ERI BIOTECMED, and Department of Genetics, Universitat de València. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain.
ERI BIOTECMED, and Department of Genetics, Universitat de València. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain.
Int J Biol Macromol. 2018 Dec;120(Pt A):59-65. doi: 10.1016/j.ijbiomac.2018.08.067. Epub 2018 Aug 16.
Vip3 proteins are secretable proteins from Bacillus thuringiensis with important characteristics for the microbiological control of agricultural pests. The exact details of their mode of action are yet to be disclosed and the crystallographic structure is still unknown. Vip3 proteins are expressed as protoxins that have to be activated by the insect gut proteases. A previous study on the peptidase processing of Vip3Aa revealed that the protoxin produced artefactual band patterns by SDS-PAGE due to the differential stability of this protein and the peptidases to SDS and heating (Bel et al., 2017 Toxins 9:131). To determine whether this phenomenon also applies to other Vip3A proteins, here we chose a different Vip3A protein (Vip3Af) and subjected it to commercial trypsin and midgut juice from a target insect species (Spodoptera frugiperda). The misleading degradation patterns were also observed with Vip3Af, both with trypsin and midgut juice. However, gel filtration chromatography showed that, under native conditions, Vip3Af is found as a tetramer and that peptidases only act upon primary cleavage sites. The proteolytic cleavage renders two fragments of approximately 20 kDa and 65 kDa which remain together in the tretameric structure and that are no further processed even at high peptidase concentrations.
Vip3 蛋白是苏云金芽孢杆菌分泌的蛋白，具有控制农业害虫的重要特性。其确切作用机制尚不清楚，晶体结构仍未知。Vip3 蛋白以原毒素的形式表达，需要被昆虫肠道蛋白酶激活。先前对 Vip3Aa 肽酶加工的研究表明，原毒素在 SDS-PAGE 中产生了人为的条带模式，这是由于该蛋白和肽酶对 SDS 和加热的不同稳定性（Bel 等人，2017 毒素 9:131）。为了确定这种现象是否也适用于其他 Vip3A 蛋白，我们选择了一种不同的 Vip3A 蛋白（Vip3Af），并用商业胰蛋白酶和目标昆虫物种（Spodoptera frugiperda）的中肠汁液进行了处理。用胰蛋白酶和中肠汁液处理 Vip3Af 时，也观察到了误导性的降解模式。然而，凝胶过滤层析表明，在天然条件下，Vip3Af 以四聚体的形式存在，并且肽酶仅作用于初级切割位点。蛋白水解切割产生两个大约 20 kDa 和 65 kDa 的片段，它们在四聚体结构中保持在一起，即使在高肽酶浓度下也不会进一步加工。
