CLIPA protein pairs function as cofactors for prophenoloxidase activation in Anopheles gambiae.

Insect prophenoloxidases (proPO) are activated during immune responses by a proPO activating protease (PAP) in the presence of a high molecular weight cofactor assembled from serine protease homologs (SPH) that lack proteolytic activity. PAPs and the SPHs have a similar architecture, with an amino-terminal clip domain and a carboxyl-terminal protease domain. The SPHs belong to CLIPA subfamily of SP-related proteins. In Manduca sexta, a well characterized biochemical model system for insect immunity, the functional SPH cofactor contains one molecule each from two SPH subfamilies, SPH-I and SPH-II. In Anopheles gambiae, three SPHI-SPHII pairs (CLIPs A4-A6, A4-A7Δ, and A4-A12) were previously reported as cofactors for CLIPB9-mediated activation of proPO2 and proPO7. In this study, we produced recombinant proteins for two splicing variants of CLIPA7, proCLIPA7s (s for short), proCLIPA7f (f for full-length) and proCLIPA14. We cleaved each along with proCLIPA4 using M. sexta PAP3 and found that the CLIPA pairs A4-A7s and A4-A14 are better than A4-A7f in generating highly active PO2 or PO7. CLIPA7f and CLIPA7s, products of alternative splicing, have different strengths as cofactors in combination with CLIPA4. Because mRNA for CLIPA7f is expressed at a significantly higher level than CLIPA7s, cofactors with the weaker combination A4-A7f may predominate in hemolymph, resulting in a potential dampening effect on proPO activation as a regulatory mechanism for altering the strength of the melanization response. A. gambiae CLIPB10x is involved in proPO activation but its role as a PAP was not established using mosquito proPOs. Here we showed that factor Xa-treated proCLIPB10 activated proCLIPs A7s, A7f, A14, A4 (poorly), and proPO2. At higher concentrations, CLIPB10x efficiently activated proPO2 in the absence of a cofactor, but at low concentrations it required a CLIPA cofactor, suggesting that highly active PO2 can be generated at low concentration of CLIPB10 in cooperation with an SPH cofactor in vivo. Using cofactors generated by PAP3, we demonstrated the order of efficacy for proPO2 activation by B10 is A4-A6 > A4-A14 or A4-A7s > A4-A7f > A4-A12. This agrees with their relative strengths as cofactors for proPO2 and proPO7 activation by M. sexta PAP3. In summary, we further developed an in vitro assay system to elucidate biochemical details of the complex process of proPO activation in A. gambiae.