Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism, mediates immune responses.

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.