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 cockroachpurified 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-ProThr) with a K m value of 3.8^1.1 mM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC 50 ¼ 0.68 mM). 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.Keywords: dipeptidyl aminopeptidase; enkephalinase; insect; neuropeptide; proctolin.Proctolin (Arg-Tyr-Leu-Pro-Thr) was the first neuropeptide isolated and characterized in insects [1]. This neuropeptide has been detected throughout the nervous system of insects [2][3][4] and identified in neurones with a widespread distribution within the central nervous system of arthropods [2][3][4][5]. Proctolin is considered to have a neurotransmitter/ neuromodulator role in visceral and skeletal muscles [1,3,4,6,7]. Moreover, the demonstration that proctolin can excite neurones of the ventral nerve cord ganglia [8] suggests a neuroeffector role in insect nervous system [3,4,7]. By contrast, a neurohormonal function for proctolin is unlikely as it is rapidly degraded by soluble haemolymph peptidases [9][10][11].In vitro studies using different insect tissue homogenates showed that soluble aminopeptidase and soluble dipeptidyl aminopeptidase (DPP) activities mediated the majority of the proctolin degradation [9]. Both enzyme activities were also measured in a lower range from unwashed membranes prepared from proctolin-rich tissues [9]. In those obtained from the hindgut of Schistocerca gregaria, a membrane DPP activity was finally identified as the major and initial degradation pathway of proctolin, liberating the N-terminal dipeptide Arg-Tyr and characterized by a low K m value of 0.15 mM for proctolin. The concomitant presence of a membrane aminopeptidase that degrades the former ArgTyr dipeptide was also demonstrated in these preparations. In a further study, an aminopeptidase activity exhibiting a K m value of 23 mM was shown to be located mainly within...
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 S 2 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 (K m 4 lM). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC 50 ¼ 0.62 ± 0.15 lM). 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.Keywords: enkephalinase; genome sequencing; insects; neuropeptides; proctolin.Mammalian DPP III was first discovered in the bovine anterior pituitary gland [1] and it has been recently cloned from rat liver as a 738-residue (82 kDa) cytosolic protein [2,3]. This enzyme (EC 3.4.14.4) is a zinc metallopeptidase containing a specific domain HELLGH-18X-E where a zinc molecule is bound to both histidines [4]. DPP III is mainly identified as a cytosolic peptidase, but DPP III was also detected on membranes prepared from the brain of guineapig [5] and rat [6]. Angiotensins and enkephalins constitute the preferred substrates of the rat brain cytosolic DPP III [7]. The routes of degradation of the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) have been compared to those of enkephalins. Indeed, a dipeptidyl aminopeptidase activity appears as one major proctolindegrading peptidase, liberating the N-terminal Arg-Tyr dipeptide [8][9][10][11]. This dipeptidyl aminopeptidase activity was compared to the vertebrate DPP III [11] and is mainly recovered as a cytosolic enzyme [8]. Interestingly, a proctolin-degrading DPP activity is also measured on membranes [8,9] especially those obtained from insect proctolin-rich tissues such as hindgut [10]. None of the presumed proctolinases has been fully characterized yet.We recently purified [12] from hindgut membranes of the cockroach, Blaberus craniifer, a proctolin-degrading protein (76 and 80 kDa) that removes the N-terminal dipeptide from proctolin (K m ¼ 3.8 ± 1.1 lM) and enkephalins (K m ¼ 4.2 ± 0.8 lM). The partial sequencing of this purified protein revealed a significant homology with the rat liver cytosolic ...
Dipeptidyl-peptidase (DPP) III (EC 3.4.14.4) has been characterized in rat [1,2] and human [3] as a soluble enzyme (molecular mass 82 kDa, SwissProt accession numbers O55096 and Q9NY33-1), confirming the results of their cloning and sequencing. This zinc metallopeptidase has also been reported to contain a specific HELLGH domain [4] which cleaves the second bound peptide of enkephalins.Current functional analyses of genomes have allowed the identification of putative DPP IIIs in about 20 species. In most cases, they are deduced as 700-amino acid proteins containing the specific catalytic motif HELLGH-52X-E. However, in a few cases, DPP IIIs have been predicted despite the shorter presumed DPP III and ⁄ or the lack of the specific HELLGH domain. In particular, the HELLGH domain is missing from the hypothetical Caenorhabditis elegans DPP III (NP492288, 682 residues). In humans, a truncated 317-residue DPP III isoform (SwissProt accession number Q9NY33-2), lacking 420 amino acids including the 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 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 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 (K m 4 lm) 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.Abbreviations DPP III, dipeptidyl-peptidase III.
Proctolin was incubated in vitro with crude membrane preparations derived from hind gut and ovary of Locusta rnigratoria. The metabolites of proctolin were separated and identified by reversed phase-high performance liquid chromatography which allowed the determination of the time course of degradation.At pHs 9.2 and 7.4, aminopeptidase activity predominated but some evidence of a carboxypeptidase could be inferred. At pH 6, aminopeptidase activity was slightly reduced compared to the level of aminopeptidase activity at the two former pHs; by contrast the carboxypeptidase was more important. At this pH, the presence of a membrane bound endopeptidase was detected.Phenanthroline, actinonin, amastatine, bestatin, and (2S,3R)-3-amino-2-hydroxy-4-(4-nitrophenyl)-butanoyl-L-leucine (HNBL) were tested at the concentration of 0.1 mM at pHs 8.2 and 6 and their ability to prevent the degradation of proctolin was estimated. A both pHs, HNBL and amastatin were revealed to be the most efficient inhibitors (SO to 75% inhibition) and o-phenanthroline appeared particularly efficacious at pH 6 (70% inhibition), whereas it was relatively poor inhibitor at pH 8.2. Furthermore, the use of these inhibitors confirmed the importance of carboxypeptidase activity at pH 6. These data are the first to describe proctolin degradation in ovary and hindgut muscle, important targets of proctolin action.
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