Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 +/- 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 +/- 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 +/- 16 vs. 146 +/- 19 pg/ml; TNFalpha 3.8 +/- 1.0 vs. 6.6 +/- 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFalpha and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 +/- 4 vs. 7 +/- 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.
BACKGROUND: Dipeptidyl peptidase IV (DPPIV, DPP4) is a serine protease that releases N-terminal dipeptides. It is a validated drug target for type 2 diabetes and DPPIV inhibitors are currently evaluated for other therapeutic applications. Various assays are used for DPPIV activity measurements in biological samples. Highly sensitive methods are needed to measure also very low activities in inhibited samples. METHODS: Here, the three most extensively used substrates to quantify DPPIV activity are compared using in-house methods. A luminescent kit was also included. In addition, one of the in-house fluorometric assays was elaborated for use in biological samples containing reversible DPPIV inhibitors to estimate residual DPPIV activity which is usually underestimated due to sample dilution. RESULTS: The in-house methods showed a good precision, linearity and specificity. Both fluorometric substrates had a 10-fold higher sensitivity compared to the colorimetric assay. The luminescent kit was found to be the most sensitive. CONCLUSIONS: All three in-house methods can be used to measure DPPIV activity in non-inhibited biological samples. The more sensitive fluorometric assays are recommended when sample volumes are limited or when using inhibited samples. The elaborated fluorometric method can be used to estimate the residual in vivo DPPIV activity in inhibitor treated subjects.
Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.
The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.
Title: Dipeptidyl peptidases and related proteins: multifaceted markers and therapeutic targets De Meester I, Scharpe S, Lambeir AM Ref: Clinical Chemistry & Laboratory Medicine, 47:245, 2009 : PubMed
The mRNA expression pattern of dipeptidyl peptidase (DPP) 8 and DPP9, two DPP4 homologs, was studied previously and showed a broad tissue distribution. In this study, protein expression and activity of DPP8 and DPP9 were investigated in male reproductive tissues of different mammals. Based on specific DPP activities and inhibition profiles, the proline-selective DPP activity in the bovine and rat testis could predominantly be attributed to DPP8/9 and not to DPP4. This is in contrast to the epididymis, where most of the activity was caused by DPP4. Bovine sperm preparations had very low or undetectable DPP8/9 activity. After characterization of polyclonal antibodies specific for DPP8 or DPP9, we could localize both enzymes in seminiferous tubules of the testis. A specific staining for DPP9 was found associated with spermatozoids embedded in the epithelium, just before their release into the lumen, and in spermatids. DPP8 was localized in spermatozoids in an earlier stage of maturation. These findings help to provide insight into the physiological role of DPP4-like enzymes in the male reproductive system. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Dipeptidyl peptidase 4 (DPP4) inhibitors represent a novel class of oral anti-hyperglycemic agents. The complete pharmacological profile of these protease inhibitors remains unclear. In order to gain deeper insight into the in vivo effects caused by DPP4 inhibition, two different DPP4 inhibitors (vildagliptin and AB192) were analyzed using differential peptide display. Wistar rats were treated with the DPP4 inhibitors (0.3mgkg(-1); 1mgkg(-1) or 3mgkg(-1) body weight) and DPP4 activity was measured before and at the end of the experiment. One hour after compound administration, blood plasma samples were collected to generate peptide displays and to subsequently identify differentially regulated peptides. A dose-dependent decrease in blood plasma DPP4 activity was measured for both inhibitors. DPP4 inhibition influenced collagen metabolism leading to depletion of collagen derived peptides (e.g. collagen alpha 1 (III) 521-554) and accumulation of related N-terminally extended collagen derived peptides (e.g. collagen alpha 1 (III) 519-554). Furthermore, the intact amyloid rat BRI (1-23) peptide was detected in plasma following in vivo DPP4 inhibition. DPP4 catalyzed cleavage kinetics of the BRI peptide were determined in vitro. The k(cat) and K(m) for cleavage by DPP4 were 5.2s(-1) and 14microM, respectively, resulting in a specificity constant k(cat)/K(m) of 0.36 x 10(6)s(-1)M(-1). Our results demonstrate that differential peptide analysis can be applied to monitor action of DPP4 inhibition in blood plasma. For the first time effects on basal collagen metabolism following DPP4 inhibition in vivo were demonstrated and the BRI amyloid peptide was identified as a novel DPP4 substrate.
