Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

• Isolation of a major processing enzyme from Manduca sexta hemolymph, which cuts proSPH1 and proSPH2 within the clip domains.
• Confirmation of the complex of SPH1 and SPH2 as cofactor for proPO activation by PAPs.
• Discovery of PAP3 as a proPAP3 activating enzyme besides HP21.
• Indirect activation of HP6, PAP1, and HP8 in plasma to enhance melanization and Spatzle processing.

Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta.

In Manduca sexta hemolymph, PAP3 activates proPAP3 to generate more PAP3, which cleaves proSPH1 and proSPH2 to form an SPH complex. In the presence of this complex, PAP3 activates proPOs to form active POs. PAP3 may indirectly activate proHP6, HP6 activates proPAP1, PAP1 activates proSPH2 and proPOs to further enhance melanization.Figure optionsDownload high-quality image (119 K)Download as PowerPoint slide