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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)

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

Exposure of isolated mouse hemidiaphragms to botulinum neurotoxins, 0.1 nM BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak isometric twitch tension to 50% of control values at 55 min (BoNT-A) to 68 min (BoNT-B) after application. Either coincubation of BoNT with the heavy metal chelator TPEN, preincubation with TPEN followed by BoNT, or application of TPEN after BoNT but before neuromuscular block, delayed the onset of muscle failure in a dose-dependent manner by up to five-fold. TPEN doses between 2 and 10 microM were required to antagonize significantly the muscle block produced by BoNT, and the delay in onset was maximal between 10 and 50 microM TPEN. Treatment of muscles with a Zn(2+)-TPEN coordination complex, rather than TPEN alone, eliminated any beneficial effects of TPEN on BoNT intoxication, indicating that these effects were mediated by chelation of Zn2+. Other metal chelators that were not as membrane permeant as TPEN were ineffective in delaying BoNT paralysis, suggesting that TPEN acts by chelating intraterminal Zn2+. In the absence of BoNT, TPEN caused a dose-dependent increase in nerve-elicited twitch tension with a half-maximal concentration at 8 microM. There was no corresponding change in twitches from direct electrical stimulation of the muscle. After BoNT (A or B serotype) had reduced the muscle twitch by 20 to 70%, however, subsequent application of TPEN rapidly depressed nerve-elicited twitches. The shift from potentiation to depression after BoNT treatment suggests that presynaptic vesicle mobilization and/or release involve Zn(2+)-dependent enzymes and that BoNTs interact with these enzyme pathways.
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PMID:Interactions between heavy metal chelators and botulinum neurotoxins at the mouse neuromuscular junction. 757 Jun 39

Zinc-dependent metalloprotease inhibitors phosphoramidon, captopril and a peptide hydroxamate were studied as potential pretreatment compounds by examining their ability to delay the onset or to prolong the time to 50% block of nerve-elicited muscle twitch tension in the mouse phrenic-nerve diaphragm (in vitro at 36 degrees C) after botulinum neurotoxin serotypes A and B (BoNT-A, BoNT-B). Addition of BoNT-A or BoNT-B (1 x 10(-10) M) produced 50% block of the twitch response at 56 +/- 9 min and 76 +/- 4 min, respectively. Preincubation (45 min) of muscles with phosphoramidon (0.2 mM) prolonged the time to 50% block by 15 min in BoNT-B-poisoned muscles with no effect on the time-course of paralysis in BoNT-A exposed muscles. When the same quantities of BoNT-A or BoNT-B (equivalent to 1 x 10(-10) M bath concentration) were preincubated for 2 hr with phosphoramidon (equivalent to 0.2 mM final bath concentration), and the incubation mixture was added to the muscle chamber, the times to 50% block were prolonged by 38 min and 18 min for BoNT-B and BoNT-A, respectively. Preincubation of diaphragms with captopril (up to 10 mM) or peptide hydroxamate (75 microM) failed to antagonize BoNT-A or BoNT-B-induced neuromuscular block. Among the three metalloprotease inhibitors examined here, only phosphoramidon showed a significant protection against both serotypes of BoNT. A search for better inhibitor compounds specifically tailored to match the active site on BoNT molecule deserves attention.
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PMID:A study of zinc-dependent metalloendopeptidase inhibitors as pharmacological antagonists in botulinum neurotoxin poisoning. 757 Jun 40

Tetanus and botulinum neurotoxins are produced by several Clostridia and cause the paralytic syndromes of tetanus and botulism by blocking neurotransmitter release at central and peripheral synapses, respectively. They consist of two disulfide-linked polypeptides: H (100 kDa) is responsible for neurospecific binding and cell penetration of L (50 kDa), a zinc-endopeptidase specific for three protein subunits of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxin serotypes B, D, F and G cleave at single sites, which differ for each neurotoxin, VAMP/synaptobrevin, a membrane protein of the synaptic vesicles. Botulinum A and E neurotoxins cleave SNAP-25, a protein of the presynaptic membrane, at two different carboxyl-terminal peptide bonds. Serotype C cleaves specifically syntaxin, another protein of the nerve plasmalemma. The target specificity of these metallo-proteinases relies on a double recognition of their substrates based on interactions with the cleavage site and with a non-contiguous segment that contains a structural motif common to VAMP, SNAP-25 and syntaxin.
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PMID:The metallo-proteinase activity of tetanus and botulism neurotoxins. 758 Dec 98

