Dipeptidyl Aminopeptidase IV Research Articles

Enzymatic activity of DPIV and renin–angiotensin system (RAS) proteases in patients with left ventricular dysfunction and primary prevention implantable cardioverter/defibrillator (ICD)

BackgroundPatients (pts) with severely decreased left ventricular ejection fraction (LV-EF ≤35%) are at high risk for sudden cardiac death (SCD). We sought to investigate, if pts with primary prevention ICD hold alterations in enzyme-activities of the dipeptidyl-aminopeptidase IV (DPIV) and the renin–angiotensin system (RAS) before VT/VF occurrence. Methods57 Pts (53 male, mean age 64.9 [42–84] years, mean LV-EF 26±5%) with ischemic (n=49) or non-ischemic cardiomyopathy (n=8) who had received an ICD/CRT-D for primary prevention, were included. Pts were assessed for appropriate ICD intervention for VT/VF during a mean follow-up of 365±90days. Serum levels of dipeptidyl-aminopeptidase IV (DPIV), aminopeptidase N (APN), aminopeptidase B (APB), insulin-regulated aminopeptidase (IRAP), and angiotensin-converting enzyme 2 (ACE2) were determined. ResultsPts with appropriate ICD intervention (n=16) had higher serum activities of IRAP (mean difference=12.681pkat/mL; p=0.007), and DPIV (mean difference=117.557pkat/mL; p=0.032) than pts without appropriate ICD intervention. Furthermore, ACE2 activity was significantly higher (median: 223.7RFU/smL vs. 169.10RFU/smL; p=0.037). A Cox regression analysis indicated DPIV activity >50th centile to have a hazard ratio (HR) of 5.955 (CI 95%: 1.670–21.241; p=0.006) for prediction of appropriate ICD intervention. In a multivariate Cox regression model, DPIV and IRAP >50th centile remained predictive for appropriate ICD intervention. ConclusionOur prospective study shows that pts with primary prevention ICD, who receive appropriate ICD intervention during follow-up, can be identified by elevated activities of DPIV and several RAS proteases. Hence, theses biomarkers seem to be of prognostic relevance in a primary prevention collective. Our data has to be proven in larger cohorts.

W1785 Loss of NLRP3 Signaling Results in Altered Dendritic Cells (DC) Population and Impaired Induction of Regulatory T Cells (Treg): Implications for Intestinal Inflammation

Background: We recently reported that proper blood processing using the RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls, dilution) method is of key importance before measuring plasma concentration and molecular forms of labile hormones such as acyl ghrelin, cholecystokinin-58, gastrin-releasing peptide and somatostatin-28 (Endocrinology 150:5113-8; 2009). Active glucagon-like peptide-1 (GLP1(7-36)amide and GLP-1(7-37)) can be produced in blood ex-vivo by an endopeptidase cleaving GLP-1(1-36) and GLP-1(1-37) or destroyed by the action of dipeptidyl aminopeptidase IV (DPP-IV) to produce GLP-1(9-36)amide or GLP-1(9-37). Aim: To investigate whether different blood processing influences the detection of active and total GLP-1 plasma levels. Methods: Blood was obtained between 8-9 AM from male Sprague-Dawley rats (300-350g) either fed ad libitum or 24 h fasted (n=5-6/group) and processed in three ways: 1. collected in ice cold tubes containing EDTA (7.5%, 10μl/1ml) followed by centrifugation, 2. same as 1. plus DPP-IV inhibitor (diprotin A, 50μM) or 3. RAPID method without isotopic standards (RAPD). Samples were stored at -80°C until radioimmunoassays for total and active GLP1. Results: Active GLP-1 levels did not change among metabolic conditions irrespective of the blood processing (P 0.05). Plasma formation and DPP-IV inhibition gave comparable results for circulating total GLP-1 plasma levels of ad libitum fed rats while RAPD processing resulted in lower levels (P 0.05). In a preliminary experiment ~50% of iodinated GLP-1(7-36) was lost in Falcon tubes as used in the RAPD method. Conclusion: Active GLP-1 does not change under the metabolic conditions tested. The two plasma formation methods yield similar results for the measurement of circulating active and total GLP-1, whereas RAPD processing unexpectedly results in lower plasma levels of total and active GLP-1. The use of isotopic exogenous standards will be important to determine recovery and peptide structure alteration ex-vivo possibly leading to changed blood levels of active or total GLP-1. NIDDK 41301/70851/73152

