Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.

动物体内的体液免疫反应通常受到蛋白水解的严格控制。这种蛋白水解作用是通过细胞外蛋白酶级联反应来实现的，其级联作用最终激活了关键免疫蛋白和酶的蛋白水解作用。这种免疫系统调节的模型是昆虫中的黑化反应，其中的前苯氧过氧化物酶（proPO）的激活导致在寄生虫，细菌和真菌等异物表面上快速生成Eumelanin。ProPO激活受到所谓的片段结构域丝氨酸蛋白酶，其蛋白水解无活性的同源物及其丝氨酸蛋白酶抑制剂的严格控制。在冈比亚按蚊，是撒哈拉以南非洲的主要疟疾传播媒介，对该蛋白酶网络的操纵会影响对多种微生物的抵抗力以及宿主的存活率。但是，到目前为止，我们对蚊子中的分子组成和蛋白酶网络调控的理解是有限的。在这里，我们结合遗传和生化分析，报告了该域中的夹结构域丝氨酸蛋白酶CLIPB10的功能。CLIPB10敲低部分逆转了无感染时Serpin 2沉默诱导的黑色素瘤形成。CLIPB10还需要用于啮齿动物疟疾寄生虫的ookinete期的黑色素化伯氏疟原虫在难治性蚊子遗传背景中。重组丝氨酸蛋白酶抑制剂2蛋白，proPO激活级联中的关键抑制剂一个。冈比亚，与活化的重组CLIPB10形成SDS稳定的蛋白复合物，并有效抑制CLIPB10活性 体外化学计量比为1.89：1。重组激活的CLIPB10增加了PO中的PO活性满天星 血淋巴 离体，并直接活化纯化 六分体 脯氨酸 体外。综上所述，这些数据将CLIPB10鉴定为第二个具有前酚氧化酶激活功能的蛋白酶。一个。冈比亚除了先前描述的CLIPB9以外，还暗示了蛋白酶网络中控制黑色素化的功能冗余。此外，我们的数据表明，寄生虫的组织变黑和体液变黑至少部分由相同的蛋白酶介导。