Aguiar Juliana M, Franco Octávio L, Rigden Daniel J, Bloch Carlos, Monteiro Ana C S, Flores Victor M Q, Jacinto Tânia, Xavier-Filho José, Oliveira Antonia E A, Grossi-de-Sá Maria F, Fernandes Kátia V S
Laboratório de Química e Função de Proteínas e Peptídeos, UENF, Campos dos Goytacazes, RJ, Brazil.
Proteins. 2006 May 15;63(3):662-70. doi: 10.1002/prot.20901.
Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.
植物半胱氨酸蛋白酶抑制剂作为一种工具，在通过基因工程培育抗虫害作物方面具有巨大潜力。两个重要的潜在目标是豆象，即菜豆象（Acanthoscelides obtectus）和巴西豆象（Zabrotes subfasciatus），它们在中肠中表现出主要的消化半胱氨酸蛋白酶活性。在本研究中，豇豆半胱氨酸蛋白酶抑制剂（一种在豇豆（Vigna unguiculata）种子中发现的半胱氨酸蛋白酶抑制剂）在大肠杆菌中表达，并通过镍 - 氮三乙酸琼脂糖柱进行纯化。体外试验表明，它能强烈抑制木瓜蛋白酶以及菜豆象和巴西豆象中肠中的蛋白酶。当该蛋白以低至0.025%的浓度掺入人工种子中，且种子被豆象幼虫食用时，观察到幼虫体重显著降低，昆虫死亡率增加。豇豆半胱氨酸蛋白酶抑制剂与木瓜蛋白酶复合物的分子模拟研究表明，大豆半胱氨酸蛋白酶部分N端氨基酸序列中不存在负责酶 - 抑制剂复合物稳定化中大部分疏水相互作用的五个N端残基。我们认为，这种结构差异可能是豇豆半胱氨酸蛋白酶抑制剂比之前测试的植物半胱氨酸蛋白酶抑制剂有效性高得多的原因。本文还讨论了将这一知识应用于旨在增强植物抗虫害能力的植物蛋白质突变计划。
