin.

Tetanus toxin, a member of the family of Clostridial neurotoxins, is one of the most potent toxins known. The crystal structure of the complex of the COOH-terminal fragment of the heavy chain with an analogue of its ganglioside receptor, GT1b, provides the first direct identification and characterization of the ganglioside-binding sites. The ganglioside induces cross-linking by binding to two distinct sites on the Hc molecule. The structure sheds new light on the binding of Clostridial neurotoxins to receptors on neuronal cells and provides important information relevant to the design of anti-tetanus and anti-botulism therapeutic agents.

Binding Sites↗
PubMed1990

Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin.

Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.

Amino Acid Sequence↗
PubMed1997

Microtubules and microfilaments participate in the inhibition of synaptosomal noradrenaline release by tetanus toxin.

Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn(2+)-dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX-induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with colchicine, highlighting that this cytoskeletal component is the locus of the effect of colchicine. Replacement of Ca2+ with Ba2+ caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase (< or = 10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin-induced inhibition of the slow (10-s to > or = 5-min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.

Actin Cytoskeleton↗
PubMed1987

ATP release from pure cholinergic synaptosomes is not blocked by tetanus toxin.

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+-induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization-induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co-release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.

Acetylcholine↗
PubMed1990

Identification of two promiscuous T cell epitopes from tetanus toxin.

Tetanus toxoid-specific T cell clones were isolated from a human donor. To determine the T cell epitopes recognized by the clones, 30 peptides representing amphipathic alpha helical regions of the tetanus toxin were screened for ability to induce proliferation of the clones. Two T epitopes were identified. These occurred within peptides 12 and 21, and had the amino acid sequences NSVDDALINSTKIYSYFPSV and PGINGKAIHLVNNESSE, respectively. An unusual feature was that both peptides could be presented to their respective T cell clones by antigen-presenting cells of many HLA specificities. Further investigation of peptide 12 showed that the epitope was only seven amino acids in length and had a very hydrophobic sequence, namely YSYFPSV. The ability of the T cell epitope-containing peptides 12 and 21 to interact with many different HLA alleles means they may potentially be very useful as "universal carrier molecules" in synthetic vaccines.

Amino Acid Sequence↗
PubMed2000

Serotonin transporter phosphorylation modulated by tetanus toxin.

Tetanus toxin (TeTx) modifies Na(+)-dependent, high-affinity 5-hydroxytryptamine (5-HT, serotonin) uptake in a synaptosomal-enriched P(2) fraction from rat brain. The effect corresponds to a rapid and non-competitive uptake inhibition, and it is preceded by induction of phospholipase C (PLC) activity and translocation and down-regulation of the classical protein kinase C (PKC-alpha, -beta and -gamma) isoforms. The effects on serotonin transport and on cPKC activation were similar to the effects exhibited by phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Moreover, after treatment with TeTx, an increase in Ser- and Tyr-specific phosphorylation was found. Activation of PKC by both TeTx and TPA results in a loss of transport capacity and serotonin transporter (SERT) phosphorylation, which are abolished by coapplication of the specific PKC inhibitor bisindolylmaleimide-1. Since a specific PLCgamma1 phosphorylation prior to TeTx's inducing SERT phosphorylation was found, the studies suggest that part of the action of TeTx consists of modifying the signal cascade initiated in tyrosine kinase receptors on nerve tissue previous to its cellular internalization, resulting in transporter phosphorylation.

Animals↗
PubMed1988

Establishment of a monoclonal antibody recognizing an antigenic site common to Clostridium botulinum type B, C1, D, and E toxins and tetanus toxin.

The partial amino acid sequence of the light-chain (Lc) component of Clostridium botulinum type C1 toxin was determined. The sequence was quite similar to those of the other types of botulinum and tetanus toxins. Nine monoclonal antibodies against botulinum type E toxin were established by immunizing BALB/c mice with type E toxoid or its Lc component. Six antibodies reacted with the heavy-chain component and three reacted with the Lc component of the toxin. One of the latter three antibodies reacted with botulinum type B, C1, and D toxins and tetanus toxin, as well as botulinum type E toxin. This antibody recognized the Lc components of these toxins, indicating that there exists one common antigenic determinant on the Lc regions of these toxins.

Amino Acid Sequence↗
PubMed1984

Fragment C of tetanus toxin antagonizes the neuromuscular blocking properties of native tetanus toxin.

Tetanus toxin, fragment B and fragment C were assayed for toxicity on the mouse phrenic nerve-hemidiaphragm preparation. The native toxin was a potent blocker of neuromuscular transmission; fragment B possessed little toxicity and fragment C was atoxic. Pretreatment of tissues with fragment C antagonized the neuromuscular blocking properties of tetanus toxin, but not those of type A botulinum toxin or beta-bungarotoxin. Agents that antagonize cholera toxin (B subunit) and diphtheria toxin (CRM197) did not antagonize tetanus toxin and did not alter the ability of fragment C to antagonize tetanus toxin. Fragment C exerted its effect by competing with unbound toxin for receptor sites on the nerve membrane. The fragment did not: 1) displace bound toxin; 2) inhibit internalization of toxin; or 3) inhibit intracellular expression of toxicity. In assays on intact cells, under conditions in which toxin binding was not dissociable, fragment C binding to phrenic nerves had an apparent KD of approximately 1.4 X 10(-7) M. Homogenates of mouse cerebral cortex adsorbed tetanus toxin and these homogenates competed with phrenic nerves for unbound toxin. Homogenized cortex did not displace or promote desorption of toxin already bound to phrenic nerves. Homogenates of eel and torpedo electric organ were not very effective in adsorbing toxin.

