The importance of the dipeptidyl peptidase 4 (DPP4) gene family in regulating critical biochemical pathways continues to emerge. The two most well-studied members of the family, DPP4 and fibroblast activation protein (FAP), have been investigated both as therapeutic targets for disease and as diagnostic biomarkers. The interest in DPP4 and FAP as potential disease biomarkers has been driven primarily by observations of altered expression profiles in inflammatory diseases and cancer. Furthermore, the stability and persistence of soluble DPP4 and FAP in the serum make them attractive candidate serology markers. This review summarises investigations into DPP4 and FAP as biomarkers of autoimmune disease, gut inflammation, psychosomatic disorders and malignancy and discusses their potential likelihood as clinically useful tools.
Title: Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches Wilson CH, Zhang HE, Gorrell MD, Abbott CA Ref: Biol Chem, 397:837, 2016 : PubMed
The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation.
Title: Dipeptidyl Peptidase 10 (DPP10 789 ): A Voltage Gated Potassium Channel Associated Protein Is Abnormally Expressed in Alzheimer's and Other Neurodegenerative Diseases Chen T, Gai WP, Abbott CA Ref: Biomed Res Int, 2014:209398, 2014 : PubMed
The neuropathological features associated with Alzheimer's disease (AD) include the presence of extracellular amyloid-beta peptide-containing plaques and intracellular tau positive neurofibrillary tangles and the loss of synapses and neurons in defined regions of the brain. Dipeptidyl peptidase 10 (DPP10) is a protein that facilitates Kv4 channel surface expression and neuronal excitability. This study aims to explore DPP10789 protein distribution in human brains and its contribution to the neurofibrillary pathology of AD and other tauopathies. Immunohistochemical analysis revealed predominant neuronal staining of DPP10789 in control brains, and the CA1 region of the hippocampus contained strong reactivity in the distal dendrites of the pyramidal cells. In AD brains, robust DPP10789 reactivity was detected in neurofibrillary tangles and plaque-associated dystrophic neurites, most of which colocalized with the doubly phosphorylated Ser-202/Thr-205 tau epitope. DPP10789 positive neurofibrillary tangles and plaque-associated dystrophic neurites also appeared in other neurodegenerative diseases such as frontotemporal lobar degeneration, diffuse Lewy body disease, and progressive supranuclear palsy. Occasional DPP10789 positive neurofibrillary tangles and neurites were seen in some aged control brains. Western blot analysis showed both full length and truncated DPP10789 fragments with the later increasing significantly in AD brains compared to control brains. Our results suggest that DPP10789 is involved in the pathology of AD and other neurodegenerative diseases.
Title: Identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy metabolism Wilson CH, Indarto D, Doucet A, Pogson LD, Pitman MR, McNicholas K, Menz RI, Overall CM, Abbott CA Ref: Journal of Biological Chemistry, 288:13936, 2013 : PubMed
Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.
Title: Expression profiling of dipeptidyl peptidase 8 and 9 in breast and ovarian carcinoma cell lines Wilson CH, Abbott CA Ref: Int J Oncol, 41:919, 2012 : PubMed
Proteases, particularly serine proteases like dipeptidyl peptidase 4 (DP4) and fibroblast activation protein (FAP), play an important role in cancer invasion and angiogenesis. Aberrant expression of DP4 and FAP is associated with numerous cancers, including breast and epithelial ovarian carcinoma. We investigated the mRNA levels, protein expression and enzyme activity of the structural homologs DP8 and DP9, in addition to DP4 and FAP, in three breast carcinoma (MDA-MB-231, MDA-MB-453, MCF-7), three epithelial ovarian carcinoma (EOC) (OVCA-432, OVCA-429, SKOV3), 293T and HeLa cell lines. In addition, DP2 and prolyl endopeptidase (PEP) mRNA and enzyme levels were measured and compared in each cell line. Ubiquitous but differential expression of DP8 and DP9 mRNA and protein was observed across all cell lines. Relative to EOC, DP8 protein was lower in the breast carcinoma cell lines (p=0.057), suggesting that DP8 may play differing roles in different cancer cell types. A strong, negative, non-reciprocal relationship was identified between DP9 protein and DP4 mRNA (r=-0.903, p=0.002) and protein (r=-0.810, p=0.015). This suggests that DP4 expression plays an important role in the post-transcriptional regulation of DP9 in breast and ovarian cancer cell lines. Overall, this study suggests a potential role for DP8 and DP9 in breast and ovarian cancer and further investigations in this area are required.
