Lawrence Gary, Wang Jiafu, Chion C K N Kwo, Aoki K Roger, Dolly J Oliver
International Centre for Neurotherapeutics, Dublin City University, Dublin 9, Ireland.
J Pharmacol Exp Ther. 2007 Jan;320(1):410-8. doi: 10.1124/jpet.106.108829. Epub 2006 Oct 18.
The unique ability of a family of botulinum neurotoxins to block neuroexocytosis specifically-by selective interaction with peripheral cholinergic nerve endings, endocytotic uptake, translocation to the cytosol, and enzymic cleavage of essential proteins-underlies their increasing therapeutic applications. Although clinical use of type A is most widespread due to its prolonged inactivation of the synaptosomal-associated protein of 25 kDa, botulinum neurotoxin E cleaves this same target but at a different bond and exhibits faster onset of neuromuscular paralysis. Herein, insights were gained into the different dynamics of action of types A and E toxins, which could help in designing variants with new pharmacological profiles. Natural and recombinant type E dichain forms showed similar proteolytic and neuromuscular paralytic activities. The neuroparalysis induced by type E toxin was accelerated between 21 and 35 degrees C and attenuated by bafilomycin A1. Temperature elevation also revealed an unanticipated bipartite dose response indicative of two distinct internalization processes, one being independent of temperature and the other dependent. Although elevating the temperature also hastened intoxication by type A, a second uptake mechanism was not evident. Increasing the frequency of nerve stimulation raised the uptake of type E via both processes, but the enhanced trafficking through the temperature-dependent pathway was only seen at 35 degrees C. These novel observations reveal that two membrane retrieval mechanisms are operative at motor nerve terminals which type E toxin exploits to gain entry via an acidification-dependent step, whereas A uses only one.
肉毒杆菌神经毒素家族具有独特能力，可通过与外周胆碱能神经末梢选择性相互作用、内吞摄取、转运至胞质溶胶以及对关键蛋白进行酶切，特异性阻断神经胞吐作用，这构成了它们日益广泛的治疗应用基础。尽管A型肉毒杆菌神经毒素因能使25 kDa的突触体相关蛋白长时间失活而在临床应用最为广泛，但E型肉毒杆菌神经毒素能切割同一靶点，不过切割位点不同，且神经肌肉麻痹起效更快。在此，我们深入了解了A型和E型毒素不同的作用动力学，这有助于设计具有新药理学特性的变体。天然和重组E型双链形式显示出相似的蛋白水解和神经肌肉麻痹活性。E型毒素诱导的神经麻痹在21至35摄氏度之间加速，并被巴弗洛霉素A1减弱。温度升高还揭示了一种意想不到的双相剂量反应，表明存在两种不同的内化过程，一种与温度无关，另一种与温度有关。尽管温度升高也会加速A型毒素的中毒，但第二种摄取机制并不明显。增加神经刺激频率会通过这两种过程提高E型毒素的摄取，但仅在35摄氏度时才会看到通过温度依赖性途径增强的转运。这些新发现表明，运动神经末梢存在两种膜回收机制，E型毒素利用其中一种通过酸化依赖性步骤进入，而A型毒素仅利用一种。
