Characterization of co-factor proteins for prophenoloxidase activation in insects

Abstract

Melanization is a key part of insect innate immunity, which is mainly induced in host after pathogen invasion. Phenoloxidase (PO) is a crucial component to catalyze melanization. As PO is not stable in insects, it is always stored as prophenoloxidase (PPO), thus the activation of PPO is an important process for melanization induction. PO is generated from its precursor proPO by prophenoloxidase activatingproteases (PAPs) while PO activity can’t be highly induced, at least in some insects. It has been demonstrated that proPO activation requires additional proteins to serve as a cofactor for PAPs Serine protease homolog (SPH) in Manduca sexta and some other insects. However, it is not fully known if PAPs and cofactors are required for PPO activation in mosquito and Drosophila melanogaster. Moreover, formerly demonstrated SPH1 (1a) in M. sexta seems having a series orthologs, which need to be explored.

Here we analyzed four proSPHI proteins (proSPH4, proSPH1a, proSPH1b, proSPH101) in M. sexta including their abundance and cleavage activation and functional comparison. Based on the mRNA and protein data, we established SPH1b-2 pair as the major cofactor for proPO activation by PAPs in M. sexta. The recombinant proSPHs were tested with PAP3 and proPO to reveal the reaction mechanisms. We also identified four CLIPAs (ClipA4, ClipA6, ClipA7, and ClipA12) in An. gambiae and tested their potential to act as cofactors of CLIPB9, a PAP in An. gambiae, moreover, those ClipAs can also been cleaved by PAP3 from M. sexta. After the proteolytic activation, A. gambiae CLIPA4 formed complexes with CLIPA6, A7 or A12 to assist PPO2 or PPO7 activation. Moreover, cSPH242 and cSPH35 in drosophila were found to be cofactor proteins and work in pairs to enhance DmPPO1 PO activity. Unprecedented high levels of PO activity were achieved in the in vitro conditions, suggesting that cofactor-assisted PPO activation reaction is conserved in holometabolous insects.