Mechanism of Botulinum Toxin Action

描述（由申请人提供）：肉毒杆菌毒素是引起肉毒杆菌中毒的病原体。这种毒素通常被认为是所有毒物中毒性最大的，已知存在于七种血清型中，命名为A、B、C、D、E、F和G。在这七种血清型中，有三种（A、B和E）可解释几乎所有的人类肉毒中毒病例。对于肉毒杆菌毒素产生临床中毒，它通常必须穿过至少两个膜屏障。最初，毒素结合并经历穿过肠道或气道上皮细胞的转胞吞作用。这是毒素逸出肠道或气道腔到达全身循环的机制。随后，毒素与外周胆碱能神经细胞结合，经历受体介导的内吞作用和pH诱导的易位，然后在胞质溶胶中作为锌依赖性内切蛋白酶裂解胞吐作用所必需的多肽。这是毒素产生肌肉无力或弛缓性麻痹的机制，这是肉毒杆菌毒素中毒的特征性结果。拟议工作的目标将是选择A型肉毒杆菌毒素作为原型，选择人类肠道上皮细胞系（T-84）作为原型，然后使用这两个模型来深入了解亚细胞和分子事件，这些事件说明毒素穿透膜屏障的能力。更确切地说，将使用两种方法来帮助阐明介导结合和转胞吞作用的毒素的结构特征：1）将产生一系列截短突变体以试图鉴定保留穿过肠上皮细胞的能力的最小结构域，和2.）将产生一系列定点突变，试图将关键氨基酸定位在结合和胞吞转运所需的最小结构域内。此外，将使用两种方法来帮助鉴定人类肠道细胞中从顶端转运毒素的亚细胞组分（即，粘膜的）到基底外侧（即，（1.）药理学技术将用于区分网格蛋白包被的囊泡与脂筏囊泡，和2.）免疫学技术将用于鉴定毒素结合位点中或附近的膜多肽。预计这项拟议研究的组合产品将提供对肉毒杆菌毒素穿透人类细胞膜的清晰理解，并具有产生针对该毒素的潜在疫苗的额外益处。

DESCRIPTION (provided by applicant): Botulinum toxin is the etiologic agent responsible for the disease botulism. This toxin, which is generally regarded as the most poisonous of all poisons, is known to exist in seven serotypes designated A, B, C, D, E, F and G. Of these seven serotypes, three (A, B and E) account for virtually all cases of human botulism. For botulinum toxin to produce clinical poisoning, it must ordinarily cross at least two membrane barriers. Initially, the toxin binds and undergoes transcytosis across gut or airway epithelial cells. This is the mechanism by which the toxin escapes the lumen of the gut or airway to reach the general circulation. Subsequently, the toxin binds to peripheral cholinergic nerve cells, undergoes receptor-mediated endocytosis and pH-induced translocation, then acts in the cytosol as a zinc-dependent endoprotease to cleave polypeptides that are essential for exocytosis. This is the mechanism by which the toxin produces muscle weakness or flaccid paralysis, which is the characteristic outcome in botulinum toxin poisoning. The goal of the proposed work will be to select botulinum toxin type A as a prototype, to select a human gut epithelial cell line (T-84) as a prototype, and then use these two as models to gain insight into the subcellular and molecular events that account for the ability of the toxin to penetrate membrane barriers. More precisely, two approaches will be used to help elucidate the structural features of the toxin that mediate binding and transcytosis: 1.) a series of truncation mutants will be generated in an attempt to identify the minimal domain that retains the ability to cross gut epithelial cells, and 2.) a series of site-directed mutations will be generated in an attempt to localize critical amino acids within the minimal domain that are needed for binding and transcytosis. In addition, two approaches will be used to help identify subcellular components in human gut cells that transport the toxin from the apical (viz., mucosal) to the basolateral (viz., serosal) surfaces: 1.) pharmacologic techniques will be used to discriminate clathrin-coated vesicles from lipid raft vesicles, and 2.) immunologic techniques will be used to identify membrane polypeptides that are in or near the binding site for the toxin. It is anticipated that the combined products of this proposed research will provide a clear understanding of botulinum toxin penetration of human cell membranes, with the added benefit of generating a potential vaccine against the toxin.