Botulinum Research Center, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
Appl Environ Microbiol. 2010 Oct;76(19):6658-63. doi: 10.1128/AEM.00731-10. Epub 2010 Aug 6.
Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.
肉毒杆菌神经毒素（BoNT）血清型 B（BoNT/B）是引起致命性人类肉毒中毒的血清型之一，尽管它在临床上用于治疗许多神经肌肉疾病。BoNT/B 由肉毒梭菌产生，以 BoNT/B 复合物的形式与一组神经毒素相关蛋白（NAPs）一起分泌。该复合物在碱性 pH 值下解离成 150 kDa 的全毒素和 NAPs。150 kDa 的 BoNT/B 全毒素可被切割产生 50 kDa 的结构域，称为轻链（LC）和 100 kDa 的重链，前者具有独特的内切蛋白酶活性。两条链通过二硫键连接，该键可还原分离两条链。内切蛋白酶活性存在于毒素的所有三种形式（复合物、纯化的 BoNT/B 全毒素和分离的轻链）中，不同的研究人员使用这些形式来开发检测方法和筛选抑制剂。在这项研究中，首次在相同条件下比较了这三种形式的内切蛋白酶活性。结果表明，这三种形式的酶活性差异显著，在很大程度上取决于切割和二硫键还原条件。在使用的条件下，LC 的活性最高，复合物的活性最低。BoNT/B 全毒素的切割增强了活性，切割后二硫苏糖醇（DTT）的还原进一步增强了活性。这些信息有助于理解 BoNT 内切蛋白酶的特性，并在使用不同形式的 BoNT 内切蛋白酶进行测试时比较不同抑制剂的功效。