B-type natriuretic peptide (BNP) has emerged as a reliable biomarker in patients with congestive heart failure. The mature, biologically active B-type natriuretic peptide, BNP(1-32), is cleaved by corin from the 108 amino acid proBNP. However, in vivo as well as in vitro data demonstrated that this BNP(1-32) might be an ideal substrate for the endogenous aminopeptidase, dipeptidyl-peptidase IV (DPP IV). DPP IV removes the two amino terminal amino acids (Ser Pro) from BNP(1-32) to produce BNP(3-32), which has been detected in plasma of patients with congestive heart failure. The biological effects of BNP(3-32) remain undetermined. In cultured human cardiomyocytes and fibroblasts, equimolar concentrations of BNP(1-32) and BNP(3-32) both exert similar biological effects, as evidenced by their cGMP (cyclic guanylate monophosphate) generating capacity. However, in a canine model, intravenous BNP(3-32) infusion resulted in less natriuresis, diuresis, and vasodilation compared to intravenous infusion of BNP(1-32). The clinical relevance of these observations might be important for patients in whom the plasma BNP concentrations, measured by commercially available immunoassays, are high. Further studies exploring whether DPP IV inhibitors increase the bioavailability of BNP(1-32), delay the progression of heart failure, and increase the efficacy of exogenous administration of BNP(1-32) in decompensated heart failure are needed.
Despite its thorough enzymological and biochemical characterization the exact function of prolyl oligopeptidase (PO, E.C. 3.4.21.26) remains unclear. The positive effect of PO inhibitors on learning and memory in animal models for amnesia, enzyme activity measurements in patient samples and (neuro)peptide degradation studies link the enzyme with neurodegenerative disorders. The brain protein alpha-synuclein currently attracts much attention because of its proposed role in the pathology of Parkinson's disease. A fundamental question concerns how the essentially disordered protein is transformed into the highly organized fibrils that are found in Lewy bodies, the hallmarks of Parkinson's disease. Using gel electrophoresis and MALDI TOF/TOF mass spectrometry we investigated the possibility of alpha-synuclein as a PO substrate. We found that in vitro incubation of the protein with PO did not result in truncation of full-length alpha-synuclein. Surprisingly, however, we found an acceleration of the aggregation process of alpha-synuclein using turbidity measurements that was reversed by specific inhibitors of PO enzymatic activity. If PO displays this activity also in vivo, PO inhibitors might have an effect on neurodegenerative disorders through a decrease in the aggregation of alpha-synuclein.
Until now, only recombinant forms of dipeptidyl peptidase (DPP) 8 and 9 have been characterized. We purified non DPPII-non DPPIV enzymes from a natural source. A first DPP8/9-like enzyme was enriched 1160-fold from bovine testes and identified as 'DPP9-like enzyme' by using an anti-DPP9 antibody. A second 576-fold enriched preparation ('DPP enriched peak 3') also showed DPP8/9-like activity. SDS-PAGE analysis showed that the DPP9-like enzyme had a monomeric molecular mass of approx. 100 kDa. Size exclusion chromatography generated a native molecular mass of 164 kDa for the DPP9-like enzyme and one of 234 kDa for the DPP enriched peak 3, suggesting that both proteins appeared to be dimeric. Both enriched preparations and rDPP8 showed roughly similar substrate specificity and inhibitor profiles. The DPP9-like enzyme and the DPP enriched peak 3 possessed a neutral pH optimum and were stable at -80 degrees C. We can conclude that the natural DPP9-like enzyme and the DPP enriched peak 3 are closely related to the recombinant forms of human DPP9 and DPP8.
With vildagliptin and sitagliptin on the market for the treatment of type 2 diabetes, dipeptidyl peptidase 4 (DPP4, EC 3.4.14.5) research has entered a new era. Scientists aim to uncover the broader pharmacological profile of DPP4 inhibitors and search for therapeutic opportunities outside diabetes. During the pre-clinical and clinical evaluation of vildagliptin and sitagliptin, there has been a growing awareness of the presence of other DPP4-like peptidases in various cells and tissues. This fuelled the development of more inhibitors with defined selectivity for DPP2, 8 and 9 that were used to investigate the expression, distribution and regulation of these peptidases. In turn, these studies increased the insights in the role of DPP4 in the body's response to various insults.
To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.
Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure-activity-relationship data obtained in this way led to the preparation of a series of alpha-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.
The proline-specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9-specific activities, and DPPII- and DPPIV-specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30-fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or -9, including inhibition by allo-Ile-isoindoline and affinity for immobilized Lys-isoindoline.
Dipeptide-derived compounds, bearing various P2 residues and a diaryl pyrrolidin-2-yl phosphonate at the P1 position, were evaluated as dipeptidyl peptidase 8 (DPP8) inhibitors. With these products, irreversible inhibition of DPP8 was observed. To obtain inhibitors with an improved activity and selectivity profile, a set of selected analogues containing a diaryl isoindolin-1-ylphosphonate at P1 was synthesized and evaluated. Within this latter series, compound 2e was shown to be a potent, irreversible inhibitor of DPP8, demonstrating very low affinity for DPP IV and DPP II.
BACKGROUND: Analysis of plasma B-type natriuretic peptide (BNP) has suggested the in vivo formation of a truncated form, BNP (3-32), also called des-SerPro-BNP. The objectives of this study were to investigate (a) whether BNP and other natriuretic peptides are truncated by dipeptidyl-peptidase IV (DPP IV/CD26; EC 3.4.14.5) and (b) whether this truncation affects the susceptibility to cleavage by neutral endopeptidase (NEP; EC 3.4.24.11). METHODS: Human BNP (1-32), A-type natriuretic peptide 1-28 (ANP 1-28), and related peptides were incubated with purified DPP IV and with human plasma. In addition, BNP (1-32), BNP (3-32), and ANP (1-28) were subjected to hydrolysis by NEP. Cleavage products were analyzed by mass spectrometry. RESULTS: BNP (1-32) was cleaved by purified DPP IV with a specificity constant of 0.37 x 10(6) L.mol(-1).s(-1). The DPP IV activity in EDTA-plasma was able to truncate BNP (1-32) ex vivo. Addition of Vildagliptin, a specific DPP IV inhibitor, prevented this truncation in a concentration-dependent manner. Under in vitro circumstances in which ANP was hydrolyzed extensively, BNP (1-32) and BNP (3-32) were very resistant to NEP-mediated cleavage. CONCLUSIONS: DPP IV cleaves BNP (1-32) with an efficiency higher than or comparable to several known in vivo substrates of the enzyme. Even after loss of the amino-terminal dipeptide, BNP remains highly resistant to cleavage by NEP.
Dipeptidyl peptidase (DPP) II (E.C. 3.4.14.2) is an intracellular protease that releases, preferably at acidic pH, N-terminal dipeptides from oligopeptides with Pro or Ala in the penultimate position. The natural substrates and the physiological role of DPPII remain unclear. The aim of the present study was to investigate the involvement of DPPII activity in different forms of cell death (apoptosis, necrosis and autophagy) in human leukocytes. We determined specific DPP activities in leukocytes. Compared to other subpopulations of peripheral blood mononuclear cells (PBMC), we observed relatively high DPPII specific activity in monocytic cells, opening new perspectives for further investigation of the DPPII functions. A second intriguing finding was that DPPII specific activity increased during necrosis, whereas induction of apoptosis or autophagy did not affect any of the dipeptidyl peptidase activities. Finally, we showed that inhibition of DPPII (>90%) using the in vitro applicable, highly potent (K(i) of 0.082+/-0.048 nM) and selective DPPII inhibitor UAMC00039, did not induce any form of cell death in leukocytes. These data are of importance for a more precise interpretation of the in vitro and in vivo effects of other dipeptidyl peptidase inhibitors.
In this paper, we report the synthesis of diastereomerically pure N-(4-substituted-2,4-diaminobutanoyl)piperidines. These compounds were prepared to investigate the influence of the 4-substitution on the dipeptidyl peptidase II (DPP II) activity and selectivity of the parent N-(2,4-diaminobutanoyl)piperidine. The (4S)-methyl compound showed subnanomolar inhibition, comparable with the parent compound. The (4R)-methyl group or bigger substituents decreased the activity.
UNLABELLED: CD26/Dipeptidyl peptidase (DPP) IV is an integral membrane protein of lymphocytes that modulates the activities of chemokines, interleukins, and neuropeptides. We investigated the effect of enzymatic DPP IV inhibition on ischemia/reperfusion injury after extended ischemia prior to transplantation. MATERIALS AND METHODS: We used a syngeneic rat (Lewis) orthotopic left lung transplantation model. In the control group (group I), donor lungs were flushed and preserved in Perfadex for 18 hours at 4 degrees C, then transplanted and reperfused for 2 hours. Group II donor lungs were perfused with and stored in Perfadex +25mol/L AB192 (bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate), a small molecular weight DPP IV inhibitor. After 2-hour reperfusion, we measured blood gas, peak airway pressure, and thiobarbituric acid reactive substances. RESULTS: Grafts from group II versus group I showed a significantly increased oxygenation capacity (II: 298.4 +/- 87.6 mm Hg vs 120.9 +/- 48.0, P < .01), lower peak airway pressure (11.8 +/- 0.9 mm Hg vs 16.0 +/- 1.4, P < .01), and less lipid peroxidation (9.3 +/- 2.0 micromol/L vs 13.8 +/- 1.8, P < .01). CONCLUSION: Inhibition of intragraft DPP IV enzymatic activity significantly reduced ischemia/reperfusion-associated pulmonary injury, allowing for successful transplantation after 18 hours of ischemia.