Gender and age-dependent differences in the bradykinin-degradation within the pericardial fluid of patients with coronary artery disease

Cardiovascular diseases (CVD) and, in particular, coronary artery disease (CAD) are the leading causes of death in developed countries, especially in the elderly population. Males exhibit a higher risk for cardiovascular events than women. The pericardial fluid (PF) is in direct contact with the epicardial sections of the coronary arteries and the perimyocardium. A systematic analysis of gender-specific or age-related differences in angiotensin-related pathways like bradykinin metabolism however, has not been performed in the PF so far. Therefore, the amounts of angiotensin-converting enzyme (ACE) and the rate of the degradation of bradykinin (BK) and the amounts/activity of major BK-degrading enzymes, aminopeptidase N (APN) and dipeptidyl-aminopeptidase IV (DPIV), were assessed in the pericardial fluid (PF) of 44 patients undergoing coronary artery bypass grafting. We found BK being degraded within the PF. Interestingly, there was an age-dependent decrease in the amounts of ACE protein in women. In elderly women, ACE/APN and ACE/DPIV ratios were substantially reduced to 41.4% or 29.4% respectively (p<0.05). In contrast, an age-dependent decline of ACE protein and ACE/protease ratios were not found in men. In men and women, total BK degradation correlated with age (r=0.5; p=0.021) further supporting a switch in BK metabolising enzymes in elderly women. Thus, we can show age- and gender-dependent differences in BK metabolism within the PF in patients with coronary artery disease. The present finding that the expression of ACE is lowest in elderly women, despite the presence of similar BK degradation, might help to explain the potentially reduced therapeutic effects of ACE inhibitors in elderly women.

Recent insights into the role of dipeptidyl aminopeptidase IV (DPIV) and aminopeptidase N (APN) families in immune functions

In the past, different research groups could show that treatment of immune cells with inhibitors of post-proline splitting dipeptidyl aminopeptidases leads to functional changes in the immune system consistent with immunosuppression. This is due to the inhibition of proliferation of lymphocytes and the production of inflammatory cytokines of the TH1, TH2, and TH17, cells as well as the induction of immunosuppressive cytokines, such as transforming growth factor-beta1 (TGF-beta1) and interleukin (IL)-1RA. Until recently, most of the effects of these inhibitors on immune functions were attributed to the inhibition of dipeptidyl aminopeptidase IV (DPIV/CD26). With the identification of new peptidases of the DPIV family (DASH) with the same or similar substrate specificity [fibroblast activation protein (FAP), DP8/9], the question arose whether and to what extent the inhibition of intracellularly localized enzymes, DP8 and DP9, contribute to the observed immunosuppression. In addition, members of the aminopeptidase N (APN) family are also involved in the regulation of immune functions. Hence, the concept of a combined targeting of both families of peptidases for treatment of inflammatory diseases is a promising strategy. Summarizing data obtained from the usage of different non-selective and selective inhibitors of DPIV, DP8/9, FAP, and DPII, this review provides evidence that in addition to DPIV, DP8/9 also regulate the immune response via modulation of cell cycle progression and cytokine production. The strongest and most consistent effects in vitro were, however, observed with non-selective inhibitors for the suppression of DNA synthesis and cytokine production. Similar effects were provoked by APN inhibitors, which were also found to suppress DNA synthesis and the production of inflammatory cytokines in vitro. However, different mechanisms and signaling pathways appear to mediate the cellular effects resulting from the inhibition of either APN or DPIV family members. In particular, members of the APN family uniquely influence the function of CD4+CD25+ regulatory T-cells. Consequently, the concomitant inhibition of both APN and DPIV enzyme families by means of two separate inhibitors or by binary inhibitors with specificity for both enzyme families (PETIR, peptidase targeted immunoregulation) synergistically affects immune cells on the level of cell cycle regulation, suppression of TH1, TH2, and TH17 cytokines as well as the activation of regulatory T-cells. Besides leukocytes, dermal cells as sebocytes, keratinocytes, and fibroblasts are also targeted by these inhibitors. This strongly suggests a broad potential of the multiple anti-inflammatory effects of PETIR in treatment of chronic inflammatory diseases, such as autoimmune diseases, allergies, and transplant rejections, as well as of inflammatory skin diseases, such as acne, psoriasis, rosacea or atopic dermatitis. The first active dual inhibitor, IP10.C8, has been developed by IMTM for the treatment of inflammatory skin diseases and has just entered the first phase II study.

