Roles of Manduca sexta serpin-8 and hemolymph proteinase-1 in the prophenoloxidase activation system

Melanization (prophenoloxidase activation) is a major innate immune response in insects, which produces highly toxic and reactive compounds around the wounding or infection. It involves an extracellular serine proteinase pathway which rapidly amplifies responses to infection and stimulate killing of pathogens. Inhibitory regulation of the proteinases by serine proteinase inhibitors (serpins) is important for ensuring a localized defense response.;One immune responsive serpin and one clip domain hemolymph proteinase (HP) from M. sexta were investigated. RT-PCR was used to examine gene expression. Proteins were expressed using baculovirus/insect cell system, and purified with various types of columns. Western immunoblotting was performed to analyze protein levels and protein-protein interactions in vitro and in vivo.;M. sexta serpin-8 mRNA and protein levels increased significantly after immune challenge. Similar to Drosophila serpin Necrotic, serpin-8 has an N-terminal extension and a Leu at the predicted P1 site. The serpin-8 core domain (serpin-8-ΔN) was expressed and purified. Serpin-8-ΔN has a specificity being able to inhibit elastase- and chymotrypsin-like proteinases and formed inhibitory complexes with chymotrypsin, Cathepsin G, and elastase. Addition of recombinant serpin-8-ΔN to M. sexta plasma suppressed proPO activation. M. sexta hemolymph proteinase, HP19 and HP14 are highly possible physiological targets of serpin-8.;Two isoforms of hemolymph proteinase-1 (HP1) are present in M. sexta hemolymph. The two proteins share 90% amino acid sequence similarity. Recombinant HP1 zymogens and catalytic domains were expressed and purified. Addition of zymogens or catalytic domains of HP1 into plasma resulted in proPO activation. Proteolytic cleavage of proHP6 was observed, when catHP1a alone was added into plasma. ProHP6 cleavage occurred within the catalytic domain when proHP6 was incubated with proHP1a in the presence of detergent. Some hemolymph factor(s) may contribute to the cleavage of proHP6 at its correct activation site, although the identity of such factor(s) is not yet known. HP1 was found to be regulated by M. sexta serpin-4 and serpin-5, and the serpin-proteinase complexes formed in an uncommon way.