Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, People's Republic of China.
Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, People's Republic of China.
Int J Biol Macromol. 2018 Feb;107(Pt A):1220-1226. doi: 10.1016/j.ijbiomac.2017.09.101. Epub 2017 Sep 29.
Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys, Lys and Lys) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys, Lys, Lys and Lys with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.
Vip3Aa 被昆虫中肠蛋白酶的蛋白水解对于其对靶标昆虫的毒性是必不可少的。在本研究中，通过斜纹夜蛾中肠蛋白酶（MJ）评估了 Vip3Aa 的蛋白水解。证实胰蛋白酶参与了 Vip3Aa 的激活，并且鉴定出三个潜在的切割位点（Lys、Lys 和 Lys）。通过分别用 Ala 替换残基 Lys、Lys、Lys 和 Lys，设计并构建了四个 Vip3Aa 突变体（KKK195197198AAA、KK197198AA、KK195198AA 和 KK195197AA）。蛋白水解处理分析表明，突变体 KK197198AA、KK195198AA 和 KK195197AA 可以被胰蛋白酶或 MJ 加工成 66kDa 的激活毒素，而突变体 KKK195197198AAA 不能被胰蛋白酶切割，并且对 MJ 的敏感性降低。生物测定表明，突变体 KK197198AA、KK195198AA 和 KK195197AA 对斜纹夜蛾具有类似于野生型 Vip3Aa 的毒性，然而，突变体 KKK195197198AAA 对斜纹夜蛾的 LC 高于野生型。这些结果表明，MJ 的蛋白水解与 Vip3Aa 对斜纹夜蛾的杀虫毒性有关。它还表明胰蛋白酶在 Vip3Aa 激活毒素的形成中起重要作用。我们的研究阐明了 Vip3Aa 的蛋白水解处理，并为这种新型 Bt 毒素的激活提供了新的见解。
