Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Dr, Madison, WI 53706, United States.
Biochem Biophys Res Commun. 2011 Feb 25;405(4):673-7. doi: 10.1016/j.bbrc.2011.01.093. Epub 2011 Feb 1.
Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.
无毒型肉毒神经毒素 A（BoNT/A）衍生物具有作为神经元靶向递药载体和研究细胞内运输的试剂的潜在用途。我们设计并表达了一种 BoNT/A 的无毒衍生物（BoNT/A ad），它是一个全长 150 kDa 的分子，由一个 50 kDa 的轻链（LC）和一个 100 kDa 的重链（HC）组成，通过在 LC 中引入金属蛋白酶失活点突变而变得无毒。在神经元培养物中的研究表明，BoNT/A ad 不能切割突触相关蛋白 25（SNAP25），wt BoNT/A 的底物，并且它可以有效地与 wt BoNT/A 竞争结合内源性神经元受体。体外和体内研究表明 BoNT/A ad 在小鼠膈肌的运动终板处积累。免疫沉淀研究表明，BoNT/A ad 的 LC 与神经元胞质部分中的 SNAP25 形成复合物，表明无毒 LC 保留了 wt 毒素的 SNAP25 结合能力。BoNT/A ad 的毒性相对于 wt BoNT/A 降低了约 100,000 倍。
