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.     

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.     

肉毒毒素中和抗体的研究进展

肉毒毒素是目前已知毒性最强的细菌蛋白质,极少量便可以致人死亡,我国每年都有散发病例出现,并且它极有可能被用于恐怖行动或被一些国家用作生物战剂。肉毒毒素中和抗体是肉毒毒素中毒后惟一有效的药物。与马源的抗毒素血清相比,重组基因工程中和抗体具有很多优势,是目前肉毒毒素预防和治疗研究的主要方向。简要综述了肉毒毒素基因工程中和抗体研究现状、保护性抗原选择、体内外中和活性检测方法及研发难点、解决方法等。     

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.     

肉毒毒素抑制剂的研究进展

肉毒毒素(BoNT)是目前已知的毒性最强的生物毒素,肉毒毒素中毒的抗毒素治疗只对未进入神经细胞的毒素有效,而对中毒时间较长、轻链已进入神经系统者无效。近10年来,随着肉毒毒素晶体结构的测定、中毒机理的深入研究,以及体外高通量评价模型的建立,对小分子肉毒毒素抑制剂的研究取得了显著进展。从肉毒毒素中毒机理、结构特征出发,简要综述了肉毒毒素抑制剂的研究进展。     

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.     

Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain*

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221–231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. etal. 2003, Biochemistry 42:12539–12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.     

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]     

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.     

Cell entry strategy of clostridial neurotoxins

Tetanus neurotoxin and botulinum neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins' sophisticated access to their site of action.     

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.     

肉毒神经毒素受体的研究进展

肉毒神经毒素(botulinumneurotoxin)是世界已知毒性最强的生物毒素,它通过酶切在递质释放过程中起关键作用的SNARE蛋白,抑制神经递质释放,阻断突触传递.综述了有关肉毒受体研究的进展.这些研究表明,肉毒的结合位点有低亲和力的和高亲和力的两种.肉毒的结合过程分两步,它首先与细胞表面的神经节苷脂结合,形成低亲和力的聚合体,然后再与高亲和力的蛋白受体——synaptotagmin结合,形成牢固的三聚体结构,并由内吞进入细胞.这种解释肉毒结合过程的双受体学说得到了越来越多的支持,文中列举和评述了支持该学说的实验资料.     

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.     

Mapping of the Antibody-Binding Regions on the HN-Domain (Residues 449–859) of Botulinum Neurotoxin A with Antitoxin Antibodies from Four Host Species. Full Profile of the Continuous Antigenic Regions of the H-Chain of Botulinum Neurotoxin A

Previously, we mapped the antibody (Ab) and T-cell recognition regions on the HC domain (residues 855-1296) of the 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We have mapped here the HN-domain (residues 449-859) regions that bind protective anti-BoNT/A Abs raised in four different species. We synthesized, purified, and characterized 29 19-residue peptides that spanned the entire HN and overlapped consecutively by 5 residues, and also region L218-231 around the L-chain's substrate-binding site. Human, horse, mouse, and chicken anti-BoNT/A Abs did not bind to the L-peptide but recognized similar HN regions within peptides 519-537/533-551/547-565/561-579 (with slight left- or right-shifts), 743-761, 785-803, and 813-831/827-845 overlap. Recognition of other peptides that bound lower Ab levels showed similarities and also some differences. Peptide 463-481, strongly immunodominant with horse antisera, did not bind human, mouse, and chicken Abs. However, peptide 449-467 bound Abs in these three antisera, and the region may have shifted to the right (peptide 463-481) with horse Abs. The overlap 659-677/673-691 reacted strongly with human Abs whereas with mouse and chicken antisera, only peptide 673-691 showed low reactivity. Horse antisera had no detectable Ab binding to region(s) 659-691. The Ab-recognition regions on the H chain occupy surface locations in BoNT/A three-dimensional structure, but the great part of the surface is not immunogenic. Regions recognized by the protective antisera of the four different species are prime candidates for inclusion in synthetic vaccine designs.