The function of prolyl oligopeptidase (PO) has been associated with several disorders of the central nervous system. The purpose of this study was to identify endogenous substrates for recombinant porcine PO in porcine brain. The smaller polypeptides were extracted from total brain homogenates and fractionated by two-dimensional chromatography prior to incubation with PO. Shifts in the mass spectrum between the control and the incubated sample, marked potential substrates. Using MSMS peptide sequencing techniques, we identified several fragments of intracellular proteins as potential substrates, which opens new perspectives for finding the function of PO in the intracellular space.
Title: Inhibition of dipeptidyl-peptidase IV catalyzed peptide truncation by Vildagliptin ((2S)-{[(3-hydroxyadamantan-1-yl)amino]acetyl}-pyrrolidine-2-carbonitrile) Brandt I, Joossens J, Chen X, Maes MB, Scharpe S, De Meester I, Lambeir AM Ref: Biochemical Pharmacology, 70:134, 2005 : PubMed
Vildagliptin (NVP-LAF237/(2S)-{[(3-hydroxyadamantan-1-yl)amino]acetyl}-pyrrolidine-2-carbonitr ile) was described as a potent, selective and orally bio-available dipeptidyl-peptidase IV (DPP IV, EC 3.4.14.5) inhibitor [Villhauer EB, Brinkman JA, Naderi GB, Burkey BF, Dunning BE, Prasad K, et al.1-[[(3-Hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem 2003;46:2774-89]. Phase III clinical trials for the use of this compound in the treatment of Type 2 diabetes were started in the first quarter of 2004. In this paper, we report on (1) the kinetics of binding, (2) the type of inhibition, (3) the selectivity with respect to other peptidases, and (4) the inhibitory potency on the DPP IV catalyzed degradation of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and substance P. Vildagliptin behaved as a slow-binding DPP IV inhibitor with an association rate constant of 1.4x10(5)M(-1)s(-1) and a K(i) of 17nM. It is a micromolar inhibitor for dipeptidyl-peptidase 8 and does not significantly inhibit dipeptidyl-peptidase II (EC 3.4.11.2), prolyl oligopeptidase (EC 3.4.21.26), aminopeptidase P (EC 3.4.11.9) or aminopeptidase M (EC 3.4.11.2). There was no evidence for substrate specific inhibition of DPP IV by Vildagliptin or for important allosteric factors affecting the inhibition constant in presence of GIP and GLP-1.
The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell proline dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).
The feasibility of the fluoro-olefin function as a peptidomimetic group in inhibitors for dipeptidyl peptidase IV and II (DPP IV and DPP II) is investigated by evaluation of N-substituted Gly-Psi[CF=C]pyrrolidines, Gly-Psi[CF=C]piperidines, and Gly-Psi[CF=C](2-cyano)pyrrolidines. Of this later class, the (Z)- and (E)-fluoro-olefin analogues were prepared and chemical stability in comparison with the parent amide was checked. Most of these compounds exhibited a strong binding preference toward DPP II with IC(50) values in the low micromolar range, while only low DPP IV inhibitory potential is seen.
Title: Expression, purification and preliminary crystallographic analysis of dipeptidyl peptidase IV from Porphyromonas gingivalis Rea D, Lambeir AM, Kumagai Y, De Meester I, Scharpe S, Fulop V Ref: Acta Crystallographica D Biol Crystallogr, 60:1871, 2004 : PubMed
The asaccharolytic periodontopathogen Porphyromonas gingivalis produces membrane-anchored proteases such as dipeptidyl peptidase IV that are involved in the destruction of host periodontal tissue. The extracellular domain of this enzyme was overexpressed in Escherichia coli as an N-terminal His-tag fusion protein, purified using standard metal-affinity chromatography and crystallized using the hanging-drop vapour-diffusion technique in 40% 2-methyl-2,4-pentanediol and 100 mM Tris-HCl pH 8.0. Diffraction data to 2.7 A resolution were collected using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 117.0, b = 112.9, c = 310.0 A, beta = 95.0 degrees. There are ten molecules per asymmetric unit, indicating a solvent content of 50%. Data were also collected from selenomethionine-derived crystals and structure solution by SAD or MAD is in progress.
