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PubMed1995

Botulinum versus tetanus neurotoxins: why is botulinum neurotoxin but not tetanus neurotoxin a food poison?

Botulinum and tetanus neurotoxins, produced by Clostridium botulinum and Clostridium tetani, respectively, are the most poisonous poisons known to mankind. Although botulinum and tetanus neurotoxins share several characteristics, such as similar mol. wts, similar macrostructure, virtually identical mode of action, and a strong amino acid sequence homology, the two neurotoxins differ in one very significant way; only botulinum neurotoxin is a food poison. Factors responsible for the food poisoning potential of botulinum neurotoxins seem to be a group of complexing proteins that are also produced by C. botulinum, and are known to associate with the neurotoxin. Translation products of nucleotide sequences upstream to the neurotoxin genes of serotypes A, B, C, D, E and F botulinum neurotoxin reveal the location of genes for one of the complexing proteins that could be transcribed as polycistronic mRNA to include neurotoxin sequences. No such protein seems to be present in C. tetani, suggesting that the lack of complexing proteins might be responsible for tetanus not being a food poison.

Amino Acid Sequence↗
PubMed1993

Snake-venom phospholipase A2 neurotoxins. Potentiation of a single-chain neurotoxin by the chaperon subunit of a two-component neurotoxin.

The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).

Animals↗
PubMed1998

Two novel alpha-neurotoxins isolated from Taiwan cobra: sequence characterization and phylogenetic comparison of homologous neurotoxins.

Two novel postsynaptic neurotoxins (alpha-neurotoxins) isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct primary sequences and different neurotoxicities as compared with the most abundant and lethal component in the venom, i.e., cobrotoxin characterized before from the same venom. The complete sequences of two neurotoxin analogues were determined by N-terminal Edman degradation and comparison of amino acid compositions of proteolytic toxin fragments with other homologous toxins of known sequences. The short-chain neurotoxin consists of 61 amino acid residues with eight conserved cysteine residues and is found to show 78% sequence identity with cobrotoxin. The other toxin, consisting of 65 residues with ten cysteines, belongs to the family of long-chain neurotoxins. It is the first long-chain alpha-neurotoxin reported from the Taiwan cobra. The lethal toxicities of these two novel neurotoxins were much lower than cobrotoxin, albeit with close structural homology among the three toxins in terms of their primary sequences and tertiary structure predicted by homology modeling. Multiple sequence alignment and comparison coupled with construction of a phylogenetic tree for various alpha-neurotoxins of Naja and closely related genuses have established that all nicotinic alpha-neurotoxins present in the snake family of Elapidae are closely related to each other, presumably derived from an ancestral polypeptide by gene duplication and subsequent multiple mutational substitutions.

Amino Acid Sequence↗
PubMed1983

[1H-NMR study of the Naja naja oxiana neurotoxin II and its spin-labeled derivatives. Conformation of "short" neurotoxins].

In 1H NMR spectra of neurotoxin II N. n. oxiana the chemical shift pH-dependences in H2O and 2H2O solutions were studied, and also the deuterium exchange rates and chemical shift temperature gradients were measured for the amide protons. The spin probe method was applied to assess the degree of exposure into solvent of the amide and side chain protons. With the purpose of establishing mutual disposition of certain neurotoxin II groupings, nuclear Overhauser effect was studied in the 1H NMR spectra, along with the broadening of proton resonances induced by spin labels selectively attached to epsilon-amino groups of Lys26, Lys27, Lys45 or Lys47. The mobility of these labels was determined from the EPR spectra. The methyl resonances of Val and Leu residues were assigned to a definite position in the amino acid sequence. The following pKa were determined: alpha-NH2 Leu1 (9,2), gamma-COOH Glu2 (3,7), alpha-COOH Asn62 (1,3). The protonation of a carboxyl group(s) in neurotoxin II (alpha-COOH Asn62 seems to be involved) decreases the temperature stability of the neurotoxin II conformation. On the basis of studies on neurotoxin II and some other homologous neurotoxins, the model for the "short" neurotoxin folding in solution was proposed. Comparison of experimental data for the disposition of equivalent groups in homologous neurotoxins and in the X-ray structure of erabutoxin b Laticauda semifasciata revealed that the Val46 side chain in solution might change its orientation by 180 degrees with respect to polypeptide backbone. Binding of spin labeled neurotoxin II derivatives to the acetylcholine receptor was discussed in light of the obtained data.

Amino Acid Sequence↗
PubMed1992

Inhibition of calcium-dependent release of noradrenaline from PC12 cells by botulinum type-A neurotoxin. Long-term effects of the neurotoxin on intact cells.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin.

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)

Amino Acid Sequence↗
PubMed2005

A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins.

Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.

Amino Acid Sequence↗
PubMed1994

Inhibition of vacuolar adenosine triphosphatase antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins.

