Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
J Struct Biol. 2011 Apr;174(1):52-7. doi: 10.1016/j.jsb.2010.11.010. Epub 2010 Nov 13.
Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. BoNTs consist of three essential domains for toxicity: the cell binding domain (Hc), the translocation domain (Hn) and the catalytic domain (LC). A functional derivative (LHn) of the parent neurotoxin B composed of Hn and LC domains was recombinantly produced and characterised. LHn/B crystallographic structure at 2.8Å resolution is reported. The catalytic activity of LHn/B towards recombinant human VAMP was analysed by substrate cleavage assay and showed a higher specificity for VAMP-1, -2 compared to VAMP-3. LHn/B also showed measurable activity in living spinal cord neurons. Despite lacking the Hc (cell-targeting) domain, LHn/B retained the capacity to internalize and cleave intracellular VAMP-1 and -2 when added to the cells at high concentration. These activities of the LHn/B fragment demonstrate the utility of engineered botulinum neurotoxin fragments as analytical tools to study the mechanisms of action of BoNT neurotoxins and of SNARE proteins.
肉毒神经毒素（BoNTs）通过抑制胆碱能神经末梢的神经递质传递而导致弛缓性瘫痪。BoNTs 由三个毒性必需结构域组成：细胞结合结构域（Hc）、易位结构域（Hn）和催化结构域（LC）。亲本神经毒素 B 的功能衍生物（LHn）由 Hn 和 LC 结构域组成，通过重组生产并进行了表征。报道了 LHn/B 的晶体结构，分辨率为 2.8Å。通过底物切割测定分析了 LHn/B 对重组人 VAMP 的催化活性，结果表明它对 VAMP-1、-2 的特异性高于 VAMP-3。LHn/B 在活体脊髓神经元中也表现出可测量的活性。尽管缺乏 Hc（细胞靶向）结构域，但当以高浓度添加到细胞中时，LHn/B 仍保留了内化和切割细胞内 VAMP-1 和 -2 的能力。LHn/B 片段的这些活性证明了工程化肉毒神经毒素片段作为分析工具的实用性，可用于研究 BoNT 神经毒素和 SNARE 蛋白的作用机制。
