Functional Characterization of Botulinum Neurotoxin Serotype H as a Hybrid of Known Serotypes F and A (BoNT F/A)
Functional Characterization of Botulinum Neurotoxin Serotype H as a Hybrid of Known Serotypes F and A (BoNT F/A)
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DOI：
10.1021/ac504716v
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发表时间：
2015-04-07
影响因子：
7.4
通讯作者：
Barr, John R.
中科院分区：
文献类型：
--
作者：
Kalb, Suzanne R.;Baudys, Jakub;Barr, John R.
最近发现了一种独特的肉毒梭菌菌株（IBCA 10 -7060），其产生两种毒素：肉毒神经毒素（BoNT）血清型B和一种新的BoNT血清型H。先前的分子评估显示，新BoNT的轻链（LC）最类似于已知亚型F5的轻链的键，而重链（H-C）的C-末端最类似于血清型A的结合结构域。我们评估了这两种毒素的功能，在文化中产生的第一个纳入免疫亲和步骤，使用单克隆抗体纯化BoNT从培养上清液和测试每一个免疫捕获的神经毒素与全长基板囊泡相关膜蛋白2（VAMP-2），突触体相关蛋白25（SNAP-25），突触融合蛋白，和缩短的肽代表的基板。由该菌株产生的BoNT/B表现为典型的BoNT/B，对抗B单克隆抗体具有免疫亲和性，并在预期位置切割全长VAMP-2和基于VAMP-2序列的肽。正如预期的那样，对SNAP-25或突触融合蛋白没有活性。新的BoNT表现出对抗A单克隆抗体的免疫亲和力，但不像BoNT/A所预期的那样切割SNAP-25。相反，新的BoNT在与BoNT/F5相同的位置切割VAMP-2和基于VAMP-2的肽。这是首次发现具有BoNT/A抗原性和BoNT/F轻链功能的单一肉毒杆菌神经毒素。这项工作表明，新报告的血清型H实际上可能是以前已知的BoNT血清型A和血清型F的杂交种，特别是F5亚型。
A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (H-C) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.