Unique Biological Activity of Botulinum D/C Mosaic Neurotoxin in Murine Species
Unique Biological Activity of Botulinum D/C Mosaic Neurotoxin in Murine Species
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DOI：
10.1128/iai.00302-12
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发表时间：
2012-08-01
影响因子：
3.1
通讯作者：
Kozaki, Shunji
中科院分区：
文献类型：
--
作者：
Nakamura, Keiji;Kohda, Tomoko;Kozaki, Shunji
肉毒梭菌C型和D型通过产生肉毒梭菌型特异性或嵌合型肉毒杆菌神经毒素（BoNT）引起动物肉毒中毒。D/C嵌合型BoNT（BoNT/DC）是由日本牛肉毒中毒分离株产生的，在所有BoNT中对小鼠毒性最高。相比之下，大鼠似乎对C型和D型BoNT及其嵌合体BoNT中的BoNT/DC非常耐药。我们试图通过与C型和D型BoNT（BoNT/C和BoNT/D）的比较来表征BoNT/DC的酶活性和受体结合活性。BoNT/DC和D对小鼠小脑颗粒细胞（CGC）的毒性作用相似，但前者对大鼠CGC的毒性较低。在重组鼠源性囊泡相关膜蛋白（VAMP）中，两种BoNTs对大鼠同种型1 VAMP（VAMP 1）的酶活性均低于其他VAMP同系物。然后，我们研究了神经节苷脂作为C型和D型以及嵌合型BoNT的结合组分的生理意义。发现BoNT/DC和C在分别外源性添加GM 1a和GT 1b神经节苷脂后切割PC 12细胞的细胞内底物，这表明每个BoNT识别不同的神经节苷脂部分。BoNT/DC对CGCs谷氨酸释放的影响被霍乱毒素B亚基（CTB）阻止，但不被不与GM 1a结合的CTB定点突变体阻止。牛肾上腺嗜铬细胞对BoNT/DC比BoNT/C和D更敏感。这些结果表明，BoNT/DC的受体结合的独特机制可能差异调节其在动物中的生物活性。
Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.