Purkiss J R, Friis L M, Doward S, Quinn C P
Centre for Applied Microbiology and Research, Salisbury, Wiltshire, UK.
Neurotoxicology. 2001 Aug;22(4):447-53. doi: 10.1016/s0161-813x(01)00042-0.
We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated [3H]-noradrenaline ([3H]-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold. Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry. Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order. This indicated that the toxins differed in their efficiency of binding/endocytosis or enzymatic activity inside the cell. The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit [3H]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive. The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.
我们已经描述了在未分化的人神经母细胞瘤SH-SY5Y细胞中，肉毒杆菌神经毒素（BoNT）A-F型血清型的相对效力。刺激后的[3H]-去甲肾上腺素（[3H]-NA）释放对毒素的敏感性在暴露3天后的效力顺序为：C>D>A>B>F。最有效力的（BoNT/C：IC50 0.54 nM）和效力最低的（BoNT/F：IC50>300 nM）之间的差异约为1000倍。尽管液相内吞作用可能是低效毒素进入细胞的机制，但BoNT/C的效力要高得多，这表明其进入细胞是由受体驱动的。效力并非释放机制对特定SNARE依赖性的反映，因为底物特异性在整个效力顺序中是混合的。这表明毒素在细胞内的结合/内吞效率或酶活性方面存在差异。切割囊泡相关膜蛋白（VAMP）同工型的血清型（BoNT/B、D和F）并未完全抑制[3H]-NA释放。所有血清型均观察到了对相应底物蛋白的切割。BoNT/A对SNAP-25的切割显示为剂量依赖性，且与释放减少密切相关，支持蛋白水解是毒素抑制分泌的机制。将SH-SY5Y细胞系对BoNT/A的敏感性与释放甘氨酸的大鼠原代脊髓神经元培养物进行比较，发现效力存在巨大差异；原代培养物的敏感性大约高20万倍。使用BoNTs证明SNAP-25、VAMP和 syntaxin在SH-SY5Y细胞中的关键作用，表明可将这种神经母细胞瘤用作研究这些蛋白质在神经递质释放中作用的模型。