Title: Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8) Pitman MR, Menz RI, Abbott CA Ref: Biol Chem, 391:959, 2010 : PubMed
Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.
Title: Expression and prognostic assessment of dipeptidyl peptidase IV and related enzymes in B-cell chronic lymphocytic leukemia Sulda ML, Abbott CA, Macardle PJ, Hall RK, Kuss BJ Ref: Cancer Biol Ther, 10:180, 2010 : PubMed
Recent observations of the deregulated expression of several dipeptidyl peptidase (DP) IV-like enzymes in human cancers have led to presumptions of their pathogenic role in cancer. To further explore this concept we have characterized the expression of all DPIV-like enzymes in chronic lymphocytic leukemia (CLL). We have demonstrated the constitutive expression of DPIV, DP8, DP9, DPII and PEP mRNA and DPIV, DP8 and DP9 protein in CLL. FAP mRNA was not detected in CLL or normal B-lymphocytes. This correlated with an absence of FAP protein on the cell surface. This study also shows that DP8 mRNA expression is significantly upregulated in CLL compared to normal tonsil B-lymphocytes (p < 0.05) which may suggest biological importance in this disease. DP expression could not be correlated with any molecular or clinical prognostic markers for CLL in this cohort including IgVH mutational status, CD38, ZAP-70 or CD49d expression (n = 58). However, the constitutive expression of the DPIV-like enzymes in CLL and their emergence as potent immune regulators makes them candidate therapeutic targets in this disease.
BACKGROUND: We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection. MATERIALS AND METHODS: Wildtype (WT) and DPIV(-/-) mice consumed 2% DSS in drinking water for 6 days to induce colitis. Mice were treated with saline or the DP inhibitors Ile-Pyrr-(2-CN)*TFA or Ile-Thia. DP mRNA and enzyme levels were measured in the colon. Glucagon-like peptide (GLP)-2 and GLP-1 concentrations were determined by radioimmunoassay, regulatory T-cells (Tregs) by fluorescence activated cell sorting (FACS) on FOXp3+T cells in blood, and neutrophil infiltration assessed by myeloperoxidase (MPO) assay. RESULTS: DP8 and DP2 mRNA levels were increased (P < 0.05) in WT+saline mice compared to untreated WT mice with colitis. Cytoplasmic DP enzyme activity was increased (P < 0.05) in DPIV(-/-) mice at day 6 of DSS, while DP2 activity was increased (P < 0.05) in WT mice with colitis. GLP-1 (63%) and GLP-2 (50%) concentrations increased in WT+Ile-Pyrr-(2-CN)*TFA mice compared to day-0 controls. MPO activity was lower in WT+Ile-Thia and WT+Ile-Pyrr-(2-CN)*TFA treated mice compared to WT+saline (P < 0.001) at day 6 colitis. CONCLUSIONS: DP expression and activity are differentially regulated during DSS colitis, suggesting a pathophysiological role for these enzymes in human inflammatory bowel disease (IBD). DP inhibitors impaired neutrophil recruitment and maintenance of the Treg population during DSS-colitis, providing further preclinical evidence for the potential therapeutic use of these inhibitors in IBD. Finally, DPIV appears to play a critical role in mediating the protective effect of DP inhibitors.
