Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To define conserved domains within the light (L) chains of clostridial neurotoxins, we determined the sequence of botulinum neurotoxin type B (BoNT/B) and aligned it with those of tetanus toxin (TeTx) and BoNT/A, BoNT/C1, BoNT/D, and BoNT/E. The L chains of BoNT/B and TeTx share 51.6% identical amino acid residues whereas the degree of identity to other clostridial neurotoxins does not exceed 36.5%. Each of the L chains contains a conserved motif, HExxHxxH, characteristic for metalloproteases. We then generated specific 5'- and 3'-deletion mutants of the L chain genes of TeTx and BoNT/A and tested the biological properties of the gene products by microinjection of the corresponding mRNAs into identified presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Toxicity was determined by measurement of neurotransmitter release, as detected by depression of postsynaptic responses to presynaptic stimuli (Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844-7848). Our studies allow the following conclusions. 1) Residues Cys439 of TeTx and Cys430 of BoNT/A, both of which participate in the interchain disulfide bond, play no role in the toxification reaction. 2) Derivatives of TeTx that lacked either 8 amino- or 65 carboxyl-terminal residues are still toxic, whereas those lacking 10 amino- or 68 carboxyl-terminal residues are nontoxic. 3) For BoNT/A, toxicity could be demonstrated only in the presence of added nontoxic heavy (H) chain. A deletion of 8 amino-terminal or 32 carboxyl-terminal residues from the L chain had no effect on toxicity, whereas a removal of 10 amino-terminal or 57 carboxyl-terminal amino acids abolished toxicity. 4) The synergistic effect mediated by the H chain is linked to the carboxyl-terminal portion of the H chain, as demonstrated by injection of HC-specific mRNA into neurons containing the L chain. This finding suggests that the HC domain of the H chain becomes exposed to the cytosol during or after the putative translocation step of the L chain.
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PMID:Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A. 163 16

The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica. When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses. The reverse was true for TeTx or a mixture its constituent chains. Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type. When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived. Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx. Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin. Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.
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PMID:Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia. 167 11

1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.
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PMID:Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains. 198 13

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons. If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context. To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations. Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known. Both toxins block neurotransmitter release at nanomolar intracellular concentrations. These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced. Their active sites are located (or partly located) on the light chain. mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ.
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PMID:Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons. 223

1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
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PMID:Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process. 257 18

The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.
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PMID:Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons. 257 22

Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.
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PMID:Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain. 289 93

The action of type A and type B botulinum neurotoxin on neurotransmitter release was studied on identified ganglionic synapses of Aplysia. Using this model, we have shown that botulinum neurotoxins at concentrations used in vertebrate preparations had the same specificity of action and that both heavy and light chains of these toxins are intracellularly required to inhibit neurotransmitter release.
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PMID:[Ganglionic synapses of Aplysia as a model for the study of the mechanism of action of botulinum neurotoxins]. 290 Jun 74

mAbs were produced in mice against highly purified, renatured light chain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on nitrocellulose to avoid the undesirable use of toxoids. Subcutaneous implants of relatively high amounts (up to 10 micrograms each) of LC allowed its slow release into the systemic circulation and, thus, yielded much higher antibody titres against the underivatized antigen than had hitherto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG1 and IgG2b subclasses reactive specifically with BoNT A and LC, in native and denatured states, without showing any cross-reactivity with types B, E, F or tetanus toxin. The pronounced reactivities of three mAbs towards refolded LC or intact toxin, observed in immunobinding and precipitation assays, relative to that seen in Western blots imply a preference for conformational epitopes. Though mAbs 4, 5 and 7 failed to neutralize the lethality of BoNT in vivo, administration intraneurally of mAb7 prevented the inhibition of transmitter release normally induced by subsequent extracellular administration of BoNT A. Notably, the latter mAb reacted with a synthetic peptide corresponding to amino acids 28-53 in the N-terminus of the LC, a highly conserved region in Clostridial neurotoxins reported to be essential for maintaining the tertiary structure of the chain. Most importantly, when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transiently, the blockade of acetylcholine release by the toxin; this novel finding is discussed in relation to the nature of the zinc-dependent protease activity of the toxin.
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PMID:Antagonism of the intracellular action of botulinum neurotoxin type A with monoclonal antibodies that map to light-chain epitopes. 750 83

Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.
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PMID:A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly. 810 36


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