Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats.

The expression of dipeptidyl peptidase 4 (DP4, CD26) affects T-cell recruitment to lungs in an experimental rat asthma model. Furthermore, the gene of the structural homologous DP10 represents a susceptibility locus for asthma in humans, and the functional homologous DP8/9 are expressed in human leukocytes. Thus, although several mechanisms may account for a role of DP4-like peptidases in asthma, detailed information on their anatomical sites of expression and function in lungs is lacking. Therefore, bronchi and lung parenchyma were evaluated using immunohistochemistry and histochemical/enzymatic activity assays, as well as quantitative real-time PCR for this family of peptidases in naïve and asthmatic rat lungs derived from wild-type F344 and DP4-deficient F344 rat strains. Surprisingly, results show not only that the induction of experimental asthma increases DP4 enzymatic activity in the bronchoalveolar lavage fluid and parenchyma, but also that DP8/9 enzymatic activity is regulated and, as well as the expression of DP10, primarily found in the bronchial epithelium of the airways. This is the first report showing a differential and site-specific DP4-like expression and function in the lungs, suggesting a pathophysiologically significant role in asthma. (J Histochem Cytochem 56:147–155, 2008)     

CD26/DPPIV cell membrane expression and DPPIV activity in plasma of patients with acute leukemia

CD26/DPPIV (dipeptidil peptidase IV) displays an array of diverse functional properties, with a role in the development of several human cancers. This enzyme is found mainly anchored in the membrane of cells although it also has an enzymatically active plasma isoform. The regulation of biological activities of cytokines by DPP IV activity has a potential role in the homeostatic regulation of hematopoiesis. In this study, we analyzed the CD26 antigen cell membrane expression by flow cytometry and the DPPIV activity in plasma of patients of acute leukemia. The results showed that the plasma DPPIV activity is significantly higher in leukemia patients and could be 100% inhibited by Januvia? (Merck Sharp & Dohme) a selective DPPIV inhibitor. Although CD26 expression on immune cells were not leukemia-dependent the analysis of the correlation between CD26 expression and the DPPIV plasma activity were statistically significant (p < 0.01) in acute lymphoid leukemia (B-ALL and T-ALL).     

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.     

N-linked glycosylation of dipeptidyl peptidase IV (CD26): effects on enzyme activity, homodimer formation, and adenosine deaminase binding

The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.     

Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor

CD26 is a T cell surface molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular region. In addition to its membrane form, CD26 exists in plasma as a soluble form (sCD26), which is the extracellular domain of the molecule thought to be cleaved from the cell surface. In this paper, we demonstrate that sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPPIV enzyme activity. We also show that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. Our findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.     

The 3D structure of rat DPPIV/CD26 as obtained by cryo-TEM and single particle analysis

We present the three-dimensional structure of rat DPPIV/CD26, as determined by cryo-TEM and single particle analysis at a resolution of approximately 14A. The reconstruction confirms that the protein exists as a dimer, as predicted earlier. Since there are structural analogies to the serine peptidase POP, docking calculations of the two structures were performed. Although the docking showed a similar spatial organization (catalytic domain, beta-propeller, distal opening, central cavity), the detailed comparison revealed clear discrepancies. The most marked difference is a second (lateral) opening in DPPIV/CD26, which would enable direct access to the catalytic site. We therefore assume that substrate selectivity and binding rate are most probably driven by different mechanisms in DPPIV/CD26 and POP.     

Isolation of rugosin A,B and related compounds as dipeptidyl peptidase-IV inhibitors from rose bud extract powder

Dipeptidyl peptidase-IV (DPP-IV) is a protease responsible for the degradation of the incretin hormone. A number of DPP-IV inhibitors have been approved for use in the treatment of type 2 diabetes. While these inhibitors are effective for this treatment, methods for the prevention of this disease are also required as diabetes patient numbers are currently increasing rapidly worldwide. We screened the DPP-IV inhibitory activities of edible plant extracts with the intention of using these extracts in a functional food supplement for the prevention of diabetes. Rose (Rosa gallica) bud extract powder was a promising material with high inhibitory activity. In this study, seven ellagitannins were isolated as active compounds through activity-guided fractionations, and their DPP-IV inhibitory activities were measured. Among them, rugosin A and B showed the highest inhibitory activities and rugosin B was shown as the major contributing compound in rose bud extract powder.     

Purification and Properties of a Type II-like Dipeptidyl Peptidase from the Ruminal Peptidolytic Bacterium,Prevotella albensis M384

A dipeptidyl peptidase which hydrolyses the synthetic dipeptidyl peptidase (DPP) substrate, Ala₂- p -nitroanilide, was purified 193-fold from the ruminal peptidolytic bacterium, Prevotella albensis M384. The enzyme was a homodimer of molecular mass 91 kDa. Its activity against Ala₂- p -nitroanilide had optimal pH and temperature of 7.2 and 40°C respectively. Enzyme activity was inhibited by the serine protease inhibitors, PMSF and dichloroisocoumarin, but not by inhibitors of other categories of proteases. Synthetic substrates for DPP-1 (GlyArg- p -nitroanilide, GlyArg-4-methoxy-naphthylamide), DPP-3 (ArgArg-4-methoxynaphthylamide) and DPP-4 (GlyPro-4-methoxynaphthylamide) or for leucine or alanine aminopeptidase were not hydrolysed, nor were di- or tripeptides. N-Acetyl-Ala₂- p -nitroanilide was not hydrolysed. Oligopeptides with Ala, Ile, Ser or Val adjacent to the N-terminal amino acid were all hydrolysed, while peptides with basic or acidic residues in the same position were not. The purified DPP from P. albensis is therefore most similar in its catalytic properties to mammalian DPP-2.     

