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)

The ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b-coated plastic wells was investigated and compared with the binding characteristics of the parent approximately 150-kDa protein. Although the approximately 50-kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b-coated wells was exhibited by the approximately 50-kDa carboxy-terminal half of the approximately 100-kDa heavy chain of BTxA; the amino-terminal half of the heavy chain lacked the ability to bind. Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy-terminal half of its heavy chain. Binding of BTxA and the C-terminal half of the heavy chain was optimal in buffers of low ionic strength (mu less than or equal to 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at mu greater than or equal to 0.10. TTx and the approximately 50-kDa C-terminal half of its approximately 100-kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA. Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b. Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by approximately 60 and approximately 90%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of botulinum and tetanus neurotoxins to ganglioside GT1b and derivatives thereof. 186 Nov 41

The flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin. 251 79

Botulinum and tetanus neurotoxins are water-soluble proteins (mol. wt 150,000) produced by Clostridium botulinum and Clostridium tetani, respectively. It is believed that these neurotoxins, once internalized via receptor-mediated endocytosis, form membrane channels in order to traverse the endosomal membrane and enter the cytoplasm of the nerve terminal. Investigation of the associative properties between neurotoxin molecules could yield an understanding of this channel formation. That is, an association between neurotoxin monomers could result in an oligomeric form of the neurotoxin necessary for assembly of a channel through the hydrophobic interior of the endosomal membrane, thereby allowing passage of the neurotoxin or its active fragment through the resulting pore. Based on the native gel electrophoresis and chemical cross-linking experiments, tetanus neurotoxin exists as a dimer and a trimer, type A botulinum neurotoxin exists as a dimer, trimer, and a larger species, type E botulinum neurotoxin exists as a monomer and dimer, and type B botulinum neurotoxin appears to exist as a dimer in aqueous solution. The results imply that quaternary structures of these neurotoxins may play an important role in their mode of action during neuronal poisoning.
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PMID:Quaternary structure of botulinum and tetanus neurotoxins as probed by chemical cross-linking and native gel electrophoresis. 780 45

Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an approximately 116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the approximately 116-kDa protein more than that with GT1b. A purified preparation of the water-soluble approximately 116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the approximately 116-kDa protein. These sequences matched with the data bank sequences for beta-adducin, a cytoskeletal protein. The carboxy-terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).
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PMID:Ganglioside-induced adherence of botulinum and tetanus neurotoxins to adducin. 863 82

Predictions were made of the secondary, two-dimensional (2-D) structures and side-chain solvent accessibilities of the light (L) chains of the clostridial neurotoxins (botulinum neurotoxin serotypes A-G and tetanus neurotoxin). An artificial neural network was used to make these predictions from a multiple alignment of their primary structures and was the approach used in making successful predictions for the C-fragments of these neurotoxins (Lebeda et al., J. Prot. Chem., 17:311, 1998). We also exploited the fact that the L-chains are Zn-dependent proteases. Although no other metalloproteases were found to be sequentially homologous to these neurotoxin L-chains, a sequence clustering algorithm showed that several bacterially derived Zn-dependent proteases, including thermolysin, were the most similar. A 2-D structure topology map for the type A L-chain was constructed by using thermolysin as a design template. As in thermolysin, the region containing the Zn-binding sequence motif, which is part of the active site in these neurotoxins, was predicted to be minimally solvent accessible. On the other hand, the locations of residues with highly exposed side chains were predicted to occur in non-periodic structure elements. Together, these 2-D structure and solvent accessibility predictions can be used to identify important solvent-exposed regions of the L-chain. These regions may include sites that interact with residues of the neurotoxin heavy chain, sites that bind to vesicle-docking substrates or sites that form antibody epitopes.
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PMID:Predictions of secondary structure and solvent accessibility of the light chain of the clostridial neurotoxins. 978 61

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned. Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc. However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.
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PMID:Identifying the principal protective antigenic determinants of type A botulinum neurotoxin. 979 91

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.
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PMID:Functional characterisation of tetanus and botulinum neurotoxins binding domains. 1041 79

The clostridial neurotoxins (CNTs), comprised of tetanus neurotoxin (TeNT) and the seven serotypes of botulinum neurotoxin (BoNT A-G), specifically bind to neuronal cells and disrupt neurotransmitter release by cleaving proteins involved in synaptic vesicle membrane fusion. In this study, multiple CNT sequences were analyzed within the context of the 1277 residue BoNT/A crystal structure to gain insight into the events of binding, pore formation, translocation, and catalysis that are required for toxicity. A comparison of the TeNT-binding domain structure to that of BoNT/A reveals striking differences in their surface properties. Further, the solvent accessibility of a key tryptophan in the C terminus of the BoNT/A-binding domain refines the location of the ganglioside-binding site. Data collected from a single frozen crystal of BoNT/A are included in this study, revealing slight differences in the binding domain orientation as well as density for a previously unobserved translocation domain loop. This loop and the conservation of charged residues with structural proximity to putative pore-forming sequences lend insight into the CNT mechanism of pore formation and translocation. The sequence analysis of the catalytic domain revealed an area near the active-site likely to account for specificity differences between the CNTs. It revealed also a tertiary structure, highly conserved in primary sequence, which seems critical to catalysis but is 30 A from the active-site zinc ion. This observation, along with an analysis of the 54 residue "belt" from the translocation domain are discussed with respect to the mechanism of catalysis.
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PMID:Sequence homology and structural analysis of the clostridial neurotoxins. 1051 45

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.
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PMID:Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment. 1055 89

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.
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PMID:Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis. 1058 2


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