Mating-increases trypsin in female Drosophila hemolymph.

Male-derived accessory gland proteins (Acps) are transferred to the female reproductive tract during mating and affect female reproductive maturation and behavior. Some Acps subsequently enter the female hemolymph. We hypothesized that humoral proteases are the primary effectors of Acp bioactivity by processing (activating) and/or degrading them. To test this hypothesis we examined the fate of one Acp, Drosophila melanogaster Sex Peptide (Acp70A, DrmSP), which possesses several putative serine-protease cleavage sites, in hemolymph of unmated and mated females. In D. melanogaster, DrmSP induces post-mating non-receptivity and enhances oogenesis. To determine if serine proteases regulate the duration of DrmSP activity in mated females, we performed kinetic analysis of cleavage of a synthetic N-terminal truncated DrmSP(8-36) (T-SP) with hemolymph of unmated versus mated females. We found that T-SP is cleaved more rapidly and completely in mated female hemolymph. Using LC-MS/MS analyses, we identified its primary cleavage sites, indicating that trypsin was the major endopeptidase regulating T-SP in hemolymph. This was verified in vitro by utilizing specific chromogenic serine-protease substrates and inhibitors. We propose that post-mating cleavage of DrmSP in the female hemolymph regulates the duration of the rapidly induced post-mating responses in D. melanogaster and that this is a specific example of Acp bioactivity regulated by hemolymph serine proteases.

The presence of Drosophila melanogaster sex peptide-like immunoreactivity in the accessory glands of male Helicoverpa armigera.

In this study a highly specific polyclonal antibody to DrmSP was produced and used to develop and standardize a sensitive direct ELISA. Structure-activity studies revealed that the antiserum is specific to the N-terminal of DrmSP. This ELISA was used for the detection of DrmSP-like immunoreactivity in the reproductive tissues of male Helicoverpa armigera moths at femtomole levels. Two positive immunoreactive peaks were found in HPLC purified extracts of male accessory glands. The immunoreactive peak, which contained a higher amount of immunoreactivity, was also found to be pheromonostatic in PBAN-injected decapitated females as well as in intact female moths during their peak pheromone production. Lower levels of DrmSP-like immunoreactivity were found in younger males (1-2 day-old) when compared to older males (3-7 day-old).

Thiol-activated serine proteinases from nymphal hemolymph of the African migratory locust, Locusta migratoria migratorioides.

Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds.