Partial reconstitution of the prophenoloxidase activation system of Anopheles gambiae and maturation of Manduca sexta moricin-6 by dipeptidyl peptidase IVs

Insects rely on innate immune mechanisms to defend against pathogens, including melanization, antimicrobial peptides, and peptidoglycan recognition proteins (PGRPs). Melanization, a key immune response to wounding and microbial invasion, is regulated by serine protease cascades. In Anopheles gambiae, we selectively expressed SP217, four CLIPCs, and twelve CLIPBs, and reconstructed a branched five-step cascade with purified zymogens. SP217, orthologous to Drosophila ModSP, initiates the cascade by activating proCLIPC1, which in turn activates CLIPBs leading to prophenoloxidase (proPO) activation. CLIPB9 and CLIPB10 function as proPO-activating proteases (PAPs), generating highly active PO2 in the presence of a cofactor. This work reveals the organization of the mosquito serine protease system and highlights both evolutionary conservation and species-specific features. We further investigated dipeptidyl peptidase IV (DPP4) in Manduca sexta, which cleaves Xaa-Pro dipeptides from the N-terminus of peptides. Two isoforms, DPP4A and DPP4B, were cloned, and their expression was upregulated in fat body after bacterial challenge, suggesting an immune role. Recombinant proteins were purified and characterized, revealing that DPP4B exhibits higher catalytic activity than DPP4A. Functional assays showed that both enzymes cleave the N-terminal APEP motif from pro-moricin-6, a precursor of antimicrobial peptides, enhancing its antimicrobial activity against Escherichia coli, Bacillus megaterium, and fungi Fusarium oxysporum and Fusarium proliferatum. The N-terminal Xaa-Pro motifs in precursors may attenuate peptide activity, which DPP4s remove to unleash full antimicrobial function. Finally, we examined the function of soluble PGRPs in M. sexta hemolymph. The PGRPs preferentially recognized meso-diaminopimelic acid-peptidoglycans (DAP-PGs) and synergistically enhanced proPO activation in the presence of bacterial components. Amidase activity of PGRP2 and PGRP4 was confirmed and shown to be Zn²⁺-dependent. PGRP4 alone reduced B. megaterium colony-forming units (CFUs), whereas combinations of PGRP1–5 or 3s with lysozyme significantly enhanced bacterial killing. The synergistic effect of PGRP2 with lysozyme was further supported by electron microscopy, which revealed amplified bacterial cell wall damage. These results indicate that PGRPs contribute to both melanization and bactericidal activity through amidase-dependent and -independent mechanisms. Overall, this study elucidates multiple layers of insect immune defense, revealing mechanisms by which serine proteases, DPP4s, and PGRPs coordinate to detect, process, and neutralize microbial threats.