Adsorption↗
PubMed1984

Action of botulinum A toxin and tetanus toxin on synaptic transmission.

Intracellular recordings of the spontaneous activity from mammalian spinal cord neurons in culture demonstrated different sensitivities of excitatory and inhibitory synaptic transmission for the action of tetanus toxin (Tetx) and botulinum toxin type A (Botx). The effects of Tetx and Botx on spontaneous and nerve-evoked transmitter release were compared under identical experimental conditions in experiments on in vitro poisoned mouse diaphragms. At 37 degrees C completely paralyzed endplates are characterized by a very low frequency of spontaneous miniature endplate potentials (m.e.p.p.s) and by a 100% failure to evoke endplate potentials (e.p.p.s) in response to single nerve stimuli. Striking differences in the action of both toxins have been observed when the very low transmitter release probabilities of paralyzed nerve-muscle preparations were increased by tetanic nerve stimulation and/or application of potent K+-channel blockers and/or by reduction of temperature to 25 degrees C. While Botx did not change the short latency between nerve impulse and postsynaptic response, Tetx produced a temporal dispersion of the quantal release suggesting that the toxins act at different sites in the chain of events that result in transmitter release. To find further evidence to support the different actions of the toxins the spontaneous transmitter release was studied in more detail. Tetx blocked preferentially the release of so-called large mode m.e.p.p.s without affecting the frequency of the small mode ones. In contrast, Botx strongly inhibited both the small and large mode m.e.p.p.s.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1982

The effects of induced hypoxia on the cardiovascular system in dogs poisoned with tetanus toxin.

Tetanus toxicity was induced in dogs by injecting the toxin subcutaneously in the groin. On developing generalised toxic symptoms, these dogs were characterised by signs of increased sympathetic discharge to the cardiovascular system as evidenced by high basal values of blood pressure, heart rate and LV dP/dt max. Mild to moderate hypoxia induced by ventilation with 10% O2 in N2 had no appreciable effect on the cardiovascular variables. However, moderate to severe hypoxia induced by ventilation with 7% O2 in N2 further increased the sympathetic discharge to the heart and blood vessels resulting in increases in heart rate, LV dP/dt max and blood pressure. These responses were abolished by adrenergic blockers. The responses in the tetanus dogs were identical to those seen in dogs without tetanus toxicity. Atropine or moderate lactic acidaemia did not alter the responses to hypoxia. Beta-adrenergic blockers appear to be useful drugs in the control of tetanus patients who show evidence of increased sympathetic activity or who develop hypoxaemia.

Animals↗
PubMed1983

Botulinum A toxin and tetanus toxin do not affect presynaptic membrane currents in mammalian motor nerve endings.

The hypothesis according to which BoTx and TeTx block neuromuscular transmission by impairing Ca2+ influx was tested by recording presynaptic membrane currents at motor endplates of the mouse by means of external electrodes. The use of K-channel blockers allowed us to observe inward Ca current which was unaffected by bath application of BoTx or TeTx at doses which block neuromuscular transmission.

Animals↗
PubMed1981

Different effects of botulinum A toxin and tetanus toxin on the transmitter releasing process at the mammalian neuromuscular junction.

The quantal transmitter release of tetanus (TeTx) and botulinum A (BoTx) toxin paralyzed mouse diaphragms was studied. The very low release probability could be enhanced by increasing the frequency of nerve stimulation to 50 Hz or by the application of 4-aminopyridine. In the BoTx-muscles the endplate potentials were strongly coupled to the stimuli with synaptic delays similar to unpoisoned terminals. In contrast, in the TeTx-muscles large variations in the delay of release of quanta in response to stimulation were observed. From these findings it is suggested that TeTx and BoTx act at different sites of the depolarization-transmitter release process.

Animals↗
PubMed1997

Immunization of mice with poliovirus replicons expressing the C-fragment of tetanus toxin protects against lethal challenge with tetanus toxin.

In this study, we describe the construction of poliovirus genomes or "replicons" which contain the C fragment gene of tetanus toxin substituted for the poliovirus P1 capsid. Upon transfection of replicon RNA into cells, we immunoprecipitated a protein corresponding to the C-fragment of tetanus toxin using tetanus-specific antibodies. Using a recombinant vaccinia virus expressing poliovirus P1 capsid protein (VV-P1) to provide P1 protein, the replicon RNA was encapsidated; stocks of the replicons were generated by passage with VV-P1. The immunogenicity of the replicons was determined by immunization of transgenic mice which are susceptible to poliovirus. A serum antibody response to poliovirus and tetanus toxoid was detected in all of the immunized mice. Protection against a lethal dose of tetanus toxin generally correlated with the levels of serum anti-tetanus antibodies. To address whether pre-existing antibodies to poliovirus limit the effectiveness of the replicon as a vaccine vector, mice were first immunized with the inactivated poliovirus vaccine followed by immunization with the replicons expressing C-fragment protein. Anti-tetanus antibodies were detected in these mice after a single administration of the replicon; these antibodies conferred protection upon challenge with tetanus toxin. These results demonstrate the potential use of poliovirus replicons encoding foreign proteins to induce a protective antibody response, even in the presence of pre-existing antibodies to poliovirus.

Animals↗