Title: Dipeptidyl peptidase (DP) 6 and DP10: novel brain proteins implicated in human health and disease McNicholas K, Chen T, Abbott CA Ref: Clinical Chemistry & Laboratory Medicine, 47:262, 2009 : PubMed
Dipeptidyl peptidase (DP) 6 and DP10 are non-enzyme members of the dipeptidyl peptidase IV family, which includes fibroblast activation protein, DP8, and DP9. DP6 and DP10 proteins have been shown to be critical components of voltage-gated potassium (Kv) channels important in determining cellular excitability. The aim of this paper was to review the research to date on DP6 and DP10 structure, expression, and functions. To date, the protein region responsible for modulating Kv4 channels has not been conclusively identified and the significance of the splice variants has not been resolved. Resolution of these issues will improve our overall knowledge of DP6 and DP10 and lead to a better understanding of their role in diseases, such as asthma and Alzheimer's disease.
Title: Dipeptidyl peptidase 8 and 9--guilty by association? Pitman MR, Sulda ML, Kuss B, Abbott CA Ref: Front Biosci (Landmark Ed), 14:3619, 2009 : PubMed
Dipeptidyl peptidases (DP) 8 and 9 are members of the DPIV enzyme family. Other members include DPIV, fibroblast activation protein (FAP) and the non-enzymes DP6 and DP10. DPIV family members have diverse biological roles, and have been implicated in a range of diseases including diabetes, cancer, inflammatory bowel disease, multiple sclerosis (MS), arthritis and asthma. While DP8/9 biological functions are yet to be established, they have been predicted to have similar roles to the other DPs due to high sequence similarities within the active site of the enzymes. While there is mounting evidence towards the involvement of DP8 and/or DP9 in innate and acquired immunity, direct proof for the link between DP8 and DP9 and human disease is yet to be definitively shown, thus DP8 and 9 proteins remain guilty by association.
Title: Growth factor based therapies and intestinal disease: is glucagon-like peptide-2 the new way forward? Yazbeck R, Howarth GS, Abbott CA Ref: Cytokine Growth Factor Rev, 20:175, 2009 : PubMed
Inflammatory bowel disease (IBD) is a chronic, debilitating disease associated with severe damage to the intestinal mucosa. Glucagon-like peptide-2 (GLP-2) is a potent and specific gastrointestinal growth factor that is demonstrating therapeutic potential for the prevention or treatment of an expanding number of intestinal diseases, including short bowel syndrome (SBS), small bowel enteritis and IBD. The biological activity of GLP-2 is limited due to proteolytic inactivation by the protease dipeptidyl peptidase (DP)IV. Inhibitors of DPIV activity may represent a novel strategy to prolong the growth promoting actions of GLP-2. This review outlines evidence for the clinical application of GLP-2, its degradation resistant analogue, Teduglutide, and novel DPIV inhibitors in efficacy studies utilizing pre-clinical models of intestinal damage, in particular IBD.
Title: Dipeptidyl peptidase inhibitors, an emerging drug class for inflammatory disease? Yazbeck R, Howarth GS, Abbott CA Ref: Trends in Pharmacological Sciences, 30:600, 2009 : PubMed
Dipeptidyl peptidase (DPP)-4 is a member of the S9b serine protease family, which also includes DPP8 and DPP9. DPP4 cleaves a number of regulatory factors, including chemokines and growth factors. DPP4 inhibitors have recently emerged as an effective treatment option for type 2 diabetes. Early in vitro studies demonstrated that DPP4 inhibitors inhibit T-cell proliferation and cytokine production, leading to their investigation in numerous pre-clinical models of inflammatory diseases, including arthritis, multiple sclerosis and inflammatory bowel disease. Recent data suggest that the early DPP4-specific inhibitors might also bind DPP8 and DPP9, thus exerting their effects through non-specific binding. This review highlights recent insights into the applicability of DPP inhibitors as novel pharmacological agents for inflammatory disease.
