He Yan, Wang Yang, Zhao Picheng, Rayaprolu Subrahmanyam, Wang Xiuhong, Cao Xiaolong, 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. 2017 Nov;90:71-81. doi: 10.1016/j.ibmb.2017.09.015. Epub 2017 Oct 5.
Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg for trypsin-like proteases, Gly and Ala for the elastase and Thr for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0-50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae.
丝氨酸蛋白酶抑制剂（Serpins）是一类蛋白质超家族，其中大多数通过形成无活性的酰基 - 酶复合物来抑制同源丝氨酸蛋白酶。在烟草天蛾曼陀罗中，丝氨酸蛋白酶抑制剂 -1、-3至 -7负向调节一种血淋巴丝氨酸蛋白酶系统，该系统可激活丝氨酸蛋白酶同源物（SPHs）、酚氧化酶（POs）、斯帕茨蛋白（Spätzles）和其他细胞因子的前体。在此，我们报告了烟草天蛾丝氨酸蛋白酶抑制剂 -9和 -13的克隆与特性。丝氨酸蛋白酶抑制剂 -9是一种含有402个氨基酸残基的蛋白质，与果蝇的Spn77Ba最为相似，在切割位点前的P1位置为精氨酸（R）；丝氨酸蛋白酶抑制剂 -13是果蝇Spn28Dc的4个氨基酸残基的直系同源物，比其他7种丝氨酸蛋白酶抑制剂更长，且P1残基为精氨酸。这两种丝氨酸蛋白酶抑制剂主要在脂肪体中产生并分泌到血浆中发挥作用。虽然它们的mRNA和蛋白质水平在免疫刺激后没有上调，但它们能阻断蛋白酶活性并影响血淋巴中前酚氧化酶的激活。丝氨酸蛋白酶抑制剂 -9对人中性粒细胞弹性蛋白酶、组织蛋白酶G、胰蛋白酶和胰凝乳蛋白酶有不同程度的抑制作用；丝氨酸蛋白酶抑制剂 -13在摩尔比为4:1（丝氨酸蛋白酶抑制剂：酶）时将胰蛋白酶活性降低至约10%。丝氨酸蛋白酶抑制剂 -9被具有不同特异性的酶在精氨酸处切割，但丝氨酸蛋白酶抑制剂 -13有4个P1位点（胰蛋白酶样蛋白酶的精氨酸、弹性蛋白酶的甘氨酸和丙氨酸以及组织蛋白酶G的苏氨酸）。用重组丝氨酸蛋白酶抑制剂 -9补充诱导的无细胞血淋巴（IP，P代表血浆）对前酚氧化酶的激活没有明显影响，但在细菌引发IP的0 - 50%硫酸铵分级分离后，略微降低了酚氧化酶活性的增加。相比之下，添加重组丝氨酸蛋白酶抑制剂 -13显著抑制了IP中前酚氧化酶的激活，并且在IP分级分离中抑制作用更强。使用它们的抗体从IP中分离出含有丝氨酸蛋白酶抑制剂 -9和 -13的蛋白质复合物。发现血淋巴蛋白酶 -1前体（proHP1）、HP6和HP8与丝氨酸蛋白酶抑制剂 -9相关，而proHP1、HP2和HP6与丝氨酸蛋白酶抑制剂 -13一起被沉淀下来。这些结果表明，这两种丝氨酸蛋白酶抑制剂都调节烟草天蛾幼虫血淋巴中的免疫蛋白酶。