Bafilomycin A1, an inhibitor of vacuolar adenosine triphosphatase, was tested for its ability to antagonize botulinum neurotoxins (serotypes A-G), tetanus toxin and phospholipase A2 neurotoxins (notexin, beta-bungarotoxin, taipoxin and textilotoxin) on the mouse phrenic nerve-hemidiaphragm preparation. Bafilomycin itself produced concentration-dependent blockade of neuromuscular transmission without blocking nerve action potentials or muscle action potentials. This effect may have been due to inhibition of the proton pump that regulates acetylcholine transport into vesicles. At submaximal concentrations, bafilomycin was very effective in delaying the onset of paralysis due to all clostridial neurotoxins, but it had no protective effect against phospholipase A2 neurotoxins. Experiments were done to determine which of the three steps in clostridial neurotoxin action was antagonized by bafilomycin (e.g., binding, internalization and intracellular poisoning). Both pharmacological experiments and ligand-binding experiments showed that the drug did not block toxin binding to the plasma membrane. Similarly, pharmacological experiments on the time-dependent effects of bafilomycin showed that the drug did not antagonize the intracellular actions of toxins. The data indicated that bafilomycin acted at the intermediate step of internalization. This is in keeping with the facts that: 1) bafilomycin inhibits vacuolar adenosine triphosphatase, which in turn leads to inhibition of acidification in endosomes and 2) clostridial neurotoxins depend upon acidification of endosomes for translocation to the cytosol. The finding that bafilomycin antagonizes tetanus toxin may provide important clues for understanding how this toxin can act locally to produce flaccid paralysis. The finding that bafilomycin is a universal antagonist that protects against all clostridial neurotoxins may have important implications for developing therapeutic drugs.

Adenosine Triphosphatases↗
PubMed1997

[Action mechanisms of botulinum neurotoxins and tetanus neurotoxins].

Tetanus (TeNT) neurotoxin and botulinum (BoNT, serotypes A-G) neurotoxins are di-chain bacterial proteins of MW-150 kDa which are also termed as clostridial neurotoxins. They are the only causative agents of two severe neuroparalytic diseases, namely tetanus and botulism. The peripheral muscle spasms which characterise tetanus are due to a blockade of inhibitory (GABAergic and glycinergic) synapses in the central nervous system leading to a motor neurones desinhibition. In contrast, botulism symptoms are only peripheral. They are consequent to a near irreversible and highly selective inhibition of acetyl-choline release at the motor nerve endings innervating skeletal muscles. During the past decade, the cellular and molecular modes of action of clostridial neurotoxins has been near completely elucidated. After a binding step of the neurotoxins to specific membrane acceptors located only on nerve terminals, BoNTs and TeNT are internalized into neurons. Inside their target neurones, the intracellularly active moiety (their light chain) is translocated from the endosomal compartment to the cytosol. The neurotoxins' light chains are zinc-dependent (endopeptidases which are specific for one among three synaptic proteins (VAMP/synaptobrevin, syntaxin or SNAP-25) implicated in neurotransmitter exocytosis. The presence of distinct targets for BoNTs and TeNT correlates well with the observed quantal alterations of neurotransmitter release which characterize certain toxin serotypes. In addition, evidence for a second, non-proteolytic, inhibitory mechanism of action has been provided recently. Most likely, this additional blocking action involves the activation of neurone transglutaminases. Due to their specific action on key proteins of the exocytosis apparatus, clostridial neurotoxins are now widely used as molecular tools to study exocytosis.

Animals↗
PubMed1991

Immunological characterization of Clostridium butyricum neurotoxin and its trypsin-induced fragment by use of monoclonal antibodies against Clostridium botulinum type E neurotoxin.

We examined the reactivities of Clostridium butyricum neurotoxin to nine monoclonal antibodies against Clostridium botulinum type E neurotoxin which recognize the light chain or the amino-terminal half (H-1 fragment) or the carboxyl-terminal half (H-2 fragment) of the heavy chain of botulinum neurotoxin. Butyricum neurotoxin and its derived chains did not react to two of four monoclonal antibodies recognizing the light chain, one of three recognizing the H-1 fragment, and one of two recognizing the H-2 fragment. The results indicate that the immunological difference between the two neurotoxins is not attributable to a particular portion of the toxin molecule. The fragment of butyricum neurotoxin obtained by prolonged tryptic treatment was found to comprise the light chain and H-1 fragment linked together by a disulfide bond.

Animals↗
PubMed1996

Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms.