Synthesis of tetrapeptide p-nitrophenylanilides containing dehydroalanine and dehydrophenylalanine and their influence on cathepsin C activity.

Three dehydrotetrapeptides of rationally varying structure were prepared and tested as affectors of cathepsin C. These compounds appeared to be substrates of the enzyme, being equipotent with their classical counterparts. Thus, replacement of amino acid in a short peptide by corresponding dehydroamino acid does not prevent cathepsin C in recognizing dehydropeptide as its substrate.     

Angiostatin directly inhibits human prostate tumor cell invasion by blocking plasminogen binding to its cellular receptor, CD26

Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca(2+) signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel. We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the plasminogen 2epsilon-induced Ca(2+) signaling cascade is inhibited, the expression of matrix metalloproteinase-9 is suppressed, and invasion of Matrigel by 1-LN cells is blocked. Angiostatin 2epsilon is also the only angiostatin glycoform which is able to inhibit in vitro endothelial cell proliferation and tubule formation. These studies suggest that, in addition to its ability to inhibit tumor vascularization, angiostatin 2epsilon may also directly block tumor metastasis.     

Identification of cancer stem cell markers in human malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24⁺ cells proliferated by asymmetric cell division-like manner. In addition, CD9⁺ and CD24⁺ cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.     

Genetic variants of dipeptidyl peptidase IV are linked to the clinicopathologic development of prostate cancer

CD26/dipeptidyl peptidase IV (DPP4) is a multifunctional cell-surface glycoprotein widely found in many cell types, and a soluble form is present in body fluids. There is longstanding evidence indicating a tumour-promoting or -suppressive role of DPP4 in different cancer types. However, studies focusing on the impacts of genetic variants of DPP4 on cancers are very rare. Herein, we conducted a case–control study to evaluate whether single-nucleotide polymorphisms (SNPs) of DPP4 were associated with the risk or clinicopathologic development of prostate cancer (PCa). We genotyped four loci of DPP4 SNPs, including rs7608798 (A/G), rs3788979 (C/T), rs2268889 (T/C) and rs6741949 (G/C), using a TaqMan allelic discrimination assay in 704 PCa patients and 704 healthy controls. Our results showed that PCa patients with the DPP4 rs7608798 AG+GG genotype or rs2268889 TC+CC genotype had a higher risk of developing an advanced clinical primary tumour (cT) stage (adjusted odds ratio (AOR): 1.680, 95% confidence interval (CI): 1.062–2.659, p = 0.025; AOR: 1.693, 95% CI: 1.092–2.624, p = 0.018). Additionally, in The Cancer Genome Atlas (TCGA) database, we observed that lower DPP4 expression levels were correlated with higher Gleason scores, advanced cT and pathological stages, tumour metastasis, and shorter progression-free survival rates in PCa patients. Furthermore, overexpression of DPP4 suppressed migration/invasion of metastatic PC3 PCa cells. Our findings suggest that DPP4 levels may affect the progression of PCa, and the DPP4 rs7608798 and rs2268889 SNPs are associated with the clinicopathologic development of PCa in a Taiwanese population.     

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III

Abstract

Human dipeptidyl peptidase III (hDPP III) is a zinc-exopeptidase of the family M49 involved in final steps of intracellular protein degradation and in cytoprotective pathway Keap1-Nrf2. Biochemical and structural properties of this enzyme have been extensively investigated, but the knowledge on its contacts with other proteins is scarce. Previously, polypeptide aprotinin was shown to be a competitive inhibitor of hDPP III hydrolytic activity. In this study, aprotinin was first investigated as a potential substrate of hDPP III, but no degradation products were demonstrated by MALDI-TOF mass spectrometry. Subsequently, molecular details of the protein–protein interaction between aprotinin and hDPP III were studied by molecular modeling. Docking and long molecular dynamics (MD) simulations have shown that aprotinin interacts by its canonical binding epitope with the substrate binding cleft of hDPP III. Thereby, free N-terminus of aprotinin is distant from the active-site zinc. Enzyme-inhibitor complex is stabilized by intermolecular hydrogen bonding network, electrostatic and hydrophobic interactions which mostly involve constituent amino acid residues of the hDPP III substrate binding subsites S1, S1', S2, S2' and S3'. This is the first study that gives insight into aprotinin binding to a metallopeptidase.

Communicated by Ramaswamy H. Sarma     

3D structure of the CD26-ADA complex obtained by cryo-EM and single particle analysis

The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.     

Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.     

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.