Lawrence G W, Foran P, Dolly J O
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
Eur J Biochem. 1996 Mar 15;236(3):877-86. doi: 10.1111/j.1432-1033.1996.00877.x.
Botulinum neurotoxin (BoNT) types A and B are Zn2+-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocystosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by Ba2+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20microM Ca2+ (and to a lesser extent by 1 mM Ba2+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg.ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba2+-stimulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNT/E to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.
A型和B型肉毒杆菌神经毒素（BoNT）是需要锌离子的内蛋白酶，它们分别通过切割25 kDa的突触体相关蛋白（SNAP-25）和突触小泡蛋白来有效阻断神经递质的释放。突触小泡蛋白对于儿茶酚胺从致密核心颗粒的胞吐作用很重要，本文提供了证据表明SNAP-25参与神经内分泌细胞的这一过程。在完整的牛嗜铬细胞中比较了BoNT/A和BoNT/B对调节性分泌的影响，以研究切割不同靶点的后果。每种毒素都能阻止由Ba2+、升高的K+浓度或尼古丁引起的儿茶酚胺分泌。分别观察到SNAP-25切割程度或突触小泡蛋白切割程度与BoNT/A或BoNT/B对分泌的抑制之间有很好的相关性，这表明SNAP-25和突触小泡蛋白在调节性胞吐作用中的重要性。尽管细胞暴露于BoNT/A会使几乎整个SNAP-25库被截断，但在透化后，由20μM Ca2+（在较小程度上由1 mM Ba2+）诱导的分泌仍有一小部分残留。添加更多的BoNT/A未能降低这种分泌水平。Ca2+刺激或Ba2+刺激的BoNT/A抗性分泌需要加入Mg·ATP，Mg·ATP能极大地增强透化细胞的分泌。此外，突触小泡蛋白对于这种反应至关重要，因为在BoNT/B处理的细胞中未观察到这种反应。鉴于BoNT/E能够消除透化细胞的分泌并从SNAP-25的C末端删除26个氨基酸，可以推断BoNT/A仅切割九个残基并不会阻止所得的截短形式在透化所产生的条件下表现出减弱的活性。这种对透化细胞分泌新成分的鉴定应有助于研究SNAP-25与参与调节性胞吐作用的其他蛋白质之间的功能相互作用。
