Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.     

CD26 expression determines lung metastasis in mutant F344 rats: involvement of NK cell function and soluble CD26

The association between CD26 expression, tumor cell adhesion, metastasis, and natural killer (NK) cell function was investigated in a CD26 mutant Fischer 344 (F344/DuCrj) substrain from Japanese breeders (F344JAP) in comparison with wild-type F344 substrains from US (F344/Crl) and Hannover (HAN; F344/Ztm) breeders. F344JAP rats lack the dipeptidyl peptidase IV activity of CD26 and show a reduced cell surface expression of the mutated CD26 glycoprotein. In vivo adhesion of vital dye-labeled MADB106 tumor cells, tumor colonization, CD26 enzymatic activity, and CD26 immunoreactivity in lungs and soluble CD26-like protein expression in serum were markedly reduced in F344JAP rats. These findings demonstrate that CD26 protein expression exerts a key role in lung metastasis. In addition, NK cell cytotoxicity against MADB106 cells was diminished in the mutant F344 substrain, suggesting that CD26 enzymatic activity sustains NK cytotoxicity. Interestingly, tumor cells lacked CD26 immunoreactivity in vitro, but displayed CD26 immunoreactivity in situ after in vivo inoculation as well as after incubation with rat serum, indicating that soluble CD26-like protein assembles in tumor cells during in vivo passage, which may interact with the process of tumor adhesion and metastasis. Overall, these findings indicate that altered expression and function of a single enzyme-the CD26 protein--can drastically change the outcome of metastatic disease.     

Expression,subcellular localisation,and possible roles of dipeptidyl peptidase 9 (DPP9) in murine macrophages

Dipeptidyl peptidase 9 (DPP9) is a peptidase of the DPPIV gene family, and its role in immune responses has been reported. In this study, we compared the messenger RNA expression profile of DPP9 to that of the related DPP8 and DPPIV in murine haematopoietic and lymphatic tissues. A similar order of expression levels was observed for all 3 peptidases: peritoneal macrophages < bone marrow < spleen ≤ lymph nodes. Also, we examined the subcellular localisation of DPP9 and its possible role(s) in J774 cell line of macrophage origin. DPP9 was dominantly expressed intracellularly. DPPIV‐like enzymatic activity was mostly present in cytoplasm, but also in cell membranes and organelles/vesicles. Decreased expression of DPP9 was observed upon activation of J774 cells by combined treatment with interferon gamma and lipopolysaccharide. Changes induced by DPP9 gene silencing in J774 cells suggest possible role of DPP9 in regulation of proliferation and activation status. The colocalisation of DPP9 with endocytosed DQ‐OVA demonstrated in endosomes of J774 cells might suggest the role of DPP9 in peptide processing within endosomal/vesicular compartment.     

The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age

Neuropeptide Y (NPY)-induced modulation of the immune and inflammatory responses is regulated by tissue-specific expression of different receptor subtypes (Y1–Y6) and the activity of the enzyme dipeptidyl peptidase 4 (DP4, CD26) which terminates the action of NPY on Y1 receptor subtype. The present study investigated the age-dependent effect of NPY on inflammatory paw edema and macrophage nitric oxide production in Dark Agouti rats exhibiting a high-plasma DP4 activity, as acknowledged earlier. The results showed that NPY suppressed paw edema in adult and aged, but not in young rats. Furthermore, plasma DP4 activity decreased, while macrophage DP4 activity, as well as macrophage CD26 expression increased with aging. The use of NPY-related peptides and Y receptor-specific antagonists revealed that anti-inflammatory effect of NPY is mediated via Y1 and Y5 receptors. NPY-induced suppression of paw edema in young rats following inhibition of DP4 additionally emphasized the role for Y1 receptor in the anti-inflammatory action of NPY. In contrast to the in vivo situation, NPY stimulated macrophage nitric oxide production in vitro only in young rats, and this effect was mediated via Y1 and Y2 receptors. It can be concluded that age-dependant modulation of inflammatory reactions by NPY is determined by plasma, but not macrophage DP4 activity at different ages.     

Localization,transmission, spontaneous mutations,and variation of function of the Dpp4 (Dipeptidyl-peptidase IV; CD26) gene in rats

Dipeptidyl-peptidase IV (DPPIV) is involved in endocrine and immune functions via cleavage of regulatory peptides with a N-terminal proline or alanine such as incretins, neuropeptide Y, or several chemokines. So far no systematic investigations on the localization and transmission of the Dpp4 gene or the natural variations of DPPIV-like enzymatic function in different rat strains have been conducted. Here we mapped the Dpp4 gene to rat chromosome 3 and describe a semi-dominant mode of inheritance for Dpp4 in a mutant F344/DuCrj(DPPIV-) rat substrain lacking endogenous DPPIV-like activity. This mutant F344/DuCrj(DPPIV-) rat substrain constantly exhibits a nearly complete lack of DPPIV-like enzymatic activity, while segregation of DPPIV-like enzymatic activity was observed in another DPPIV-negative F344/Crl(Ger/DPPIV-) rat substrain. Screening of 12 different inbred laboratory rat strains revealed dramatic differences in DPPIV-like activity ranging from 11 mU/microl (LEW/Ztm rats) to 40 mU/microl (BN/Ztm and DA/Ztm rats). A lack of DPPIV-like activity in F344 rats was associated with an improved glucose tolerance and blunted natural killer cell function, which indicates the pleiotropic functional role of DPPIV in vivo. Overall, the variations in DPPIV-like enzymatic activity probably represent important confounding factors in studies using rat models for research on regulatory peptides.     

Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects

The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)?-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO?)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.     

Dose-dependent recruitment of CD25+ and CD26+ T cells in a novel F344 rat model of asthma

The ovalbumin (OVA)-induced airway inflammation in rats is a commonly used model to explore the pathobiology of asthma. However, its susceptibility varies greatly between rat strains, and presently Brown Norway (BN) rats are preferentially used. Since recruitment of T cells to the lungs depends on the CD26 (dipeptidyl peptidase IV, DPPIV) expression, Fischer 344 strain (F344) rats are a highly relevant rat strain, in particular because CD26-deficient substrains are available. To establish a F344 rat model of asthma, we challenged F344 rats using different doses of aerosolized antigen (0%, 1%, 2.5%, 5%, and 7.5% OVA) and compared these effects with intratracheal instillation of OVA (1.5 mg/0.3 ml). Asthmoid responsiveness was determined by analysis of early airway responsiveness (EAR), antigen-specific IgE levels, as well as airway inflammation including the composition of T cell subpopulations in the bronchoalveolar lavage (BAL) and lung tissue with special respect to the T cell activation markers CD25 and CD26. Even low allergen doses caused allergen-specific EAR and increases of antigen-specific IgE levels. However, EAR and IgE levels did not increase dose dependently. Higher concentrations of OVA led to a dose-dependent increase of several immunological markers of allergic asthma including an influx of eosinophils, T cells, and dendritic cells. Interestingly, a dose-dependent increase of CD4(+)/CD25(+)/CD26(+) T cells was found in the lungs. Summarizing, we established a novel F344 rat model of aerosolized OVA-induced asthma. Thereby, we found a dose-dependent recruitment of cellular markers of allergic asthma including the activated CD4(+)/CD25(+)/CD26(+) T cell subpopulation, which has not been described in asthma yet.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

E2F-1 regulation by an unusual DNA damage-responsive DP partner subunit

E2F activity is negatively regulated by retinoblastoma protein (pRb) through binding to the E2F-1 subunit. Within the E2F heterodimer, DP proteins are E2F partner subunits that allow proper cell cycle progression. In contrast to the other DP proteins, the newest member of the family, DP-4, downregulates E2F activity. In this study we report an unexpected role for DP-4 in regulating E2F-1 activity during the DNA damage response. Specifically, DP-4 is induced in DNA-damaged cells, upon which it binds to E2F-1 as a non-DNA-binding E2F-1/DP-4 complex. Consequently, depleting DP-4 in cells re-instates E2F-1 activity that coincides with increased levels of chromatin-bound E2F-1, E2F-1 target gene expression and associated apoptosis. Mutational analysis of DP-4 highlighted a C-terminal region, outside the DNA-binding domain, required for the negative control of E2F-1 activity. Our results define a new pathway, which acts independently of pRb and through a biochemically distinct mechanism, involved in negative regulation of E2F-1 activity.     

Biochemical properties and expression profile of human prolyl dipeptidase DPP9

Dipetidyl peptidase 9 (DPP9) is a prolyl dipeptidase preferentially cleaving the peptide bond after the penultimate proline residue. The biological function of DPP9 is unknown. In this study, we have significantly improved the yield using Strep·Tactin^® purification system and characterized the biochemical property of DPP9. Moreover, the dimer interaction mode was investigated by introducing a mutation (F842A) at the dimer interface, which abolished the enzymatic activity without disrupting its quaternary structure. Furthermore, DPP9 was found ubiquitously expressed in fibroblasts, epithelial, and blood cells. Surprisingly, contrary to previous report, we found that the expression levels of DPP8 and DPP9 did not change upon the activation of the PBMC or Jurkat cells. These results indicate that the biochemical property of DPP9 is very similar to that of DPP8, its homologous protease. DPP9 and DPP8 are likely redundant proteins carrying out overlapping functions in vivo.     

Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.     

Dominant Expression of Rat Prostanoid DP Receptor mRNA in Leptomeninges, Inner Segments of Photoreceptor Cells, Iris Epithelium, and Ciliary Processes

Abstract: Prostaglandin (PG) D₂ is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD₂ among the various prostanoids. In this study, we cloned the full-length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT-PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin-type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT-PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD₂.     

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.     

D-4F, an apoA-1 mimetic, decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma

Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DWFKAFYDKVAEKFKEAFNH(2) (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)β-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michael's adducts in lungs of OVA-sensitized mice. 4F decreased TGFβ-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.     

Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries

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.