Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 2009 Mar 24;48(11):2522-8. doi: 10.1021/bi801686b.
Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes, BoNT/A1 and BoNT/A2, which are approximately 95% homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified, BoNT/A3 (Loch Maree) and BoNT/A4 (657Ba), which are 81 and 88% homologous with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different from BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant light chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% of the rate of LC/A1 but cleaved SNAPtide at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide at slower rates than LC/A1. LC/A3 and LC/A4 had similar K(m) values for SNAP25 relative to LC/A1, while the k(cat) for LC/A4 was 10-fold slower than that for LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule or peptide inhibitors as therapies against botulism.
肉毒杆菌毒素（BoNT）是锌蛋白酶（血清型 A-G），通过在运动神经元内切割 SNARE 蛋白而导致弛缓性瘫痪。BoNT/A 最初分为两个亚型，BoNT/A1 和 BoNT/A2，它们大约有 95%的同源性，并具有相似的催化活性。随后，又确定了另外两个亚型，BoNT/A3（Loch Maree）和 BoNT/A4（657Ba），它们分别与 BoNT/A1 有 81%和 88%的同源性。序列比对研究预测，BoNT/A3 和 BoNT/A4 与 BoNT/A1 有足够的差异，会影响 SNAP25 的结合和切割。对 BoNT/A3（LC/A3）和 BoNT/A4（LC/A4）的重组轻链（LC）进行了生化分析。LC/A3 切割 SNAP25 的速度是 LC/A1 的 50%，但切割 SNAPtide 的速度比 LC/A1 快，而 LC/A4 切割 SNAP25 和 SNAPtide 的速度比 LC/A1 慢。LC/A3 和 LC/A4 对 SNAP25 的 K(m) 值与 LC/A1 相似，而 LC/A4 的 k(cat)比 LC/A1 慢 10 倍，这表明底物切割存在缺陷。LC/A3 和 LC/A4 均没有自身催化活性，这是 LC/A1 和 LC/A2 的特性。因此，BoNT/A 的四个亚型与 SNAP25 的结合亲和力相似，但 SNAP25 切割、SNAPtide 切割和自身催化的催化能力不同。LC/A 亚型之间确定的催化特性可能会影响小分子或肽抑制剂作为抗肉毒中毒疗法的开发策略。