An array of brain protease activities and their modulation by the toxic effects of cyanamide

Brain proteolytic enzymes have been implicated in a variety of disorders though most studies have focused on a few components such as the ATP-ubiquitin pathway. In this study we assayed a broad range of representative proteolytic enzyme types, to address the hypothesis that their activities are perturbed in cyanamide toxicity. We dosed maleWistar rats (approximately 0.1 kg BW) with either saline (0.15 mol/l NaCl) or cyanamide, an inhibitor of aldehyde dehydrogenase (ALDH) (0.5 mmol/kg BW; ip). After 3 h rats were killed and cerebellum dissected. Using fluorimetry, we measured the activities of the cytoplasmic enzymes alanyl-aminopeptidase (ALAAP), arginyl-aminopeptidase (ARGAP), leucylaminopeptidase (LEUAP), dipeptidyl-aminopeptidase IV (DIA-IV), tripeptidyl aminopetidase (TRI-AP) and proline endopeptidase (PROAP), as well as the lysosomal enzymes cathepsins B (CATHB), D (CATHD), H (CATHH) and L (CATHL), dipeptidyl aminopeptidases I (DIA-I) and II (DIA-II) and proteasomal chymotrypsin-like (PRCHY) and trypsin-like (PRTRP) activities (Table 1). There was a wide range of proteolytic activities (Table 1). The highest activity was found in arginyl aminopeptidase whereas proteosomal activities were low. In response to cyanamide dosage in vivo, there were significant increases in the in vitro activities of cerebellum DIA-IV (P < 0.05). There were also reductions in DIA-I and CATHL (P < 0.001). In conclusions, impaired proteolysis may contribute to, or reflect, the cellular toxicity of cyanamide.

Molecular Mechanism for Connective Tissue Destruction by Dipeptidyl Aminopeptidase IV Produced by the Periodontal PathogenPorphyromonas gingivalis

Porphyromonas gingivalis is a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion of P. gingivalis to fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.

Peptidase activity of dipeptidyl aminopeptidase IV produced by Porphyromonas gingivalis is important but not sufficient for virulence.

Porphyromonas gingivalis is a pathogen associated with adult periodontitis, which is a chronic inflammatory disease characterized by breakdown of the periodontal tissue. Dipeptidyl aminopeptidase IV (DPPIV) produced by P. gingivalis has been considered to be a potential virulence factor based on the finding that the virulence was reduced by disruption of the gene (dpp ) coding for DPPIV. In the present study, we constructed a shuttle vector that is mobilized from Escherichia coli to P. gingivalis and is maintained stably in both bacteria, and we showed that the virulence was restored by introducing the cloned wild-type dpp gene into the null mutant of P. gingivalis using our vector system. To assess the implications of the peptidase activity in the virulence, mutant DPPIV with the catalytic Ser mutagenized to Ala (DPPSA) was produced. The P. gingivalis strain expressing DPPSA exhibited an intermediate virulence between the strain expressing wild-type DPPIV and the strain harboring a vector. From these results, it is suggested that peptidase activity is very important but not sufficient for virulence.