Using 1-[(S)-2,4-diaminobutanoyl]piperidine as lead compound, we developed a large series of highly potent and selective dipeptidyl peptidase II (DPP II) inhibitors. gamma-Amino substitution with arylalkyl groups, for example, a 2-chlorobenzyl moiety, resulted in a DPP II inhibitor with an IC(50) = 0.23 nM and a high selectivity toward DPP IV (IC(50) = 345 microM). Furthermore, it was shown that the basicity of the gamma-amino is important and that alpha-amino substitution is not favorable. Piperidine-2-nitriles did not show an increase in potency but rather reduced the selectivity. Introduction of a 4-methyl or a 3-fluorine on piperidine improved selectivity and preserved the high potency.
Title: Dipeptidyl peptidase IV substrates. An update on in vitro peptide hydrolysis by human DPPIV De Meester I, Lambeir AM, Proost P, Scharpe S Ref: Advances in Experimental Medicine & Biology, 524:3, 2003 : PubMed
Title: Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV Lambeir AM, Durinx C, Scharpe S, De Meester I Ref: Crit Rev Clin Lab Sci, 40:209, 2003 : PubMed
Dipeptidyl-peptidase IV/CD26 (DPP IV) is a cell-surface protease belonging to the prolyloligopeptidase family. It selectively removes the N-terminal dipeptide from peptides with proline or alanine in the second position. Apart from its catalytic activity, it interacts with several proteins, for instance, adenosine deaminase, the HIV gp120 protein, fibronectin, collagen, the chemokine receptor CXCR4, and the tyrosine phosphatase CD45. DPP IV is expressed on a specific set of T lymphocytes, where it is up-regulated after activation. It is also expressed in a variety of tissues, primarily on endothelial and epithelial cells. A soluble form is present in plasma and other body fluids. DPP IV has been proposed as a diagnostic or prognostic marker for various tumors, hematological malignancies, immunological, inflammatory, psychoneuroendocrine disorders, and viral infections. DPP IV truncates many bioactive peptides of medical importance. It plays a role in glucose homeostasis through proteolytic inactivation of the incretins. DPP IV inhibitors improve glucose tolerance and pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients. The role of DPP IV/ CD26 within the immune system is a combination of its exopeptidase activity and its interactions with different molecules. This enables DPP IV/CD26 to serve as a co-stimulatory molecule to influence T cell activity and to modulate chemotaxis. DPP IV is also implicated in HIV-1 entry, malignant transformation, and tumor invasion.
In this paper, we present a parallel synthesis of several series of dipeptide diphenyl phosphonates that are known to be irreversible inhibitors of serine proteases. Polymer-assisted solution-phase synthesis (PASP) is used for the rapid and clean coupling between various alpha-aminoalkyl diphenyl phosphonate ester building blocks and commercially available or easily accessible amino acids. These compounds were used for the rapid profiling of dipeptidyl peptidase II (DPP II) and the closely related dipeptidyl peptidase IV (DPP IV). A highly selective DPP II inhibitor was identified, N-cyclopentylglycyl-NHCH(C(6)H(5))PO(OPh)(2) (9.35), that will be useful to discriminate between DPP II and DPP IV in biological systems in order to further elucidate the biological function of DPP II.
In this paper we report the systematic search for new, potent, and selective DPP II inhibitors. A study of the structure-activity relationship was conducted starting from aminoacyl pyrrolidides as lead compounds. Rational exploration of the P(1) and P(2) building blocks led to the discovery of some very potent DPP II inhibitors which can be characterized by their high selectivity for DPP II with regard to DPP IV. Dab-Pip and Dab-Pip-2-CN were selected as the most promising inhibitors (IC(50) nM range) and will enable us to study the physiological role of DPP II and to differentiate between DPP II and DPP IV in biological systems.
Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)).
Chemokines coordinate many aspects of leukocyte migration. As chemoattractants they play an important role in the innate and acquired immune response. There is good experimental evidence that N-terminal truncation by secreted or cell surface proteases is a way of modulating chemokine action. The localization of CD26/dipeptidyl peptidase IV on cell surfaces and in biological fluids, its primary specificity, and the type of naturally occurring truncated chemokines are consistent with such a function. We determined the steady-state catalytic parameters for a relevant selection of chemokines (CCL3b, CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11, and CXCL12) previously reported to alter their chemotactic behavior due to CD26/dipeptidyl peptidase IV-catalyzed truncation. The results reveal a striking selectivity for stromal cell-derived factor-1alpha (CXCL12) and macrophage-derived chemokine (CCL22). The kinetic parameters support the hypothesis that CD26/dipeptidyl peptidase IV contributes to the degradation of certain chemokines in vivo. The data not only provide insight into the selectivity of the enzyme for specific chemokines, but they also contribute to the general understanding of CD26/dipeptidyl peptidase IV secondary substrate specificity.
Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.
Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.
The previously reported diphenyl 1-(S)-prolylpyrrolidine-2(R, S)-phosphonate (5) was used as a lead compound for the development of potent and irreversible inhibitors of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). The synthesis of a series of diaryl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonates with different substituents on the aryl rings (hydroxyl, methoxy, acylamino, sulfonylamino, ureyl, methoxycarbonyl, and alkylaminocarbonyl) started from the corresponding phosphites. A good correlation was found between the electronic properties of the substituent and the inhibitory activity and stability. The most striking divergence of this correlation was the high potency combined with a high stability of the 4-acetylamino-substituted derivative 11e. This compound shows low cytotoxicity in human peripheral blood mononuclear cells and also has favorable properties in vivo. Therefore bis(4-acetamidophenyl) 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate (11e) is considered as a major improvement and will be a highly valuable DPP IV inhibitor for further studies on the biological function of the enzyme and the therapeutic value of its inhibition.
Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as CD26, is a membrane-bound serine protease that cleaves off aminoterminal dipeptides from peptides with a penultimate proline (or, at a much slower rate, a penultimate alanine). Recently, we synthesized and characterized a number of dipeptide-derived diphenylphosphonates. Out of the resulting series of slow-binding irreversible inhibitors of DPP IV, diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate hydrochloride (Pro-Pro-diphenylphosphonate or Prodipine) was selected for further study. We investigated the in vivo applicability of Prodipine. Male rabbits weighing 3-4 kg received a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity had decreased to less than 20% of the preinjection value and remained unchanged during a 24-hr observation period. In a next step, we aimed to study (i) the dose dependency and (ii) the duration of the effect after a single intravenous dose of Prodipine. A profound and long-lasting inhibition of plasma DPP IV activity was observed in the treated animals (1, 5 or 10 mg). It took 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery. Systemic treatment with Prodipine not only led to inhibition of plasma DPP IV activity but also decreased tissue DPP IV activity in circulating mononuclear cells, kidney cortex, thymus, spleen, lung, and liver. No differences in activities of the related peptidases aminopeptidase P (APP, EC 3.4.11.9), prolyl oligopeptidase (PO, EC 3.4.21.26), or aminopeptidase M (mAAP, EC 3.4.11.2) were detected between Prodipine-treated and control rabbits. The in vivo applicability of this chemically stable, irreversible inhibitor of DPP IV opens new possibilities, not only to further unravel the biological functions of this intriguing ectopeptidase, but also to explore this enzyme as a new target in various fields of pharmacological research.
Title: Development and evaluation of peptide-based prolyl oligopeptidase inhibitors--introduction of N-benzyloxycarbonyl-prolyl-3- fluoropyrrolidine as a lead in inhibitor design Goossens F, Vanhoof G, De Meester I, Augustyns K, Borloo M, Tourwe D, Haemers A, Scharpe S Ref: European Journal of Biochemistry, 250:177, 1997 : PubMed
The current study has been undertaken to develop new and biocompatible inhibitors for prolyl oligopeptidase, a highly specific endopeptidase, proposed to be involved, through its affinity for neuropeptides and kinins, in the processes of learning and memory and in the control of blood pressure. For in vitro evaluation of the inhibitors, human platelet prolyl oligopeptidase was purified to homogeneity and characterized. Northern blot analysis showed that mRNA coding for prolyl oligopeptidase was present in all tissues examined and only one transcript of 3.1 kb was detected. In addition to the human platelet enzyme, we also purified rat brain prolyl oligopeptidase, which proved to have the same characteristics as the human enzyme. In a series of tested peptides, bradykinin was found to be the best substrate. Based on this information, peptides bearing pseudopeptide bonds were generated and evaluated as inhibitors. The experiments clearly demonstrated that changes to the scissile peptide bond significantly decrease the affinity of prolyl oligopeptidase for the peptide derivatives. In our series of synthetic N-terminal blocked dipeptides, N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine was the most potent compound. Inhibition was reversible, but the inhibitor was bound tightly. Calculation of its Ki according to Henderson [Henderson, J. P. (1972) Biochem. J. 127, 321-333] yielded a value of 0.8 nM. This compound was not cytotoxic in a cell culture system and inhibited the purified prolyl oligopeptidase from rat as well as from human origin. In vivo evaluation in male Whistar rats showed no acute toxicity. 5 h after administration, the most profound decrease in prolyl oligopeptidase activity was found in the thymus, brain, and testis. This study demonstrates that N-benzyloxycarbonyl-prolyl-3-fluoropyrrolidine is a potent inhibitor and a promising compound suitable to investigate the physiologic function of the enzyme in vitro and in vivo.
