Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

Fukasawa, Katsuhiko; Fukasawa, Masayoshi; Kanai, Masashi; Fujii, Shingo; Hirose, Junzo; Harada, Minoru

doi:10.1042/bj3290275

Cited by 68 publications

(91 citation statements)

References 35 publications

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“…In addition, the sequencing of cockroach proteins allowed us to identify in D. melanogaster a deduced protein of 723 amino-acid residues, sharing a 50% overall homology with rat and human DPP III. The putative Drosophila DPP has an expected mass of 82 kDa and a pI of 5.3, close to the values measured for the purified cockroach proteins and also for several vertebrate DPP III [17,20,21]. Even though the differences of molecular mass between the two purified proteins (80 and 76 kDa) in cockroach were not further investigated, it is interpreted as a different rate of glycosylation between the proteins.…”

Section: Discussionsupporting

confidence: 75%

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Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin

Mazzocco¹,

Fukasawa

Raymond³

et al. 2001

European Journal of Biochemistry

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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...

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Section: Discussionsupporting

confidence: 75%

“…The specific DPP III inhibitor tynorphin (ValVal-Tyr-Pro-Trp) was a gift from K. Fukasawa (Matsumoto Dental University, Nagano, Japan). The anti-(rat liver DPP III) Ig was prepared as described previously by Fukasawa et al [17].…”

Section: Methodsmentioning

confidence: 99%

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin

Mazzocco¹,

Fukasawa

Raymond³

et al. 2001

European Journal of Biochemistry

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“…Molecular cloning and sequencing of the rat and human enzyme 2,13 revealed a characteristic zinc-binding motif, hexapeptide HELLGH, enabling the recognition of a distinct DPP III family 14 , also known as metallopeptidase family M49 15 .…”

Section: Introductionmentioning

confidence: 99%

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

Cvitešić¹,

Sabljić

Makarević³

et al. 2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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Human dipeptidyl peptidase III (hDPP III), a zinc-metallopeptidase of the family M49, is an activator of the Keap1-Nrf2 cytoprotective pathway involved in defense against oxidative stress. Pathophysiological roles of DPP III have not been elucidated yet, partly due to the lack of specific inhibitors. We showed that substrate analog H-Tyr-Phe-NHOH is a strong competitive inhibitor of hDPP III, while H-Tyr-Gly-NHOH expresses much weaker inhibition. To investigate the effects of amino acid substitutions in inhibitor P1 position, we synthesized three new dipeptidyl hydroxamates and examined their influence on the activity of hDPP III and DPP III from the human gut symbiont Bacteroides thetaiotaomicron. The extent of inhibition of hDPP III, but not of bacterial enzyme, was dependent on the amino acid in P1. H-Phe-Phe-NHOH is recognized as one of the strongest inhibitors of hDPP III (K i ¼ 0.028 mM), and H-Phe-Leu-NHOH discriminated between human and bacterial ortholog of the M49 family.

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“…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].…”

mentioning

confidence: 99%

“…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].…”

mentioning

confidence: 99%

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

Mazzocco¹,

Fukasawa²,

Auguste³

et al. 2003

European Journal of Biochemistry

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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 ...

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Dipeptidyl peptidase III is a zinc metallo-exopeptidase: Molecular cloning and expression

Cited by 68 publications

References 35 publications

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster

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