Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae
Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae
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DOI：
10.1002/ps.5866
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发表时间：
2020-05-20
影响因子：
4.1
通讯作者：
Wu, Songqing
中科院分区：
文献类型：
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作者：
Guo, Yajie;Wang, Yafang;Wu, Songqing
背景苏云金芽孢杆菌Cry3毒素对几种鞘翅目幼虫表现出特异性毒性。然而，由于 Cry3A 毒素对松墨天牛的毒性较低，因此不能用于控制松墨天牛幼虫。在这里，我们评估了松斑潜蝇幼虫中肠中Cry3Aa毒素的蛋白水解活化，并通过分子修饰增加了其毒性。结果我们的结果表明，松斑潜蝇幼虫中肠中Cry3Aa原毒素的加工不充分和Cry3Aa毒素的非特异性酶消化导致了低毒性。转录组分析、荧光底物酶学分析和多重底物质谱分析结果表明，松毛霉幼虫中肠的主要消化酶是类胰蛋白酶，优先裂解含有精氨酸和赖氨酸残基的肽。因此，胰蛋白酶识别位点被引入到 Cry3Aa 原毒素的结构域 I 的 α 螺旋 3 和 α 螺旋 4 之间的环区域中，以促进蛋白水解激活。 Cry3Aa 蛋白中远离螺旋片和功能区的多个潜在胰蛋白酶切割位点也突变为丙氨酸，以防止非特异性酶消化。生物测定表明，与天然 Cry3Aa 毒素相比，改良的 Cry3Aa-T 毒素（K65A、K70A、K231A、K468A 和 K596A）对松叶松线虫幼虫的毒性增加了 9.5 倍（LC50 = 12.3 mu g/mL）。 结论 这项研究强调了增加 Cry3Aa 毒素毒性的有效方法M. alternatus，这可能适合管理转基因植物对其他害虫的抗性，包括农业中一些最重要的害虫。
BACKGROUND Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus, Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification.RESULTS Our results indicated that insufficient processing of Cry3Aa protoxin and non-specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin-like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between alpha-helix 3 and alpha-helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non-specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa-T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5-fold (LC50 = 12.3 mu g/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin.CONCLUSION This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus, which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture.