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Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming approximately 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at approximately 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km approximately 4 microm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts.

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (Km approximately 4 micro m). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 +/- 0.15 micro m). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.

Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III.

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.