The DNA fragment common to the genes encoding botulinum neurotoxin types C1 (BN/C1) and D (BN/D) was amplified by PCR from the culture supernatant of Clostridium botulinum type C strain 6813 (C6813) that was treated with either DNase I or proteinase K but not from the supernatant that was treated with both DNase I and proteinase K, suggesting the neurotoxin gene is located on a certain bacteriophage DNA. Thus, to isolate the neurotoxin gene, we performed PCR with the culture supernatant of C6813 and seven primer pairs designed from the genes encoding BN/C1 and BN/D. The coding region in the connected sequence encodes a neurotoxin composed of 1,280 amino acids with a molecular weight of 147,817. The neurotoxin from C6813 has 95% amino acid identity to BN/C1, except for its C-terminal one-third, which is quite similar to the C-terminal one-third of BN/D (95% identity). When we performed PCRs with four primer pairs designed from the 5'-terminal two-thirds of the BN/C1 gene and two primers from the 3'-terminal one-third of the BN/D gene, DNA fragments of the expected sizes (0.5 to 1.3 kbp) could be amplified from C. botulinum type C strains 6812 and 6814. These results suggest that some strains of C. botulinum type C contain the gene encoding the mosaic neurotoxin composed of parts of BN/C1 and BN/D.

Amino Acid Sequence↗
PubMed2006

Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

Amino Acid Sequence↗
PubMed1983

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor-bound neurotoxin.

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor bound neurotoxin. Toxicon 21, 219-229, 1983--Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50-150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residue Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.

Acetylation↗
PubMed1996

Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa.

Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.

Animals↗
PubMed1993

Nucleotide sequence of the gene coding for Clostridium barati type F neurotoxin: comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium barati ATCC43756 was cloned as a series of overlapping polymerase chain reaction (PCR) generated fragments using primers designed to conserve toxin sequences previously published. The toxin gene has an open reading frame (ORF) of 1268 amino acids giving a calculated molecular mass of 141,049 Da. The sequence identity between the C. barati ATCC43756 and non-proteolytic C. botulinum 202F neurotoxins is 64.2% for the light chain and 73.6% for the heavy chain. This is much lower than reported identities for the type E neurotoxins from C. botulinum and C. butyricum (96% identity between light chains and 98.8% between the heavy chains). Previously identified conserved regions in other botulinal neurotoxins were also conserved in that of C. barati. An ORF upstream of the toxin coding region was revealed. This shows strong homology to the 3' end of the gene coding for the nontoxic-nonhemagglutinin (NTNH) component of the progenitor toxin from C. botulinum type C neurotoxin.

Amino Acid Sequence↗
PubMed1983

[Preparation of neurotoxin and hemagglutinin from Clostridium botulinum A and characterization of its neurotoxin].

A procedure for preparation of electrophoretically and serologically homogeneous neurotoxin and a highly purified hemagglutinin from the culture fluid of Cl. botulinum A, strain 501 is described. The yield of neurotoxin with specific activity of 80-100 X 10(6) DLM/mg of protein is 5-20%. Neurotoxin has a molecular weight of 150,000, sedimentation coefficient of 7.1S, pI of 6.2-6.3; the maximum of its fluorescence corresponds to 332 nm. The toxin molecule contains 4 SH-groups. Neurotoxin consists of two subunits with molecular weights of 98,000 and 56,000. The storage of neurotoxin at -20 degrees C causes inactivation and electrophoretical heterogeneity of the protein. The inactivation leads to an alteration of the toxin molecule charge, a shift of the lambda max of fluorescence and a loss of the SH-group reactivity without affecting the molecular weight or serological properties of the protein. The data obtained suggest that the conformational state of toxin molecules is essential for its biological activity.

Clostridium botulinum↗
PubMed2004

Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins.

In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum. Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin. Type A C. botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs). Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes. BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively. Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions. Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs. We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.

Animals↗
PubMed1997

Nonidentity of the alpha-neurotoxin binding sites on the nicotinic acetylcholine receptor revealed by modification in alpha-neurotoxin and receptor structures.

alpha-Neurotoxins constitute a large family of polypeptides that bind with high affinity to the nicotinic acetylcholine receptor (nAChR). Using a recombinant DNA-derived alpha-neurotoxin (Naja mossambica mossambica, NmmI) and mouse muscle nAChR expressed transiently on the surface of HEK 293 cells, we have delineated residues involved in the binding interaction on both the alpha-neurotoxin and the receptor interface. Several of the studied NmmI mutations, including two residues conserved throughout the alpha-neurotoxin family (K27 and R33), resulted in substantial decreases in the binding affinity. We have also examined 23 mutations located on the receptor alpha subunit and have identified 4 positions that appear to be important to NmmI recognition. These determinants represent a conserved aromatic residue (Y190), two positions where neuronal and muscle receptors differ (V188 and P197), and a negatively charged residue (D200). Unlike many of the nAChR agonists and antagonists which bind to the alphadelta and alphagamma binding sites on the receptor with different affinities, the wild-type NmmI-wild-type nAChR interaction showed a single affinity. However, by mutating critical toxin or receptor residues, we were able to produce site-selectivity between the alphagamma and alphadelta interfaces. These results suggest a nonequivalence in the binding interaction at the two sites, sensitive to discrete structural changes at key contact points on either the toxin or the receptor protein, and underscore the importance of delta and gamma receptor subunits in governing binding affinity.

Amino Acid Sequence↗