舟山黄海葵兴奋性毒素AX-1的分离纯化及鉴定

Purification and identification of excitatory toxins AX-1 from Anthopleura xanthogrammica in Zhoushan

目的从舟山黄海葵(AnthopLeura xanthogrammica)的刺细胞中分离纯化多肽类毒素分子并进行功能鉴定。方法通过反复冻融法以及多步高效液相色谱分离技术从舟山黄海葵毒素中分离到一种新型多肽类毒素,进行质谱鉴定和三维结构模拟,并运用膜片钳技术检测海葵毒素多肽对大鼠背根神经节(DRG)细胞的河豚毒素-敏感型(TTX-S)和河豚毒素-不敏感型(TTX-R)钠离子通道的影响。结果获得由48个氨基酸组成,相对分子质量为5018.2的多肽毒素分子,命名为AX-1,含3对二硫键;三维结构模拟表明该毒素多肽的结构以反平行β-折叠片以及loop结构为主,该毒素能抑制大鼠背根神经节细胞的钠离子通道的失活并显著增加钠离子通道的电流。结论AX-1是一种兴奋性多肽毒素,可作为潜在的强心肽药物。

Objective To isolate and purify peptide toxin from stinging cells of Anthopleura xanthogrammica in Zhoushan of the East China Sea and to study its function. Methods The toxin was purified by repeated freeze-thaw method and multi-step HPLC separation, identified by mass spectrometry and three-dimensional structure modeling. The effects of AX-1 on tetrodotoxinsensitive （TTX-S） and tetrodotoxin -resistant （TTX-R） sodium ion channels in rat dorsal root ganglion （DRG） cells were tested by patch-clamp techniques. Results A novel toxin named AX-1 was isolated from Anthopleura xanthogrammica. AX-1 consists of 48 amino acids, with three disulfide bonds and relative molecular mass of 5018. 2. Three-dimensional structure modeling indicated that the structure of AX-1 was mainly antiparallel y-sheets and loops. It could inhibit sodium channel inactivation and significantly increased sodium channel current in rat dorsal root ganglion cells. Conclusion AX-1 was an excitatory peptide toxin, and can be used as a potential cardiac peptide drug.