Human DPP IV, isolated from seminal plasma by means of immobilised adenosine deaminase, occurs in different forms which are distinguishable by net charge and native molecular weight. Charge differences arise primarily from different degrees of glycosylation containing various amounts of sialic acid. The majority of DPP IV isolated from total seminal plasma consists of the extracellular part of the protein starting at Gly-31. It is a very stable protein resisting high concentrations of denaturant. Unfolding experiments under reducing conditions are indicative of the existence of at least two domains which function independently. One of these domains is highly stabilised by disulfide bonds. Disruption of the disulfide bonds does not affect the activity, the dimeric state nor the adenosine deaminase binding properties of the protein but renders it more susceptible to proteolysis. The low-angle X-ray scattering spectrum is consistent with a model for a protein containing two subunits, each composed of three domains linked by flexible regions with low average mass. The secondary structure composition, determined by FTIR spectrometry, indicates that 45% of the protein consists of beta-sheets, which is higher than expected from computed secondary structure predictions. Our results provide compelling experimental evidence for the three-domain structure of the extracellular part of DPP IV.
A number of dipeptide diphenyl phosphonate esters were studied as inhibitors of dipeptidyl peptidase IV, focusing on the role of the P2 residue in the inactivation process. The active compounds were slow irreversible inhibitors of the catalytic activity of the enzyme. With proline (or alanine) in the P1 position, the rate constants of inactivation correlated with the acylation rate constants reported for homologous dipeptide derived substrates. The kinetic data indicate that the mechanism of inhibition consists of the formation of a fairly weak initial complex, followed by a slow irreversible inactivation step. This indicates that, as in the case of trypsin-like proteinases, dipeptide diphenyl phosphonate esters form a covalent adduct with the catalytic site of DPP IV, even though this enzyme belongs to a completely distinct class of serine peptidases. Enantioselectivity and secondary specificity further support the evidence that diphenyl phosphonate esters are mechanism-based inhibitors. The dipeptide diphenyl phosphonate esters had a half-life of 3-10 h at 37 degrees C in Tris buffer. The inhibitors were degraded in human plasma, depending on the type of amino-terminal amino acid. The compound with proline in the P2 position was the most resistant to degradation in plasma. Due to their stability and the irreversible nature of the inhibition, the diphenyl phosphonate esters promise to be useful tools in the continuing investigation of the physiological function of dipeptidyl peptidase IV.
Title: Use of immobilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of native human CD26/dipeptidyl peptidase IV (EC 3.4.14.5) De Meester I, Vanhoof G, Lambeir AM, Scharpe S Ref: Journal of Immunological Methodsods, 189:99, 1996 : PubMed
The leukocyte differentiation antigen CD26 identified as dipeptidyl peptidase IV.(EC 3.4.14.5), cleaves off N-terminal dipeptides from peptides when a proline or alanine is located at the penultimate position. Seminal plasma and especially prostasomes, prostate-derived organelles which occur freely in seminal plasma, contain high amounts of CD26/dipeptidyl peptidase IV and therefore are suitable sources for the purification of the protein. The use of adenosine deaminase (EC 3.5.4.4) affinity chromatography for its purification is described. CD26/dipeptidyl peptidase IV was purified from human seminal plasma and prostasomes by a two step procedure. Ion exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase-Sepharose resulted in the pure, native protein with an overall yield ranging from 35 to 55%. The N-terminal sequence of the amphiphilic enzyme purified from human prostasomes was determined to be Met-Lys-Thr-Pro-Trp-Lys-Val-Leu. The preparation obtained was free of contaminating aminopeptidase activity and proved to be very stable (up to 1 month at 37 degrees C). The calf intestinal adenosine deaminase we used is commercially available and can be employed for the purification of human, bovine and rabbit CD26/dipeptidyl peptidase IV. High affinity binding of porcine dipeptidyl peptidase IV was not observed. The availability of a source with high specific activity and the introduction of adenosine deaminase affinity chromatography permits the rapid purification of milligram quantities of natural mammalian CD26/dipeptidyl peptidase IV.
