Wang Yang, Lu Zhiqiang, Jiang Haobo
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Insect Biochem Mol Biol. 2014 Jul;50:82-91. doi: 10.1016/j.ibmb.2014.04.005. Epub 2014 Apr 24.
Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta.
黑化参与了包括抗菌免疫反应在内的多种昆虫生理过程。酚氧化酶（PO）是催化黑色素形成的酶系统的关键组成部分，它以前体酚氧化酶原（proPO）的形式产生，并通过酚氧化酶原激活蛋白酶（PAP）的特异性蛋白水解切割而变得活跃。在烟草天蛾中，三种PAP可以在两种丝氨酸蛋白酶同源物（SPH1和SPH2）存在的情况下激活proPO。虽然已知产生活性PAP的血淋巴蛋白酶（HP），但尚不清楚proSPH（尤其是proSPH1）是如何被激活的。在本研究中，我们从烟草天蛾幼虫蛹期的血浆中分离出一种能切割proSPH的异亮氨酸-谷氨酸-丙氨酸-精氨酸-对硝基苯胺水解酶。这种蛋白酶PAP3产生了活性SPH1和SPH2，它们在proPO激活中作为PAP3的辅因子发挥作用。PAP3对纯化的重组proSPH的切割产生了迁移率与体内形成的SPH相似的38 kDa条带。令人惊讶的是，PAP3还可以激活proPAP3，以在直接的正反馈回路中刺激黑化。proPO激活的增强与proHP6、proHP8、proPAP1、proPAP3、proSPH1、proSPH2、proPO的切割激活同时发生，但不包括proHP14或proHP21。这些结果表明，PAP3与PAP1一样，是proPO激活系统中自我强化机制的关键因素，该机制与烟草天蛾的其他免疫反应相关。
