Li Junlan, Ma Li, Lin Zhe, Zou Zhen, Lu Zhiqiang
Department of Entomology, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100, China.
State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
Insect Biochem Mol Biol. 2016 Jun;73:27-37. doi: 10.1016/j.ibmb.2016.04.003. Epub 2016 Apr 12.
The prophenoloxidase (PPO) activation pathway and Toll pathway are two critical insect immune responses against microbial infection. Activation of these pathways is mediated by an extracellular serine protease cascade, which is negatively regulated by serpins. In this study, we found that the mRNA abundance of silkworm serpin-5 (BmSpn-5) increased dramatically in the fat body after bacterial infection. The expression level of antimicrobial peptides (AMPs), gloverin-3, cecropin-D and -E decreased in the silkworm larvae injected with recombinant BmSpn-5 protein. Meanwhile, the inhibition of beads melanization, systemic melanization and PPO activation by BmSpn-5 was also observed. By means of immunoaffinity purification and analysis by mass spectrometry, we identified that the silkworm clip domain serine proteases BmHP6 and BmSP21 form a complex with BmSpn-5, which suggests that BmHP6 and SP21 are the cognate proteases of BmSpn-5 and are essential in the serine protease cascade that activates the Toll and PPO pathways. Our study provides a comprehensive characterization of BmSpn-5 and sheds light on the multiple pathways leading to PPO activation and their regulation by serpins.
酚氧化酶原（PPO）激活途径和Toll途径是昆虫针对微生物感染的两种关键免疫反应。这些途径的激活由细胞外丝氨酸蛋白酶级联介导，丝氨酸蛋白酶抑制剂对其起负调控作用。在本研究中，我们发现家蚕丝氨酸蛋白酶抑制剂-5（BmSpn-5）的mRNA丰度在细菌感染后在家蚕脂肪体中显著增加。在注射重组BmSpn-5蛋白的家蚕幼虫中，抗菌肽（AMPs）、gloverin-3、天蚕素-D和-E的表达水平下降。同时，还观察到BmSpn-5对珠黑化、全身黑化和PPO激活的抑制作用。通过免疫亲和纯化和质谱分析，我们鉴定出家蚕clip结构域丝氨酸蛋白酶BmHP6和BmSP21与BmSpn-5形成复合物，这表明BmHP6和SP21是BmSpn-5的同源蛋白酶，在激活Toll和PPO途径的丝氨酸蛋白酶级联中至关重要。我们的研究全面表征了BmSpn-5，并揭示了导致PPO激活的多种途径及其受丝氨酸蛋白酶抑制剂的调控。
