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.     

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.     

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。     

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.     

Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.     

重组肉毒神经毒素A轻链(BoNT/A LC)突变体的构建、表达与活性分析

A型肉毒神经毒素的轻链(BoNT/A LC)是一种锌依赖性的金属内肽酶.通过X射线分析其结构并结合一些文献报道表明,轻链上的Arg362和Tyr365直接参与了酶的催化作用,而Glu350则处于其活性位点的中心位置.采用定点突变技术,对编码这3个关键性的氨基酸位点的碱基进行突变(Arg362Ala、Tyr365Phe、Glu350Ala),获得了BoNT/A LC突变体.突变体蛋白与BoNT/A的底物蛋白SNAP-25进行切割反应,结果表明,未经突变的BoNT/A轻链蛋白能够特异性地识别SNAP-25蛋白上的Q197-R198位点,而突变体蛋白则完全无法识别该位点,不具有金属内肽酶活性,成功地去除了肉毒神经毒素的毒力,为下一步的全长肉毒神经毒素重组疫苗的研究打下了基础.     

TCEP treatment reduces proteolytic activity of BoNT/B in human neuronal SHSY‐5Y cells

The light chain (LC) of botulinum neurotoxin B (BoNT/B) is unable to enter target neuronal cells by itself. It is brought into the cell in association with the BoNT/B heavy chain (HC) through endocytosis. The BoNT HC‐LC subunits are held together by a single disulfide bond. Intracellular reduction of this bond and separation of the two subunits activates the endopeptidase activity of the LC. This requirement suggests a strategy to prevent uptake by prophylactic reduction to disrupt the disulfide bond prior to endocytosis of the complex. We examined the utility of tris‐(2‐carboxyethyl)‐phosphine hydrochloride (TCEP), a relatively non‐toxic, non‐sulfur containing disulfide bond reducing agent that lacks the undesirable properties of mercapto‐containing reducing agents. We found that TCEP was as effective as DTT with maximal LC endopeptidase activation occurring at 1 mM, a concentration not toxic to the human neuronal cell line, SHSY‐5Y. In these cells, 1 mM TCEP maximally protected against BoNT/B inhibition of [³H]‐NA release, achieving 72% of the release from un‐intoxicated controls. This effect appears to be due to the sparing of SNARE proteins as the levels of VAMP‐2, the specific target of BoNT/B, were protected. These results show that TCEP disrupts the structure of BoNT/B by reduction of the LC and HC bridging disulfide bond and prevents neuronal intoxication. Since disulfide bond coupling between toxin subunits is a general motif for many toxins, e.g., ricin, snake venom, and all BoNT serotypes, this suggests that TCEP is a promising means to protect against these toxins by preventing cell penetration. J. Cell. Biochem. 107: 1021–1030, 2009. Published 2009 Wiley‐Liss, Inc.     

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.     

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 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.

     

A label-free biosensor assay for botulinum neurotoxin B in food and human serum

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5–9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3 h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.     

The biological activity of ubiquitinated BoNT/B light chain in vitro and in human SHSY‐5Y neuronal cells

BoNT/B light chain is a zinc‐dependent endopeptidase. After entering its target, the neuronal cell, BoNT/B is responsible for synaptobrevin‐2 (VAMP‐2) cleavage. This results in reduced neurotransmitter (acetylcholine) release from synaptic vesicles, yielding muscular paralysis. Since the toxin persists in neuronal cells for an extended period, regeneration of VAMP‐2 is prevented. We evaluated therapeutic targets to overcome botulinum persistence because early removal would rescue the neuronal cell. The ubiquitination/proteasome cellular pathway is responsible for removing “old” or undesirable proteins. Therefore, we assessed ubiquitination of BoNT/B light chain in vitro, and characterized the effects of ubiquitination modulating drugs, PMA (phorbol 12‐myristate 13‐acetate) and expoxomicin, on ubiquitination of BoNT/B light chain in neuronal cells. Both drugs altered BoNT/B light chain ubiquitination. Ubiquitination in vitro and in cells decreased the biological activity of BoNT/B light chain. These results further elucidate BoNT protein degradation pathways in intoxicated neuronal cells and mechanisms to enhance toxin removal. J. Cell. Biochem. 108: 660–667, 2009. Published 2009 Wiley‐Liss, Inc.     

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.     

Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes

Botulinum neurotoxins are highly potent toxins capable of rapid and specific interaction with the presynaptic membrane. We have hypothesised that: (1) these neurotoxins possess an electric dipole with the positive pole on receptor binding domain Hc-C and that (2) on approaching the negatively charged presynaptic membrane, they reorient themselves and hit the membrane surface with Hc-C; this electrostatic effect would contribute efficient binding. Electrostatic calculations confirm these hypotheses and strongly indicate that electrostatics effects can play an important role in the unique presynaptic membrane binding properties of these neurotoxins and generally on the interaction of other plasma membrane protein ligands.     

A型肉毒毒素表位多肽免疫和人源噬茵体单链抗体文库的构建

采用表位合成多肽免疫获得编码人源抗体的基因材料,抗体轻重链可变区基因通过剪接重叠延伸(SOE)技术进行组装后,亚克隆入噬菌体粒中构建免疫库。以重组制备的A型肉毒毒素保护性抗原为配体筛选结合子,ELISA鉴定,获得具有较高抗原结合力的噬菌体克隆,进行基因测序和家族定位分析。结果显示,表位合成多肽免疫,成功构建了库容大于10^8的重组抗体库。体外3轮免疫淘筛,筛选到人源A型肉毒抗毒素克隆,基因结构分析表明,其中ScFvB17全长750bp,可编码250个氨基酸残基：VH属于VH4家族,369bp,编码123个氨基酸残基;Vκ属于κ链Ⅱ家族,336bp,编码112个氨基酸残基。基因的同源分析表明,这是一株特异的单链抗体新基因。     

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.     

Specificity of Factor Xa in the cleavage of fusion proteins

The precursor protein honey bee prepromelittin has been expressed as a fusion protein inEscherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor Xa. Factor Xa was found to cleave poorly at the engineered site, giving a low yield of the required prepromelittin. In contrast, cleavage on the C-terminal side of the sequence VLGR at residue 67 in the gene 10 sequence proceeded in high yield. Factor Xa may be inhibited by adjacent hydrophobic sequences on the C-terminal side of a potential cleavage site.     

Enzymatic autocatalysis of botulinum A neurotoxin light chain

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.