Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25 - Identification of the minimal essential C-terminal residues
Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25 - Identification of the minimal essential C-terminal residues
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DOI：
10.1074/jbc.274.52.36897
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发表时间：
1999-12-24
影响因子：
4.8
通讯作者：
Dolly, JO
中科院分区：
文献类型：
--
作者：
O'Sullivan, GA;Mohammed, N;Dolly, JO
A型和B型肉毒神经毒素（BoNT）分别通过切割SNAP-25和小突触泡蛋白选择性地阻断胞吐作用;在人类中，从所导致的神经肌肉麻痹中完全恢复需要数月。为了破译这种长期中毒的分子基础，在肾上腺嗜铬细胞中毒监测超过2个月。BoNT/B处理的细胞的胞吐作用在56天后恢复，因为出现了完整的小突触泡蛋白。然而，在BoNT/A处理的细胞中，在整个相同的时间间隔内，抑制持续存在，切割的SNAP-25-（1-197）相对于完整蛋白质持续占优势。当用编码全长SNAP-25-（1-206）的基因转染后一种细胞试图从中毒中恢复时，甚至在3周后也没有恢复胞吐作用。为了确定这种失败是否是因为毒素的蛋白酶活性的持久性，用BoNT/A抗性SNAP-25构建体转染细胞;重要的是，胞吐被拯救。毒素不敏感的SNAP-25的C-末端截短揭示残基1-201、1-202、1-203提供胞吐作用的显著恢复，不像较短形式1-197、-198、-199或-200;因此，全长SNAP-25的突变体M202 A或L203 A拯救分泌。这些发现提供了SNAP-25的C-末端功能结构域的见解，证明了BoNT/A蛋白酶的寿命，并提供了肉毒杆菌中毒治疗的前景。
Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforded a significant return of exocytosis, unlike shorter forms 1-197, -198, -199, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.