CLIPB4 is a central node in the protease network that regulates humoral immunity inAnopheles gambiaemosquitoes

Abstract

AbstractInsect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist ofclip domainserineproteases (cSPs) and/or their non-catalytichomologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquitoAnopheles gambiaeas a central node in this protease network.CLIPB4is tightly co-expressed withSRPN2and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network inAn. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.