酚氧化酶(PO)催化的黑化和其他昆虫免疫反应是由丝氨酸蛋白酶(SP)及其非催化同系物(SPH)介导的。这些SP样蛋白中的许多具有调节夹域并且被称为CLIP。在迄今为止研究的大多数昆虫中,PO前体被PAP激活(即,PPO激活蛋白酶)及其夹域SPHs的辅因子。尽管黑色素包裹是蚊子对抗疟疾寄生虫的一种众所周知的难治性机制,尚不清楚PPO激活是否需要辅因子。冈比亚按蚊,CLIPA4与ManducasextaSPH2为1:1直系同源;CLIPsA5-7,A12-14,A26,A31,A32,E6和E7与M.sextaSPH1a为11:4直系同源,1b,如图4和101所示,SPH2在辅因子中配对。在这里,我们制作了proCLIPsA4,A6,A7Δ,A12,并用CLIPB9或M.sextaPAP3激活它们。在大肠杆菌中表达冈比亚PPO2和PPO7用作PAP底物。通过包括因子Xa切割位点将CLIPB9突变为CLIPB9Xa。CLIPA7Δ是去除了低复杂度区域的缺失突变体。PAP3或CLIPB9Xa处理后,CLIPA4与CLIPA6,A7Δ或A12形成了高Mr复合物,有助于PPO2和PPO7的激活。体外检测到高水平的比PO活性(PO2为55-85U/μg,PO7为1131-1630U/μg),表明辅因子辅助的PPO激活也发生在该物种中。复合物形成和辅因子功能的切割位点和机制类似于在S型双歧杆菌和黑腹果蝇中报道的那些。总之,这些数据表明,3对(或许更多)SPHI-II可能形成CLIPB9介导的PPOs激活的辅因子,用于冈比亚黑色素细胞包裹.
Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5-7, A12-14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55-85 U/μg for PO2 and 1131-1630 U/μg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.