Insight into the structural hierarchy of the protease cascade that regulates the mosquito melanization response

Abstract

AbstractSerine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic,clip domainserineproteases (cSPs) and their non-catalytichomologs (cSPHs). Here, we define partially the structural hierarchy ofAn. gambiaecSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Usingin vivogenetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanizationin vivoin response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBsin vivois efficiently cleaved itself by several CLIPBsin vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.