Abstract：
Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, has insecticidal activity, but its molecular mechanism of action is still not clear. Three proteins with modification-related functions, calreticulin (BmCRT), dipeptidyl peptidase Ⅲ (BmDPP3), and protein disulfide isomerase A5 (BmPDIA5), were selected to verify the interactions with DA in this study. The kinetic data of the interactions were measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) in vitro. The KD values of DA with BmCRT, BmDPP3, and BmPDIA5 ranged from 10-4 to 10-5 mol/L, which suggested that the three proteins all had fairly strong interactions with DA. Then, it was found that DA in a dose-dependent manner affected the interactions of the three proteins with their partners in insect two-hybrid tests in SF-9 cells. Furthermore, the results of enzyme activities by ELISA indicated that DA could inhibit the activity of BmDPP3 but had no significant effect on BmPDIA5. In addition, DA induced the upregulation of BmDPP3 and the downregulation of BmCRT. The results prove that BmCRT, BmDPP3, and BmPDIA5 are all binding proteins of DA. This study might provide new insights to elucidate the molecular mechanism of DA.
摘要：
DestruxinA（DA），昆虫病原真菌金叶绿霉菌产生的六环缩肽霉菌毒素，具有杀虫活性，但其分子作用机制尚不清楚。三种具有修饰相关功能的蛋白质，钙网蛋白（BmCRT），二肽基肽酶Ⅲ(BmDPP3),和蛋白质二硫键异构酶A5（BmPDIA5），在本研究中选择验证与DA的相互作用。通过表面等离子体共振（SPR）和体外生物层干涉法（BLI）测量相互作用的动力学数据。DA与BmCRT的KD值，BmDPP3和BmPDIA5的范围为10-4至10-5mol/L，这表明这三种蛋白质都与DA有相当强的相互作用。然后，在SF-9细胞的昆虫双杂交试验中，发现DA以剂量依赖的方式影响三种蛋白质与其伴侣的相互作用。此外，酶活性的ELISA检测结果表明，DA能抑制BmDPP3的活性，但对BmPDIA5无明显影响。此外,DA诱导BmDPP3的上调和BmCRT的下调。结果证明,BmCRT,BmDPP3和BmPDIA5都是DA的结合蛋白。本研究可能为阐明DA的分子机制提供新的见解。
