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)

A scheme based on the zinc binding site [1992, FEBS Lett. 312, 110-114] has been extended to classify zinc metalloproteases into distinct families. The gluzincins, defined by the HEXXH motif and a glutamic acid as the third zinc ligand, include the thermolysin, endopeptidase-24.11, aminopeptidase, angiotensin converting enzyme, endopeptidase-24.15, and tetanus and botulinum neurotoxin families. The metzincins, defined by the HEXXH motif, a histidine as the third zinc ligand and a Met-turn, include the astacin, serralysin, reprolysin and matrixin families. The inverted zincin motif, HXXEH, defines the inverzincin family of insulin-degrading enzymes, the HXXE motif defines the carboxypeptidase family, and the HXH motif DD-carboxypeptidase.
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PMID:Families of zinc metalloproteases. 795 88

The clostridial neurotoxins responsible for tetanus and botulism are eight different proteins, composed of two disulfide-linked polypeptide chains. They bind specifically to the presynaptic membrane via the heavy chain, while the light chain enters the cytosol of the neurons, where it displays a zinc-endopeptidase activity directed to proteins of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxin serotypes B, D, F and G cleave specifically and at single different peptide bonds VAMP/synaptobrevin, a component of small synaptic vesicles. In contrast, the other neurotoxins catalyze the hydrolysis of proteins of the presynaptic membrane. Serotypes A and E of botulinum neurotoxin cleave SNAP-25, at different sites located within the carboxyl-terminus, while the specific target of serotype C is syntaxin.
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PMID:Clostridial neurotoxins as tools to investigate the molecular events of neurotransmitter release. 799 6

Susceptibilities of Mg.ATP-independent and Mg.ATP-requiring components of catecholamine secretion from digitonin-permeabilised chromaffin cells to inhibition by Clostridial botulinum type A and tetanus toxins were investigated. These toxins are Zn(2+)-dependent proteases which specifically cleave the 25-kDa synaptosomal-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) II, respectively. When applied to permeabilised chromaffin cells they rapidly inhibited secretion in the presence of Mg.ATP but the catecholamine released in the absence of Mg.ATP, thought to represent fusion of primed granules, was not perturbed. The toxins can exert their effects per se in the absence of the nucleotide complex; therefore, Mg.ATP-requiring steps of secretion are implicated as roles for their targets. Primed release was lost rapidly after permeabilisation of the cells but could be maintained by including Mg.ATP during the incubation before stimulating release with Ca2+. This ability of Mg.ATP to maintain primed release was only partially inhibited by botulinum neurotoxin A whereas it was abolished by tetanus toxin, consistent with the distinct substrates for these toxins. This study reveals a component of release within which these proteins are either resistant to cleavage by these toxins or in such a position that degradation can no longer prevent granule fusion. Differences in the steps of release at which these toxins can affect inhibition are also revealed.
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PMID:Botulinum A and the light chain of tetanus toxins inhibit distinct stages of Mg.ATP-dependent catecholamine exocytosis from permeabilised chromaffin cells. 802 Apr 71

Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
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PMID:Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. 810 14

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.
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PMID:Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. 817 89

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc-binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc-dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxin's neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins' degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc-dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins' effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.
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PMID:Botulinum A like type B and tetanus toxins fulfils criteria for being a zinc-dependent protease. 824 89

Botulinum neurotoxin types A, B (unactivated and activated), C, D, E, F and G, as well as tetanus toxin, paralyzed transmission in mouse phrenic nerve-hemidiaphragm preparations. Toxin-induced blockade of transmission was antagonized by chelators [e.g., ethylenediamine tetraacetic acid, tetrakis(2-pyridylmethyl)ethylenediamine or diethylene-triaminepentaacetic anhydride], but this effect was dependent on incubation conditions. Pretreatment of toxin with chelators failed to produce antagonism, but pretreatment of tissues did produce antagonism. Of the various chelators tested, tetrakis(2-pyridylmethyl)ethylenediamine produced the greatest effect. Antagonism of toxin-induced neuromuscular blockade could be partially reversed by washing chelators from tissues and could be fully reversed by adding an excess of zinc. The ability of chelators to antagonize clostridial neurotoxins was specific and did not extend to phospholipase A2 neurotoxins. Ligand-binding studies with radioiodinated toxin and brain membrane preparations showed that chelators did not antagonize toxicity by inhibiting toxin association with receptors. Similarly, pharmacological experiments with unlabeled toxin- and type-specific antibodies demonstrated that chelators did not act by blocking receptor-mediated internalization of toxin. The chelators appeared to exert their effects by antagonizing the intracellular actions of clostridial neurotoxins. Electrophysiological studies showed that chelators, at concentrations relevant to antagonism of botulinum neurotoxin and tetanus toxin, did not enhance transmitter release.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chelation of zinc antagonizes the neuromuscular blocking properties of the seven serotypes of botulinum neurotoxin as well as tetanus toxin. 824 47

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.
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PMID:Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. 829 7

Experiments were conducted on mouse hemidiaphragm preparations using five phospholipase A2 neurotoxins of differing chain structures and antigenicities [notexin (one chain); crotoxin (two chains not covalently bound), beta-bungarotoxin (two chains covalently bound); taipoxin (three chains), and textilotoxin (five chains; one copy each of three chains and two copies of a fourth chain)]. Three clostridial neurotoxins (botulinum neurotoxin types A and B, and tetanus toxin) were used in comparison experiments. Phospholipase A2 neurotoxins produced concentration-dependent blockade of neuromuscular transmission. There was no obvious relationship between chain structure and potency, but there was an indication of a relationship between chain structure and binding. The binding of notexin was substantially reversible, the binding of crotoxin was slightly reversible, and the binding of beta-bungarotoxin, taipoxin and textilotoxin was poorly reversible. Experiments with neutralizing antibodies indicated that phospholipase A2 neurotoxins became associated with binding sites on or near the cell surface. This binding did not produce neuromuscular blockade. When exposed to physiological temperatures and nerve stimulation, bound toxin disappeared from accessibility to neutralizing antibody. This finding suggests that there was some form of molecular rearrangement. The two most likely possibilities are: (1) there was a change in the conformation of the toxin molecule, or (2) there was a change in the relationship between the toxin and the membrane. The molecular rearrangement step did not produce neuromuscular blockade. At a later time there was onset of paralysis; the amount of time necessary for onset of blockade was a function of toxin concentration. Phospholipase A2 neurotoxins were not antagonized by drugs that inhibit receptor-mediated endocytosis. In addition, phospholipase A2 neurotoxins did not display the pH-induced conformational changes that are typical of other endocytosed proteins, such as clostridial neurotoxins. However, phospholipase A2 neurotoxins were antagonized by strontium, and this antagonism was expressed against toxins that were free in solution and toxins that were bound to the cell surface. Limited antagonism was expressed after toxins had undergone molecular rearrangement, and no antagonism was expressed after toxin-induced neuromuscular blockade. The cumulative data suggest that phospholipase A2 neurotoxins are not internalized to produce their poisoning effects. These toxins appear to act on the plasma membrane, and this is the site at which they initiate the events that culminate in neuromuscular blockade.
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PMID:Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. 844 59

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.
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PMID:Calcium-regulated exocytosis is required for cell membrane resealing. 855 42


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