Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites
Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites
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DOI：
10.1104/pp.108.115741
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发表时间：
2008-03-01
期刊：
影响因子：
7.4
通讯作者：
Michaud, Dominique
中科院分区：
文献类型：
--
作者：
Goulet, Marie-Claire;Dallaire, Cindy;Michaud, Dominique
植物半胱氨酸蛋白酶抑制剂，类似于其他防御蛋白，包括高变，积极选择的氨基酸位点，推测影响其生物活性。使用29个单一的突变体的第八个结构域的番茄（番茄）multicystatin，SlCYS8，我们在这里评估的潜力，定点诱变在积极选择的氨基酸位点，以产生半胱氨酸蛋白酶抑制剂的变体具有改善的抑制效力和特异性对草食性昆虫消化半胱氨酸（Cys）蛋白酶。与SlCYS8相比，几种突变体（29种中的22种）表现出针对不同模型Cys蛋白酶的提高或降低的效力，强烈表明正选择的氨基酸作为靶位点来调节半胱氨酸蛋白酶抑制剂对具有农学意义的Cys蛋白酶的抑制特异性的潜力。因此，在正选择位点的突变强烈影响SlCYS8对昆虫食草动物科罗拉多马铃薯甲虫（马铃薯甲虫）的消化Cys蛋白酶的抑制效力。特别地，几种变体表现出针对该昆虫的半胱氨酸蛋白酶抑制剂敏感性和半胱氨酸蛋白酶抑制剂不敏感性消化Cys蛋白酶的改善的效力。其中，一些变体还显示出对宿主植物（马铃薯[ Solanum tuberosum]）的叶Cys蛋白酶和对双斑蝽Perillus bioculatus（科罗拉多马铃薯甲虫的昆虫捕食者）的主要消化Cys蛋白酶的较弱活性，所述双斑蝽Perillus bioculatus显示出生物防治的潜力。总体而言，这些观察结果表明，在积极选择的氨基酸位点的定点诱变的有用性工程的重组半胱氨酸蛋白酶抑制剂具有改善的抑制效力对目标食草动物的消化蛋白酶和较弱的效力对非目标Cys蛋白酶在宿主植物或环境。
Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [ Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.