Title: The purification, characterization and analysis of primary and secondary-structure of prolyl oligopeptidase from human lymphocytes. Evidence that the enzyme belongs to the alpha/beta hydrolase fold family Goossens F, De Meester I, Vanhoof G, Hendriks D, Vriend G, Scharpe S Ref: European Journal of Biochemistry, 233:432, 1995 : PubMed
Prolyl oligopeptidase was isolated and purified to homogeneity from human lymphocytes, yielding a specific activity of 7780 mU/mg. The molecular mass using size-exclusion chromatography matches the 76 kDa obtained by SDS/PAGE. This provides evidence that prolyl oligopeptidase is a monomer. The isoelectric point is 4.8 as judged by isoelectric focusing in free solution. Di-isopropyl fluorophosphate and phenylmethylsulphonyl fluoride completely abolish the activity, classifying the enzyme as a serine proteinase. The inhibition by p-chloromercuribenzoic acid indicates the importance of a free sulfhydryl group near the active-site. alpha 1-Casein and ornithine decarboxylase, two proteins containing a PEST sequence, inhibit prolyl oligopeptidase, but were not hydrolyzed. This demonstrates that prolyl oligopeptidase is not participating in the metabolism of proteins according to a PEST-dependent pathway. alpha 1-Antitrypsin partially inhibits the enzyme but in contrast, aprotinin does not. Its inability to cleave corticotropin-releasing factor, ubiquitin, albumin and aprotinin, together with the hydrolysis of bradykinin between Pro7-Arg8 confirms the affinity of prolyl oligopeptidase for small peptides. Multiple sequence alignment does not reveal any similarity with proteases of known tertiary structure. Secondary-structure prediction displays striking similarity with dipeptidyl peptidase IV and acylaminoacyl peptidase. Two characteristic features of the members of the prolyl oligopeptidase family of serine proteases are high-lighted: the linear arrangement of the catalytic triad is nucleophile-acid-base and the proteolytic cleavage releasing the catalytically active C-terminal region of around 500 amino acids from the N-terminal sequence. Secondary structure prediction and comparison of the active-site of serine proteinases with known three-dimensional coordinates prove that Asp641 is the third member of the catalytic triad. The secondary structural organization of the protease domain of prolyl oligopeptidase is in accordance with the alpha/beta hydrolase fold.
Title: Purified and cell-bound CD26: enzymatic inhibition, antibody binding profile, and expression on T cells in relation to other surface markers Scharpe S, De Meester I Ref: Verh K Acad Geneeskd Belg, 56:537, 1994 : PubMed
The CD26 activation antigen (Ag), which is expressed on a subpopulation of human T cells, has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). We investigated some molecular and inhibition characteristics as well as the monoclonal antibody (mAb)-binding profile of this molecule purified from human lymphocytes. Among the antibodies we explored, two, anti-BT5/9 and anti-TA5.9, exhibited a high affinity for both purified and cell-bound CD26 Ag. Their significance in the study of immunologic memory and lymphocyte activation is discussed in relation to other markers of lymphocyte activation. Among 4 types of inhibitors studied, Pro-boroPro proved to be the most promising substance for further research on the physiological role of dipeptidyl peptidase IV (CD26).
The human cDNA encoding prolyl endopeptidase, a cytoplasmic endoprotease which hydrolyses the peptide bond at the C-terminal side of proline, was sequenced. After the isolation of the 3' terminal fragment of the pep cDNA sequence from a human lymphocyte cDNA library, an approach based on the polymerase chain reaction (PCR) was undertaken to obtain the complete pep cDNA. Overlapping DNA fragments were generated by PCR from cDNA synthesized from human lymphocyte mRNA. The DNA fragments were subcloned and sequenced. The complete cDNA is 2562 nucleotides (nt) in length and contains an open reading frame coding for a protein of 710 amino acids (aa). Comparison of the primary PEP sequences from human lymphocyte and pig brain shows 97% identify. The aa sequence analysis shows homology with bacterial PEPs and with protease II from Escherichia coli. Asp641 probably participates in the active site of PEP.
An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), produced by the fungus Rhizopus javanicus was purified to homogeneity using an expeditious two-step isolation method. The enzyme, with a molecular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, consists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, respectively. The purified lipase was digested using chemical and enzymatical procedures: CNBr cleavage, partial acid hydrolysis, and proteolytic cleavage by means of trypsin. Amino-acid sequencing of the resulting peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenzymes but processed differently. These Rhizopus lipases show 54% identity with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus lipase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably similar distribution of hydrophobic amino acids at their surface. The tryptophan in the center of the helical lid covering the active site of Rhizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.
