Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin.
Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin.
复制标题
假定结构域II和表面残基的诱变分析苏云金芽孢杆菌CRY19AA毒素。
DOI：
10.1111/j.1574-6968.2009.01583.x
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发表时间：
2009-06
影响因子：
2.1
通讯作者：
Dean DH
中科院分区：
文献类型：
--
作者：
Roh JY;Nair MS;Liu XS;Dean DH
杀蚊晶体蛋白 Cry19Aa，来自苏云金芽孢杆菌亚种。 jegathesan 对斯氏按蚊和淡色库蚊毒性较高，但对埃及伊蚊毒性较低。为了研究推定结构域 II 和表面残基在蚊子毒性中的功能作用，将 16 个丙氨酸取代突变引入 Cry19Aa。所有突变构建体均表达为 65 kDa 原毒素，随后用胰蛋白酶消化以产生 40 和 25 kDa 的进一步片段化多肽。然而，使用胰凝乳蛋白酶，大多数原毒素被消化至 60 kDa 和较小的条带。 Cry19Aa和突变蛋白Y324A、W357A、Y412A、Y414A、W416A、D418A和F485A的胰凝乳蛋白酶激活毒素的圆二色光谱表明它们的结构没有显着变化。在蚊子生物测定中，Y324A、W357A、Y410A、W416A、D418A 和 F485A 突变蛋白对埃及伊蚊和尖色小念珠菌的杀蚊活性显着降低。这些突变蛋白还显示出与野生型荧光素 5-异硫氰酸酯标记的 Cry19Aa 与 C. pipiens 刷状缘膜囊泡结合的竞争减少。这些数据表明毒性的降低是结合亲和力降低的结果。从这些研究中，我们鉴定了结构域 II 的环残基，这些残基对于库蚊幼虫中肠的毒性和受体结合很重要。
The mosquitocidal crystal protein, Cry19Aa, from Bacillus thuringiensis ssp. jegathesan, has high toxicity to Anopheles stephensi and Culex pipiens but is less toxic to Aedes aegypti. To study the functional role of putative domain II and surface residues in mosquito toxicity, 16 alanine substitution mutations were introduced into Cry19Aa. All mutant constructs were expressed as 65-kDa protoxins and subsequently digested by trypsin to produce further fragmented polypeptides of 40 and 25 kDa. With chymotrypsin, however, most protoxins were digested to 60 kDa and minor bands. The circular dichroism spectra of the chymotrypsin-activated toxins of Cry19Aa and muteins, Y324A, W357A, Y412A, Y414A, W416A, D418A and F485A indicated that there was no significant variation in their structure. In mosquito bioassays, Y324A, W357A, Y410A, W416A, D418A and F485A muteins showed substantial reductions in mosquitocidal activity toward A. aegypti and C. pipiens. These muteins also showed reduced competition with wild-type fluorescein 5-isothiocyanate-labeled Cry19Aa for binding to C. pipiens brush border membrane vesicles. These data suggest that the reduction of toxicity was a result of the reduced binding affinity. From these studies we have identified loop residues of domain II that are important in toxicity and receptor binding to Culex larval midgut.