The effect of endomorphins on the release of 3H-norepinephrine from rat nucleus tractus solitarii slices

We used two, 3-min field stimulation cycles 30 min apart (S1, S2) in 3H-norepinephrine-loaded, superfused rat nucleus tractus solitarii-dorsal motor vagal nucleus (NTS-DVN) slices. The stimulation-induced release was expressed as the area above the baseline. Drugs were introduced 12 min before S2 and drug actions were characterized in terms of alterations of S2/S1 ratios. The S2/S1 ratio was 1.047 (0.946–1.159, n=4, geometric mean and 95% confidence interval) in controls and 0.336 (0.230–0.490, n=3), 0.726 (0.590–0.892, n=4), 0.613 (0.594–0.683, n=4) and 0.665 (0.500–0.886, n=4) in the presence of 10 −6 M clonidine, d-Ala 2,MePhe 4,Gly 5-ol-enkephalin (DAMGO), endomorphin-1 (Tyr–Pro–Trp–Phe–NH 2, EM-1) and -2 (Tyr–Pro–Phe–Phe–NH 2, EM-2) [the latter two in the presence of 10 −4 M diprotin A, an inhibitor of dipeptidyl-aminopeptidase IV (DAP-IV) enzyme]. The effect of DAMGO at 10 −5 M was significantly higher than at 10 −6 M, whereas the effect of endomorphins did not differ at the two concentration levels. Diprotin A potentiated only very modestly the action of endomorphins. These data (a) confirm the presence of functional μ-opioid receptors in the vagal complex, (b) render it likely that the enzymic degradation of endomorphins is not a highly effective process in brain slices and (c) may suggest that the apparent ceiling in the effect of endomorphins might be related to their partial agonist property.

Histopathological studies on virulence of dipeptidyl aminopeptidase IV (DPPIV) of Porphyromonas gingivalis in a mouse abscess model: use of a DPPIV-deficient mutant.

To elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence of Porphyromonas gingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells.

Effects of renal sorbitol accumulation on urinary excretion of enzymes in hyperglycaemic rats.

We studied the effects of epalrestat, a specific inhibitor of aldose reductase, on renal sorbitol accumulation and the resulting urinary enzyme excretion in hyperglycaemic rats. The activities of proximal tubule-derived enzymes such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), gamma-glutamyltranspeptidase (GGT) and dipeptidyl aminopeptidase IV (DAPIV) in urine were determined in five groups of male Wistar rats (each n = 7): (a) 0.9% saline-loaded, (b) 10% glucose-loaded, (c) 10% glucose-loaded with epalrestat pretreatment, (d) 10% mannitol-loaded and (e) 10% mannitol-loaded with epalrestat pretreatment. Epalrestat was given mixed in chow at a dose of 50 mg/kg body weight. Urinary NAG, AAP, GGT and DAPIV activities were significantly increased (P<0.005, P<0.05, P<0.01, P<0.01, respectively) by the induction of hyperglycaemia. In contrast, enzyme excretion was not increased in the mannitol- or saline-loaded groups. Pre-treatment with epalrestat completely prevented the increased urinary excretion of NAG, AAP and GGT. At the end of the infusion study, renal cortical glucose concentrations of the glucose-loaded groups with and without epalrestat pretreatment were approximately fivefold higher than those of the mannitol- or saline-loaded groups (P<0.005 each). Renal cortical sorbitol concentrations of the glucose-loaded group was also approximately twofold higher than those of the mannitol- or saline-loaded groups (P<0.01 each). However, in the group that received both glucose and epalrestat, renal cortical sorbitol concentration was not increased. These results suggest that accumulation of intracellular sorbitol leads to proximal tubular cell dysfunction and abnormal enzymuria.

Enzymatic properties of dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis and its participation in virulence.

Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) from P. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated from P. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

The activities of six exo-and endopeptidases in the substantia nigra, neostriatum, and cortex of the rat brain.

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and gamma-Glutamyl transpeptidase (gammaGTP), and two endopeptidases: Postproline endopeptidase (PEP) and "Trypsin-like" peptidase ("T-L" P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) gammaGTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a "Trypsin-like" peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.

Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function.

