Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.     

Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes.

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.     

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.     

Binding ability of Clostridium botulinum neurotoxin to the synaptosome upon treatment of various kinds of the enzymes

The binding ability of Cl. botulinum neurotoxin to synaptosomes upon treatment with various enzymes (neuraminidase, trypsin, and β-bungarotoxin containing phospholipase A₂ activity) was studied. When synaptosomes were treated with neuraminidase, their ability to bind toxin decreased; trypsin and β-bungarotoxin had slightly week or no effect. The decrease in toxin-binding ability of synaptosomes was paralleled by a release of sialic acid from the synaptosomes by the neuraminidase treatment. The toxin-binding ability of synaptosomes treated with neuraminidase was lower than untreated ones at a high concentration of sodium chloride. The binding of the toxin to synaptosomes occurred at least at the two types of structural sites, one site which contained sialic acid, and other site which was sensitive to high ionic strength. It may be possible that another binding state except these is present at the synapse.     

Differential Effects of Presynaptic Phospholipase A2 Neurotoxins on Torpedo Synaptosomes

The effects of several phospholipase A2 neurotoxins from snake venoms were examined on purely cholinergic synaptosomes from Torpedo electric organ. The noncatalytic component A of crotoxin had no effect, whereas its phospholipase component B, used alone or complexed to component A, elicited a rapid and dose-dependent acetylcholine (ACh) release and a depolarization of the preparation. Subsequent ACh release evoked by high K+ levels or calcium ionophore was identical to the control after the action of component A but reduced after the action of crotoxin or of component B. These effects were not observed when the phospholipase A2 activity of the toxin was blocked either by replacing Ca2+ by Ba2+ (respectively, activator and inhibitor of phospholipase A2) or by alkylation of component B with p-bromophenacyl bromide. beta-Bungarotoxin, another very potent phospholipase A2 neurotoxin, induced release of little ACh, did not affect ionophore-evoked ACh release, but significantly reduced depolarization-induced ACh release. The single-chain phospholipase A2 neurotoxin agkistrodotoxin behaved like crotoxin component B. A nonneurotoxic phospholipase A2 from mammalian pancrease induced release of an amount of ACh similar to that released by crotoxin but did not affect the evoked responses. The obvious differences in effect of the various neurotoxins suggest that they exert their specific actions on the excitation-secretion coupling process at different sites or by different mechanisms.     

Characterization of the functional activity of botulinum neurotoxin subtype B6

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A–G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C‐terminal heavy chain (H_C) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously‐characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H_C/B1 and H_C/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H_c.domain.

     

Microtubule-Dissociating Drugs and A23187 Reveal Differences in the Inhibition of Synaptosomal Transmitter Release by Botulinum Neurotoxins Types A and B

The inhibitory effects of botulinum neurotoxins types A and B on Ca2(+)-dependent evoked release of [3H]noradrenaline from rat cerebrocortical synaptosomes were compared and their molecular basis investigated. A23187, a Ca2+ ionophore, proved more efficacious in reversing the blockade produced by type A than that by B, whereas the actions of neither were changed by increasing intraterminal cyclic GMP levels using 8-bromo-cyclic GMP of nitroprusside. Disruption of the actin-based cytoskeleton with cytochalasin D did not alter the inhibition seen subsequently with either toxin. However, prior disassembly of microtubules with colchicine, nocodazole, or griseofulvin reduced the potency of type B toxin, but not that of type A toxin; stabilization of the microtubules with taxol counteracted this effect of colchicine. Because colchicine treatment of synaptosomes did not interfere with the measurable binding of type B toxin or its apparent uptake, it appears to act intracellularly. Collectively, these data suggest that botulinum neurotoxins types A and B inactivate transmitter release by interaction at different sites in the process. Based on the consistent results observed with four different drugs known to affect selectively microtubules, their involvement in the action of the type B neurotoxin is proposed.     

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.     

Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?

Botulinum neurotoxin, produced by Clostridium botulinum as a approximately 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The approximately 50- and approximately 100-kDa chains of the dichain molecule remain held together by an interchain disulfide bridge and noncovalent interactions. The neurotoxin binds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the approximately 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state. The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing. The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and DasGupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-830), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel.     

The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin

The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.     

Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates

Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules. Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol. Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step. Here, we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D (BoNT/D). The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol. Dihydrofolate reductase and BoNT type A (BoNT/A) light chain (LC) were efficiently conveyed into the cytosol, whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity. Luciferase and BoNT/A LC retained their catalytic activity as evidenced by luciferin conversion or SNAP-25 hydrolysis in the cytosol of synaptosomes, respectively. Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane. Thus, enzymatically inactive clostridial neurotoxins may serve as effective, safe carriers for delivering proteins in functionally active form to the cytosol of neurones.     

Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences

Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.     

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]     

The coupling of Na,K-ATPase with the sodium channels in the plasma membranes of the brain synaptosomes of rats]

Effect of neurotoxins veratrine (100 micrograms/ml) and tetrodotoxin (1 microM) on the binding of 3H-ouabain (10(-8) M) with Na,K-ATPase of intact synaptosomes and isolated synaptic membranes was studied. The persistent opening of sodium channels in synaptosomes by veratrine results in an increase of specific binding of the labeled ligand by 20%. A similar effect was caused by Na/H exchanger monensin. Destruction of microtubules with vinblastine and colchicine has no influence on veratrine action, while depolymerization of microfilaments with cytochalasin B reverses the neurotoxin effect. In isolated synaptic membranes veratrine and tetrodotoxin stimulate ouabain binding, the absolute veratrine-induced increment being several times higher in the presence of ATP than in its absence. Since the closed vesicles of any type are not permeable to ATP and ouabain, it means that in the isolated membranes an interaction between sodium channels and Na,K-ATPase molecules takes place. In intact nerve endings such a mechanism may be operative along with the known ways of control of sodium pump and its ouabain-binding site.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

外源性蛋白酶对肉毒毒素的作用初探

将B型肉毒毒素在毒素粗提阶段用胰蛋白酶处理,再经浓缩、柱层析和结晶得到纯化的B型肉毒毒素复合物。结果表明:B型肉毒神经毒素经胰蛋白酶处理后单链裂解为双链,在非还原条件下SDS-PAGE显示神经毒素条带,在还原条件下SDS-PAGE只显示轻(L)、重(H)二链条带,而不显示神经毒素条带;纯化后毒素复合物的比活性提高了5.9倍,达到1.60×108LD50/mgPr;HPLC显示活性成分峰面积所占比例增加了9.83%。     

Effect of tetranitromethane on the biological activities of botulinum neurotoxin types A,B and E

Botulinum neurotoxin serotypes A, B and E were modified at pH 7.9 with tetranitromethane, a reagent highly specific for tyrosine residues. The type B and E neurotoxins were completely detoxified without significant damage to their serological activities. Under similar modification conditions, the type A neurotoxin was incompletely detoxified with some alteration in its serological reactivity. Modification of only tyrosine residues to nitrotyrosine was evident from amino acid analysis of the acid hydrolysates of the modified proteins. The completely detoxified type B and E neurotoxins, used as toxoid, elicited antibodies in rabbits. The antisera precipitated and neutralized the homologous neurotoxin. The two toxoids, type B and E, were prepared with >99% pure neurotoxins as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis whereas the traditional toxoids produced with formaldehyde are very crude preparations of the neurotoxin ( 90% impure). Chemical modification using tetranitromethane is more specific than products that form during 7 days of reaction between a protein and formaldehyde. The toxoids produced with tetranitromethane may be considered second-generation toxoids, compared with the first-generation toxoids (crude preparation of neurotoxins detoxified with formaldehyde).     

Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.