de Paiva A, Poulain B, Lawrence G W, Shone C C, Tauc L, Dolly J O
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
J Biol Chem. 1993 Oct 5;268(28):20838-44.
Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.
A型肉毒杆菌神经毒素由一条通过二硫键连接的轻链和重链组成，重链的C端一半内存在一个内部二硫键。研究了还原这些键并烷基化硫醇的功能后果。游离半胱氨酸残基的修饰对小鼠生物测定中的毒性以及对小鼠神经-膈肌和加州海兔颊神经节中的乙酰胆碱释放均无影响。然而，在烷基化之前还原毒素会大幅降低神经麻痹效力；然而，如果直接注射到海兔神经节的突触前神经元中或添加到牛透化肾上腺嗜铬细胞中，这种衍生物会抑制递质释放。它对肉毒杆菌神经毒素A在哺乳动物运动神经末梢和海兔神经元作用的拮抗作用表明其保留了与毒素的有效细胞外受体结合的能力。因此，通过切断两个二硫键以及对相关半胱氨酸进行烷基化来消除细胞外应用的A型肉毒杆菌神经毒素的毒性，是由于摄取无效所致。利用A型肉毒杆菌神经毒素A分离链的修饰形式来确定哪些二硫键对于内化是必需的。对轻链和重链中的半胱氨酸进行烷基化，包括那些参与链间键的半胱氨酸，但不包括重链中完整二硫键的半胱氨酸，结果表明，为了使A型肉毒杆菌神经毒素A能够进入海兔神经元的膜转运，分子间键必须存在，或者相关硫醇未被修饰。
