Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, 74078, USA.
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, 66506, USA.
Insect Biochem Mol Biol. 2018 Aug;99:27-36. doi: 10.1016/j.ibmb.2018.05.004. Epub 2018 May 23.
Insect prophenoloxidase activation is coordinated by a serine protease network, which is regulated by serine protease inhibitors of the serpin superfamily. The enzyme system also leads to proteolytic processing of a Spätzle precursor. Binding of Spätzle to a Toll receptor turns on a signaling pathway to induce the synthesis of defense proteins. Previous studies of the tobacco hornworm Manduca sexta have revealed key members of the protease cascade, which generates phenoloxidase for melanogenesis and Spätzle to induce immunity-related genes. Here we provide evidence that M. sexta serpin-12 regulates hemolymph protease-14 (HP14), an initiating protease of the cascade. This inhibitor, unlike the other serpins characterized in M. sexta, has an amino-terminal extension rich in hydrophilic residues and an unusual P1 residue (Leu) right before the scissile bond cleaved by a target protease. Serpins with similarities to serpin-12, including Drosophila Necrotic, were identified in a wide range of insects including flies, moths, wasps, beetles, and two hemimetabolous species. The serpin-12 mRNA is present at low, constitutive levels in larval fat body and hemocytes and becomes more abundant after an immune challenge. We produced the serpin-12 core domain (serpin-12ΔN) in insect cells and in Escherichia coli and demonstrated its inhibition of human cathepsin G, bovine α-chymotrypsin, and porcine pancreatic elastase. MALDI-TOF analysis of the reaction mixtures confirmed the predicted P1 residue of Leu. Supplementation of larval plasma samples with the serpin-12ΔN decreased prophenoloxidase activation elicited by microbial cells and reduced the proteolytic activation of the protease precursors of HP6, HP8, PAPs, and other serine protease-related proteins. After incubation of plasma stimulated with peptidoglycan, a 72 kDa protein appeared, which was recognized by polyclonal antibodies against both serpin-12 and HP14, suggesting that a covalent serpin-protease complex formed when serpin-12 inhibited HP14. Together, these data suggest that M. sexta serpin-12 inhibits HP14 to regulate melanization and antimicrobial peptide induction.
昆虫酚氧化酶原的激活受丝氨酸蛋白酶网络的协调，该网络受丝氨酸蛋白酶抑制剂超家族的丝氨酸蛋白酶抑制剂调节。该酶系统还导致 Spätzle 前体的蛋白水解加工。Spätzle 与 Toll 受体结合会开启信号通路，诱导防御蛋白的合成。以前对烟草天蛾 Manduca sexta 的研究揭示了蛋白酶级联反应的关键成员，该级联反应产生酚氧化酶以进行黑色素生成和 Spätzle 以诱导免疫相关基因。在这里，我们提供证据表明 M. sexta serpin-12 调节血淋巴蛋白酶-14（HP14），这是级联反应的起始蛋白酶。与 M. sexta 中表征的其他丝氨酸蛋白酶抑制剂不同，这种抑制剂在靠近靶蛋白酶切割的位置具有富含亲水残基的氨基末端延伸和不寻常的 P1 残基（亮氨酸）。与 serpin-12 具有相似性的丝氨酸蛋白酶抑制剂，包括果蝇 Necrotic，在包括苍蝇、飞蛾、黄蜂、甲虫和两种半变态物种在内的广泛昆虫中被鉴定出来。serpin-12 mRNA 在幼虫脂肪体和血细胞中以低水平组成型存在，在免疫挑战后变得更加丰富。我们在昆虫细胞和大肠杆菌中产生了 serpin-12 核心结构域（serpin-12ΔN），并证明其抑制人组织蛋白酶 G、牛α-糜蛋白酶和猪胰弹性蛋白酶。MALDI-TOF 分析反应混合物证实了亮氨酸的预测 P1 残基。用 serpin-12ΔN 补充幼虫血浆样品可降低微生物细胞引起的原酚氧化酶激活，并减少 HP6、HP8、PAPs 和其他丝氨酸蛋白酶相关蛋白的蛋白酶前体的蛋白水解激活。在用肽聚糖刺激血浆孵育后，出现了一种 72 kDa 的蛋白质，该蛋白质被针对 serpin-12 和 HP14 的多克隆抗体识别，这表明当 serpin-12 抑制 HP14 时形成了共价丝氨酸蛋白酶复合物。这些数据表明，M. sexta serpin-12 抑制 HP14 以调节黑色素生成和抗菌肽诱导。
