A型肉毒神经毒素(BoNT/A)基因序列分析及其B细胞表位预测

比较GenBank中4个不同毒株的A型肉毒神经毒素（BoNT／A）的基因组序列,发现其基因序列的一致性高迭92．2％-99．9％。基于保守性高的BoNT／A氨基酸序列,根据BioSun和LaserGene软件包中的表住分析相关参数,辅以对BoNT／A蛋白的二级结构的分析,综合预测BoNT／A的B细胞表位。结果表明,BoNT／A轻链的142-150、284-292区段,轻链的284-292,重链的440-450、465-480、538-549、699-710、751-760、1087-1095、1224-1231、1263-1270区段是B细胞表位优势区的可能性较大。多参数方案井结合不同软件综合预测BoNT／A蛋白的B细胞表位,为进一步实验鉴定BoNT／A的B细胞表位及其多表位疫苗设计和研究奠定了基础。     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Production and purification of Clostridium botulinum type C and D neurotoxin

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.     

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.     

A capillary electrophoresis technique for evaluating botulinum neurotoxin B light chain activity

Botulinum neurotoxin B (BoNT/B) produces muscle paralysis by cleaving synaptobrevin/vesicle-associated membrane protein (VAMP), an 18-kDa membrane-associated protein located on the surface of small synaptic vesicles. A capillary electrophoresis (CE) assay was developed to evaluate inhibitors of the proteolytic activity of BoNT/B with the objective of identifying suitable candidates for treatment of botulism. The assay was based on monitoring the cleavage of a peptide that corresponds to residues 44-94 of human VAMP-2 (V51) following reaction with the catalytic light chain (LC) of BoNT/B. Cleavage of V51 generated peptide fragments of 18 and 33 amino acids by scission of the bond between Q76 and F77. The fragments and parent peptide were clearly resolved by CE, allowing accurate quantification of the BoNT/B LC-mediated reaction rates. The results indicate that CE is suitable for assessing the enzymatic activity of BoNT/B LC.     

Reductive methylation of lysine residues of botulinum neurotoxin types A and B

Summary Reductive methylation of botulinum neurotoxin (NT) serotypes A and B at various ratios of protein to reagent modified up to 75° 10 of the lysine residues. Amino acid analysis of the modified proteins (HCl hydrolysed) confirmed selective modifications of lysine. The derivative N,N-dimethyl lysine was more abundant than monomethyl lysine; trimethyl lysine was not detected. Distribution of modified lysine residues among the heavy and light chains (M_r 100000 and 50000, respectively) of the dichain type A NT (M_r 150000) was approximately proportional to the lysine contents of the two subunit chains of the NT. Toxicity (mouse lethality) and serological reactivity (polyclonal antibody) of serotype A NT were not (or insignificantly) damaged following methylation of up to 72 lysine residues. Modification of 3 additional residues caused precipitous loss in toxicity. Toxicity of serotype B NT, unlike type A, appeared more sensitive to lysine modification. The large number of lysine residues that can be methylated without damaging toxicity of type A NT can be exploited to a) radiolabel the dichain protein exclusively in one chain keeping the other chain unlabelled, b) restrict the number of tryptic cleavage sites of the NT, and c) tag the protein with various markers or reactive ligands.     

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (H_N and H_C) differ by 13%. The LC acts as a zinc-dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.     

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]     

Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B

Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.     

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E‐cadherin binding. It is known that HA forms a three‐arm structure, in which each of three protomers has three carbohydrate‐binding sites and one E‐cadherin‐binding site. A three‐arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier‐disrupting activity. We prepared type B full‐length HA (three‐arm form) and mini‐HA, which is a deletion mutant lacking the trimer‐forming domain. Size‐exclusion chromatography analysis showed that mini‐HA exists as dimers (two‐arm form) and monomers (one‐arm form), which are then separated. We examined the multivalency effect of HA on the barrier‐disrupting activity, the E‐cadherin‐binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco‐2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier‐disrupting activity in Caco‐2 cells. We found that basal cell surface attachment and binding ability to immobilized E‐cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier‐disrupting activity.

     

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).     

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).     

Molecular topography and secondary structure comparisons of botulinum neurotoxin types A,B and E

Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (-helix, -sheet, -turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantuin yield). The proteins are high in -pleated sheet content (41–44%) and low in -helical content (21–28%). About 30–36% of the amino acids are in random coils. The -sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and ENT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low -helix and high -sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities.     

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C‐terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis‐dependent mechanism within 10–20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.     

Circular dichroic and fluroescence spectroscopic study of the conformation of botulinum neurotoxin types A and E

Summary Botulinum neurotoxin (NT) is synthesized byClostridium botulinum in any of seven antigenically distinct forms, called types A through G. Protease(s) endogenous to the bacteria, or trypsin, nicks the single chain protein to a dichain molecule which generally is more toxic. The conformation of dichain type A (nicked by endogenous protease), single chain type E, and dichain type E NT (nicked by trypsin) have been determined using circular dichroism (CD) and fluorescence spectroscopy. The high degree of ordered secondary structure (α helix 28%, β sheet 42%, total 70%) found in type A NT at pH 6.0 was similar to that found at pH 9.0 (α 22%, β 47%, total 69%). The secondary structure of the single chain type E NT at pH 6.0 (α 18%, β 37%, total 55%) differed somewhat from these values at pH 9.0 (α 22%, β 43%, total 65%). The dichain type E NT at pH 6.0 assumed a secondary structure (α 20%, β 47%, total 67%) more similar to that of dichain type A than the single chain type E NT. Examination with the fluorogenic probe toluidine napthalene sulfonate revealed that the hydrophobicity of the type A and E NTs were higher at pH 9.0 than at pH 6.0. Also, the hydrophobicity of the dichain type E NT was higher than its precursor the single chain protein and appeared similar to that of the dichain type A NT. The CD and fluorescence studies indicate that conversion of the single chain type E NT to the dichain form (i.e. nicking by trypsin) induced changes in conformation. The ordered secondary structure (a + β contents) of botulinum NT, 70% for type A and 67% for dichain type E, agree well with 65% of α + β contents of tetanus toxin [21] that is produced byClostridium tetani.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.     

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

The peptide N-acetyl-CRATKML-amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61-64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl-containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin-converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2-mercapto-3-phenylpropionyl yielded a peptide with K(i) of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1' arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.