Abstract
Manduca sexta hemolymph protease-6 (HP6) plays a central role in coordinating antimicrobial responses, such as prophenoloxidase (PPO) activation and Toll signaling. Our previous studies indicated that HP5 and GP6 activate proHP6 in larval hemolymph and extraembryonic tissues, respectively. Here, we report the characterization of HP17b as another HP6 activating enzyme and its regulation by multiple serpins in hemolymph. The precursor of HP17b expressed in baculovirus infected Sf9 cells became spontaneously cleaved at two sites, and these products were purified together in one preparation named HP17b', a mixture of proHP17b, a 35 kDa intermediate, and HP17b. HP17b' converted proHP6 to HP6. As reported before, HP6 converted precursors of PPO activating protease-1 (PAP1) and HP8 to their active forms. HP8 activates proSpӓtzle-1 to turn on Toll signaling. We found HP17b' directly activated proSPHI and II to form a cofactor for PPO activation by PAP1. Supplementation of larval hemolymph with HP17b', HP17b, or proHP17b significantly increased PPO activation. Adding Micrococcus luteus to the reactions did not enhance PPO activation in the reactions containing HP17b', HP17b, or proHP17b. Using HP17b antibodies, we isolated from induced plasma HP17b fragments and associated proteins (e.g., serpin-4). Serpin-1A, 1J, 1J', 4, 5, or 6 reduced the activation of proHP6 by HP17b' through formation of covalent complexes with active HP17b. We detected an activity for proHP17b cleavage in hemolymph from bar-stage pharate pupae but failed to purify the protease due to its high instability. Other known HPs did not activate proHP17b in vitro. Together, these results suggest that HP17b is a clip-domain protease activated by an unknown endopeptidase in response to a danger signal and regulated by multiple serpins.