Studies on the dissociation of botulinum neurotoxin type A complexes
Studies on the dissociation of botulinum neurotoxin type A complexes
复制标题
DOI：
10.1016/j.toxicon.2010.12.019
复制
发表时间：
2011-03-15
期刊：
影响因子：
2.8
通讯作者：
Taylor, Harold V.
中科院分区：
文献类型：
--
作者：
Eisele, Karl-Heinz;Fink, Klaus;Taylor, Harold V.
肉毒梭菌的各种菌株产生的神经毒素自然与复合蛋白有关，在细菌入侵宿主的过程中，复合蛋白起到保护神经毒素免受哺乳动物胃肠道恶劣环境的作用。从A型肉毒杆菌培养物中可分离到19S(900 kDa，L1复合体)、165(500 kDa，L复合体)和12S(300 kDa，M复合体)三种不同的复合体。然而，为了影响靶细胞，这些复合体必须解离释放出150 kDa的游离神经毒素。本研究评估了这些A型肉毒梭菌神经毒素复合体(BONT/A)的稳定性，并确定了影响它们解离的因素。从纯化的毒素复合体中获得的知识随后被用来分析冷冻或喷雾干燥的商业BONT/A产品肉毒杆菌毒素和DySPORT中的这种复合体的存在，并与无复合蛋白的产品Xeomin进行比较。纯化的900 kDa和500 kDa的毒素复合体制剂显示出150 kDa的神经毒素在pH值为7.0下的半衰期不到一分钟的pH和时间依赖的释放。在pH值为6.25以下时，络合物是稳定的。此外，稀释900 kDa的浓缩络合物会导致解离成500 kDa的含有神经毒素的络合物。在等渗盐水中加入氯化钠会进一步破坏这些复合体，导致游离150 kDa神经毒素的释放。使用相同的分析方法对商品化的肉毒杆菌神经毒素产品Botox和DySPORT进行检测，得出相同的结论：这些产品的稀释、干燥和重建过程导致900 kDa复合体完全解离，85%或更多的神经毒素以游离形式存在。结论：BoNT A毒素复合体通过有效地释放神经毒素来快速响应特定环境的变化。在药物生产和BoNT A产品的重建过程中，相同的原理影响900 kDa复合体的定量解离和在注射到靶组织之前释放游离神经毒素。(C)2011爱思唯尔有限公司。保留所有权利。
The neurotoxins produced by the various strains of the anaerobic bacterium Clostridium botulinum naturally occur associated with complexing proteins which serve to protect the neurotoxins from the harsh environment of the mammalian gastrointestinal tract during bacterial invasion of the host. Three different complex species with the discrete sizes 19S (900 kDa, LL complex), 165 (500 kDa, L complex) and 12S (300 kDa, M complex) may be isolated from C. botulinum type A cultures. However, to affect their target cells these complexes must dissociate releasing the free 150 kDa neurotoxin.This study assesses the stability of these Clostridium botulinum neurotoxin serotype A (BoNT/A) complexes and identifies factors which influence their dissociation. The knowledge gained with purified toxin complexes was subsequently employed to analyze the presence of such complexes in the freeze or spray-dried commercial BoNT/A products Botox and Dysport in comparison to the complexing protein free product Xeomin.Purified 900 kDa and 500 kDa toxin complex preparations show a pH and time dependent release of the 150 kDa neurotoxin with a half-life of less than a minute at pH values >7.0. At pH values of 6.25 or less, the complexes are stable. Furthermore, dilution of concentrated 900 kDa complexes leads to dissociation into 500 kDa, neurotoxin containing complexes. Addition of sodium chloride as contained in isotonic saline leads to further disruption of these complexes resulting in the release of the free 150 kDa neurotoxin.Examination of the commercial botulinum neurotoxin products Botox and Dysport using the same analytical procedures leads to the same conclusion: the dilution, drying and reconstitution processes of these products lead to a complete dissociation of 900 kDa complexes and 85% or more of neurotoxin are present in free form.Conclusion: BoNT A toxin complexes have evolved to quickly respond to specific environmental changes by efficient release of the neurotoxin. During pharmaceutical production and reconstitution of BoNT A products, the same principles effect the quantitative dissociation of 900 kDa complexes and release of free neurotoxin prior to injection into target tissues. (C) 2011 Elsevier Ltd. All rights reserved.