Botulinum neurotoxin serotype C (BoNT/C) is a 150-kDa protein produced by Clostridium botulinum, which causes animal botulism. In contrast to the other botulinum neurotoxins that contain one atom of zinc, highly purified preparations of BoNT/C bind two atoms of zinc per toxin molecule. BoNT/C is a zinc-endopeptidase that cleaves syntaxin 1A at the Lys253-Ala254 and syntaxin 1B at the Lys252-Ala253 peptide bonds, only when they are inserted into a lipid bilayer. The other Lys-Ala bond present within the carboxyl-terminal region is not hydrolyzed. Syntaxin isoforms 2 and 3 are also cleaved by BoNT/C, while syntaxin 4 is resistant. These data suggest that BoNT/C recognizes a specific spatial organization of syntaxin, adopted upon membrane insertion, which brings a selected Lys-Ala peptide bond of its carboxyl-terminal region to the active site of this novel metalloproteinase.
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PMID:Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. 773 92

Botulinum neurotoxin serotype B (BoNT/B) and tetanus toxin (TeTx) block neuroexocytosis through selective endoproteolysis of vesicle-associated membrane protein (VAMP). The enzymological properties of both toxins were compared for the first time in their cleavage of VAMP and various sized fragments using a sensitive chromatographic assay. The optimal substrate sizes for the zinc-dependent protease activities of the light chains of TeTx and BoNT/B were established using synthetic peptides corresponding to the hydrophilic core of VAMP (30-62 amino acids in length). TeTx was found to selectively cleave the largest peptide at a single site, Gln76-Phe77. It exhibited the most demanding specificity, requiring the entire hydrophilic domain (a 62-mer) for notable hydrolysis, whereas BoNT/B efficiently cleaved the much smaller 40-mer. Thus, an unusually long N-terminal sequence of 44 amino acids upstream of the scissile bond is required for the selective hydrolysis of VAMP by TeTx. Using the largest peptide, BoNT/B and TeTx exhibited approximately 50% and 35%, respectively, of the activities shown toward intact VAMP, detergent solubilized from synaptic vesicles. Given the large size of the smallest substrates, it is possible that these neurotoxins recognize and require a three-dimensional structure. Although both toxins were inactivated by divalent metal chelators, neither was antagonized by phosphoramidon or ASQFETS (a substrate-related peptide that spans the cleavage site), and TeTx was only feebly inhibited by captopril; also, they were distinguishable in their relative activities at different pHs, temperatures, and ionic strengths. These collective findings are important in the design of effective inhibitors for both toxins, as well as in raising the possibility that TeTx and BoNT/B interact somewhat differently with VAMP.
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PMID:Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of vesicle-associated membrane protein and various sized fragments. 780 99

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. 790 Oct 2

Tetanus toxin and botulinum neurotoxins are di-chain proteins of 150 kD molecular weight. They are produced by bacteria of the Clostridium genus. These toxins act on the nervous system by inhibiting neurotransmitter release (glycine and GABA in the case of tetanus toxin; acetylcholine in the case of botulinum neurotoxins) thus inducing the spastic or flaccid paralysis that characterizes tetanus and botulism, respectively. Their cellular mechanism of action involves three main steps, namely binding to the neurone membrane, internalization and intracellular blockade of the release mechanism for neurotransmitters. Membrane acceptors for these toxins are not yet fully identified; they would consist of membrane gangliosides and proteins. The internalization step would be achieved by endocytosis. Recent findings show that both binding and internalization are mediated only by the heavy chain of the toxins whereas the intracellular blockade of neurotransmitter release involves their light chain alone. The light chain has been identified as a zinc metalloprotease and its substrates would be proteins involved in the neurotransmitter release mechanism. The target of tetanus toxin and of botulinum neurotoxin type B is VAMP/synaptobrevin, a membrane protein of the synaptic vesicles of nerve cell terminals.
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PMID:[Molecular mechanism of action of tetanus toxin and botulinum neurotoxins]. 791 55

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


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