A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.     

Effective expression of fragments of a botulinum neurotoxin type A gene, coding for the L-chain and H-chain in E. coli, with formation of products causing protective immunity to administration of the toxin

Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

Plasmid encoded neurotoxin genes in Clostridium botulinum serotype A subtypes

Clostridium botulinum, an important pathogen of humans and animals, produces botulinum neurotoxin (BoNT), the most poisonous toxin known. We have determined by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations that the genes encoding BoNTs in strains Loch Maree (subtype A3) and 657Ba (type B and subtype A4) are located on large (approximately 280 kb) plasmids. This is the first demonstration of plasmid-borne neurotoxin genes in Clostridium botulinum serotypes A and B. The finding of BoNT type A and B genes on extrachromosomal elements has important implications for the evolution of neurotoxigenicity in clostridia including the origin, expression, and lateral transfer of botulinum neurotoxin genes.     

Hemagglutinin-33 of type A botulinum neurotoxin complex binds with synaptotagmin II

Botulinum neurotoxin type A (BoNT/A), the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. NAPs are known to protect BoNT against adverse environmental conditions and proteolytic digestion. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs that is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. However, it is not known whether Hn-33 plays any role other than the protection of BoNT. Using immunoaffinity column chromatography and pull-down assays, we have now discovered that Hn-33 binds to synaptotagmin II, the putative receptor of botulinum neurotoxin. This finding provides important information relevant to the design of novel anti-botulism therapeutic agents targeted to block the entry of botulinum neurotoxin into nerve cells.     

Clostridial neurotoxins as a drug delivery vehicle targeting nervous system

Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues.     

Characterization of the subunits of botulinum neurotoxin type A

A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out. The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold. In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified. Alanine was found to be the N-terminus of the heavy chain. The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains. The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.     

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.     

Ganglioside-Induced Adherence of Botulinum and Tetanus Neurotoxins to Adducin

Abstract: 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 ∼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 ∼116-kDa protein more than that with GT1b. A purified preparation of the water-soluble ∼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 ∼116-kDa protein. These sequences matched with the data bank sequences for β-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).     

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.     

Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.     

Binding of Clostridium botulinum neurotoxin to the presynaptic membrane in the central nervous system

Large synaptosome fractions were isolated from the cerebellar and cerebral cortices of rats and were incubated with Clostridium botulinum type A neurotoxin in vitro. The binding of the neurotoxin to the synapses was observed by electron microscopy, using the double-sandwich immunocytochemical method. Botulinum neurotoxin was preferentially bound to the presynaptic membrane in the large synaptosome fraction. The binding regions for the neurotoxin were localized on both the extrajunctional and junctional areas of the presynaptic membranes and appeared as patches of various sizes. However, they did not exist on the postsynaptic membranes. Botulinum neurotoxin is proposed to be a useful analytical tool for understanding the characteristics of the presynaptic membranes in the central nervous system.     

The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.     

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.     

Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.     

Evaluation of a botulinum fragment C-based ELISA for measuring the humoral immune response in primates.

An enzyme-linked immunosorbent assay (ELISA) using botulinum neurotoxin serotype B recombinant fragment C (rBoNTB(HC)) was developed to measure specific humoral immune responses of monkeys vaccinated with a vaccine consisting of rBoNTB(HC). Several fundamental parameters for a bioassay were evaluated. The evaluation results demonstrated that using BoNTB(HC) as the capture antigen led to a specific and sensitive ELISA for botulinum type B antibody with excellent precision, accuracy, and linearity. There was a good correlation (r=0.91) between ELISA titers and neutralization bioassay titers. Experimental results suggested that the ELISA could be useful for detecting botulinum type B antibody levels and may supplement mouse neutralization bioassays during planned clinical manufacturing and clinical trials of rBoNTB(HC) vaccine.