Phenoloxidase-catalyzed melanization is a universal defense mechanism of insects against pathogen and parasite infections. In mosquitos such as Anopheles gambiae, melanotic encapsulation is effective against certain malarial and filarial parasites. Phenoloxidases (POs) are produced as inactive prophenoloxidases (PPOs), which are activated by a serine protease cascade upon recognition of the invading organisms. POs generate reactive intermediates and melanin to kill and sequester the parasites. Having nine PPO genes that can be expressed as functional proteins in E. coli, A. gambiae provides opportunities to study their structures, activation modes, and catalytic mechanisms. We accumulated useful knowledge on PPOs, PPO activating proteases, and cofactors in studies of the Manduca sexta PPO activation system. We expressed A. gambiae PPO1?9 and active PO2 and PO7, solved the PPO8 structure, and proposed a new catalytic mechanism of insect POs. Kinetic analysis revealed differences in substrate binding and catalytic efficiency of detergent-activated PPO2, 7 & 8. We examined defense proteins and their abundances in hemolymph of larval and adult mosquitos. We established reliable phylogenetic relationships of CLIPs (serine proteases and their homologs with a clip domain) in five model insects. Based on these advances, we now propose to study the structures, activation, and catalysis of A. gambiae POs. Acquired knowledge and reagents will be useful for exploring how parasites evade or suppress the host immune mechanism.
The success of pathogen transmission by mosquitoes is partly determined by the insect immune system. This project aims at elucidating the active conformation and catalytic mechanism of phenoloxidases in a major vector species, which yield reactive chemicals to kill and sequester parasites and nematodes. Acquired knowledge on these enzymes may be applied for disrupting human disease transmission in mosquitoes.
