Dipeptidyl peptidase (DPP) 4 (CD26, DPP4) is a multi-functional protein involved in T cell activation by co-stimulation via its association with adenosine deaminase (ADA), caveolin-1, CARMA-1, CD45, mannose-6-phosphate/insulin growth factor-II receptor (M6P/IGFII-R) and C-X-C motif receptor 4 (CXC-R4). The proline-specific dipeptidyl peptidase also modulates the bioactivity of several chemokines. However, a number of enzymes displaying either DPP4-like activities or representing structural homologues have been discovered in the past two decades and are referred to as DPP4 activity and/or structure homologue (DASH) proteins. Apart from DPP4, DASH proteins include fibroblast activation protein alpha (FAP), DPP8, DPP9, DPP4-like protein 1 (DPL1, DPP6, DPPX L, DPPX S), DPP4-like protein 2 (DPL2, DPP10) from the DPP4-gene family S9b and structurally unrelated enzyme DPP2, displaying DPP4-like activity. In contrast, DPP6 and DPP10 lack enzymatic DPP4-like activity. These DASH proteins play important roles in the immune system involving quiescence (DPP2), proliferation (DPP8/DPP9), antigen-presenting (DPP9), co-stimulation (DPP4), T cell activation (DPP4), signal transduction (DPP4, DPP8 and DPP9), differentiation (DPP4, DPP8) and tissue remodelling (DPP4, FAP). Thus, they are involved in many pathophysiological processes and have therefore been proposed for potential biomarkers or even drug targets in various cancers (DPP4 and FAP) and inflammatory diseases (DPP4, DPP8/DPP9). However, they also pose the challenge of drug selectivity concerning other DASH members for better efficacy and/or avoidance of unwanted side effects. Therefore, this review unravels the complex roles of DASH proteins in immunology.
BACKGROUND: Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. METHODS: In the present study, a novel congenic rat model of DP4 deficiency on a "DP4-high" DA rat genetic background was generated (DA.F344-Dpp4(m)/ SvH rats) and comprehensively phenotyped. RESULTS: Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4(m)/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. CONCLUSIONS: While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immuneregulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo.
Chemokines mediate the recruitment of leukocytes to the sites of inflammation. N-terminal truncation of chemokines by the protease dipeptidyl peptidase IV (DPPIV) potentially restricts their activity during inflammatory processes such as allergic reactions, but direct evidence in vivo is very rare. After demonstrating that N-terminal truncation of the chemokine CCL11/eotaxin by DPPIV results in a loss of CCR3-mediated intracellular calcium mobilization and CCR3 internalization in human eosinophils, we focused on the in vivo role of CCL11 and provide direct evidence for specific kinetic and rate-determining effects by DPPIV-like enzymatic activity on CCL11-mediated responses of eosinophils. Namely, it is demonstrated that i.v. administration of CCL11 in wild-type F344 rats leads to mobilization of eosinophils into the blood, peaking at 30 min. This mobilization is significantly increased in DPPIV-deficient F344 rats. Intradermal administration of CCL11 is followed by a dose-dependent recruitment of eosinophils into the skin and is significantly more effective in DPPIV-deficient F344 mutants as well as after pharmacological inhibition of DPPIV. Interestingly, CCL11 application leads to an up-regulation of DPPIV, which is not associated with negative feedback inhibition via DPPIV-cleaved CCL11((3-74)). These findings demonstrate regulatory effects of DPPIV for the recruitment of eosinophils. Furthermore, they illustrate that inhibitors of DPPIV have the potential to interfere with chemokine-mediated effects in vivo including but not limited to allergy.
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 naive 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.
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.
        
Title: Distribution of dipeptidyl peptidase IV-like activity enzymes in canine and porcine tissue sections by RT-PCR Wagner L, Hoffmann T, Rahfeld JU, Demuth HU Ref: Advances in Experimental Medicine & Biology, 575:109, 2006 : PubMed
The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.
        
Title: Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme Bar J, Weber A, Hoffmann T, Stork J, Wermann M, Wagner L, Aust S, Gerhartz B, Demuth HU Ref: Biol Chem, 384:1553, 2003 : PubMed
Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.
The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 A crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated beta-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed beta-propeller with open Velcro topology and the C-terminal alpha/beta-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the beta-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P(2)-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.
The NIH Xenopus Initiative is establishing many of the genetic and genomic resources that have been recommended by the Xenopus research community. These resources include cDNA libraries, expressed sequence tags, full-length cDNA sequences, genomic libraries, pilot projects to mutagenize and phenotype X. tropicalis, and sequencing the X. tropicalis genome. This review describes the status of these projects and explains how to access their data and resources. Current information about these activities is available on the NIH Xenopus Web site (http://www.nih.gov/science/models/xenopus/).
Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).
The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.