PDF(Pubmed)
Abstract：
Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura. To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II-V.
摘要：
苏云金芽孢杆菌Vip3毒素形成对其杀虫活性至关重要的四聚体结构。每个Vip3Aa单体包含五个结构域。结构域I中的前四个α-螺旋与靶细胞膜的相互作用被认为是孔形成之前的关键步骤。在这项研究中,产生了四个Vip3Aa的N末端α-螺旋缺失截短，发现它们失去了脂质体的渗透性和对斜纹夜蛾的杀虫活性。为了进一步探讨结构域I在膜渗透中的作用，全长结构域I和N端α-螺旋截短结构域I的片段与绿色荧光蛋白（GFP）融合，分别。只有携带全长结构域I的融合体对人工脂质体表现出渗透性。此外,还通过将Vip3Aa结构域I和II的α-螺旋与Cry1Ac的结构域II和III组合，构建了七个Vip3Aa-Cry1Ac融合体。确定了七种组合中的五种在人工脂质体中显示出膜渗透性。然而,由于蛋白水解稳定性的显著降低,Vip3Aa-Cry1Ac组合都没有表现出杀虫活性。这些结果表明，Vip3Aa结构域I中的N末端螺旋α1对于杀虫活性和脂质体渗透性都是必需的，并且Vip3Aa的结构域I独立于结构域II-V保持了高脂质体渗透性。
