Mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin
Mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab δ-endotoxin
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DOI：
10.1128/aem.69.8.4648-4657.2003
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发表时间：
2003-08-01
影响因子：
4.4
通讯作者：
Chen, JS
中科院分区：
文献类型：
--
作者：
Lee, MK;Walters, FS;Chen, JS
Vip3A蛋白是芽孢杆菌在营养生长阶段分泌的一个新的杀虫蛋白家族。在我们对Vip3A作用方式的研究中，88 kda的Vip3A全长毒素（Vip3A- f）被胰蛋白酶（Vip3A- t）或鳞翅目肠液提取物（Vip3A- g）蛋白水解激活为约62 kda的核心毒素。生物素化的Vip3A-G与鳞翅目中肠刷状边界膜囊（BBMV）具有竞争性结合。此外，在烟草角虫Manduca sexta (Linnaeus) BBMV的配体印迹实验中，激活了Cry1Ab结合在120 kda的氨肽酶N （APN）样分子和250 kda的钙粘蛋白样分子上，而Vip3A-G结合在80 kda和100 kda的分子上，这与已知的Cry1Ab受体不同。此外，单独的Vip3A-G印迹实验没有显示与分离的Cry1A受体结合，如m.s sexta APN蛋白或Cry1Ab外结合域的钙粘蛋白。在对感虫中肠解剖的压紧实验中，Vip3A-G能明显形成孔，而Vip3A-F则不能形成孔。在同样的实验中，Vip3A-G不能与不敏感昆虫，黑脉金斑蝶，Danaus plexippus （Linnaeus）的幼虫形成孔。在平面脂质双分子层中，Vip3A- g和Vip3A- t在没有任何受体的情况下形成稳定的离子通道，支持孔隙形成，这是Vip3A的固有特性。Cry1Ab和Vip3A通道均具有电压不依赖性和高度阳离子选择性；然而，它们在主电导状态和阳离子特异性上有很大的不同。Vip3A的作用模式支持其作为一种新型杀虫剂的应用。
The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.