Dipeptidyl peptidase IV (DP-IV; dipeptidyl-peptide hydrolase, EC 3.4.14.5) is a serine protease with a specificity for cleaving Xaa-Pro dipeptides from polypeptides and proteins. It is found in a variety of mammalian cells and tissues, including those of lymphoid origin where it is found specifically on the surface of CD4+ T cells. Although the functional significance of this enzyme has not been established, a role in T-cell activation and immune regulation has been proposed. Here we report that Ala-boroPro and Pro-boroPro, where boroPro is the alpha-amino boronic acid analog of proline, are potent and specific inhibitors of DP-IV, having Ki values in the nanomolar range. Blocking the N terminus of Ala-boroPro abolishes the affinity of this inhibitor for DP-IV, while removal of the N-terminal residue, to give boroPro, reduces the affinity for DP-IV by 5 orders of magnitude. The dipeptide boronic acids exhibit slow-binding kinetics, while boroPro does not. We also report here that low concentrations of Pro-boroPro inhibit antigen-induced proliferation and interleukin 2 production in murine T-cell lines but do not inhibit the response of these T cells to the mitogen concanavalin A. These results indicate that DP-IV plays a role in antigen-induced, but not mitogen-induced, activation of T lymphocytes.

Comparative cytochemical study of dipeptidyl aminopeptidase (DAP) II and IV in normal and malignant haemic cells.

Modified cytochemical methods were used to show dipeptidyl aminopeptidases (DAP) II and IV in peripheral blood buffy coat preparations and bone marrow smears. In 23 normal buffy coats both enzymes were confined to lymphocytes. DAP II was found in T and B lymphocytes (about 80%) while DAP IV was restricted to T lymphocytes only (around 46%). In 11 normal bone marrows DAP II was found in 53% of the lymphocytes, as well as in plasma cells, macrophages, and occasional myeloblasts. DAP IV was found only in lymphocytes (around 32%). DAP II activity, but not DAP IV activity, was present in all of the mast cells in a case of systemic mastocytosis. Whereas DAP II was found, to a variable extent, in leukaemic myeloblasts, monoblasts, proerythroblasts, and in megakaryoblasts in 52 cases of acute myeloid leukaemia, DAP IV was not shown. Variable positivity to DAP II and DAP IV was found in the lymphoblasts in seven cases of acute lymphoblastic leukaemia, in 14 cases of B chronic lymphocytic leukaemia, and in three cases of non-Hodgkin's lymphoma. DAP II activity was variable compared with DAP IV activity, which was constantly reduced. Virtually all of the myeloma cells (96%) all of the myeloma cells (96%) in five cases of multiple myeloma and two cases of plasma cell leukaemia were DAP II positive and DAP IV negative. In 10 cases of hairy cell leukaemia most hairy cells were positive to DAP II (74%) with no demonstrable DAP IV activity. In a single case of Sézary's syndrome around 90% of the helper T cells were positive to DAP II with no DAP IV activity.

Isolation and characterization of dipeptidyl peptidase IV from human meconium: Functional role of β-casomorphins

Dipeptidyl aminopeptidase IV (DAP-IV) (EC 3.4.14.1) was purified from meconium particles sedimenting at 105000 × g. Its molecular properties and activity on synthetic and natural substrates (casomorphin and procasomorphin) were investigated.

Purification of dipeptidyl-aminopeptidase IV from human kidney by anti dipeptidyl-aminopeptidase IV affinity chromatography

Dipeptidyl-aminopeptidase IV (DAP-IV) was purified 850 fold with a yield of 16.5% from human kidney cortex. Only two chromatographic procedures of DEAE-cellulose and anti DAP-IV Sepharose were used. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Among various substrates with the structure of X-Y-p-nitroanilide, Lys-Pro-Gly-Pro- and Arg-Pro-p-nitroanilides were hydrolyzed very fast by DAP-IV. Optimum pH of DAP-IV in human kidney was pH 8.7, Km value for Gly-Pro-pNA was 2.51 +/- 0.01 x 10(-4) M and V max was 66.6 +/- 0.7 mumol/min/mg protein. Diisopropylfluorophosphate completely inhibited DAP-IV at 0.1 mM. However, the enzyme was almost unaffected by N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, phenylmethylsulfonylfluoride, and several metals. The amino acid composition of DAP-IV purified from human kidney was similar to that of DAP-IV purified from pig kidney, liver and intestine. These results indicate that the properties of DAP-IV purified from human kidney are almost same as those from pig kidney.