The dipeptidyl peptidase IV (DPIV) enzyme family contains both potential and proven therapeutic targets. Recent reports indicate the presence of DP8 and DP9 in peripheral blood lymphocytes, testis, lung, and brain. For a more comprehensive understanding of DP8 and DP9 tissue and cellular expression, mRNA and enzyme activity were examined. Many organs from C57BL/6 wild-type and DPIV gene-knockout mice were examined; DP8/9 enzyme activity was detected in the immune system, brain, testis, muscle, and epithelia. In situ hybridization localized DP8 and DP9 mRNA to lymphocytes and epithelial cells in liver, gastrointestinal tract, lymph node, spleen, and lung. DP8 and DP9 mRNA was detected in baboon and mouse testis, and DP9 expression was elevated in human testicular cancers. DP8 and DP9 mRNA were ubiquitous in day 17 mouse embryo, with greatest expression in epithelium (skin and gastrointestinal tract) and brain. Thus, DP8 and DP9 are widely expressed enzymes. Their expression in lymphocytes and epithelia indicates potential for roles in the digestive and immune systems. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.
New treatment strategies are required for the debilitating inflammatory bowel diseases (IBD), Crohn's Disease and Ulcerative Colitis. DP inhibitors can prolong the bioactivity of the potent intestinotrophic growth factor glucagon-like peptide-2 (GLP-2(1-33)). We investigated whether novel inhibitors of DP activity could modify the course of disease activity in the dextran sulfate sodium (DSS) model of colitis. C57BL/6 mice consumed 2 percent DSS in drinking water for 6 days. Mice were orally gavaged twice daily with 0.9% saline, 10 mg/kg isoleucyl-cyano-pyrrolidine (P59/99) or isoleucyl-thiazolidine (P32/98). Assessment of disease severity incorporated a disease activity index (DAI), together with histological assessment of crypt area and depth in the distal colon. DP activity was significantly inhibited at all time points. The DAI was significantly lower in the P59/99 and P32/98 treatment groups compared to saline treatment in all three time courses. Crypt hyperplasia (p<0.05) was observed in the saline group compared to P32/98 treatment at day 9. This preliminary study shows that novel inhibitors of DP activity may provide a new treatment strategy for IBD.
Title: Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity Chen T, Ajami K, McCaughan GW, Gai WP, Gorrell MD, Abbott CA Ref: Biochimica & Biophysica Acta, 1764:33, 2006 : PubMed
DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly(644)-->Ser, Lys(643)Gly(644)-->TrpSer, or Asp(561)Lys(643)Gly(644)-->TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.
Title: Prediction of dipeptidyl peptidase (DP) 8 structure by homology modelling Pitman MR, Menz RI, Abbott CA Ref: Advances in Experimental Medicine & Biology, 575:33, 2006 : PubMed
Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic growth factor that enhances repair of damaged intestinal tissue. However, its bioactivity is limited by dipeptidyl peptidase IV (DPIV)-mediated degradation. We hypothesized that DPIV(-/-) mice would display an increased resistance to, and an enhanced recovery from, dextran sulfate sodium (DSS)-induced colitis compared to DPIV(+/+) mice. DPIV(+/+) and DPIV(-/-) mice consumed 2% DSS for 6 days, followed by a 15 day recovery period. Mice were killed at days 0, 3, 6, 9, 14, and 21 (n = 6-8) and the small intestine and colon removed for histological assessment of villus height, crypt depth, and crypt area. The epithelial cell proliferative labeling index was determined by proliferating cell nuclear antigen (PCNA) immunostaining. Small intestine, colon, and total body weight did not differ between DPIV(+/+) and DPIV(-/-) mice. Distal colon crypt depth did not differ significantly between DPIV(+/+) and DPIV(-/-) mice during the development of DSS-colitis or during the recovery phase. Similarly no significant effects were apparent on distal colon crypt area or PCNA labeling index between DPIV(+/+) and DPIV(-/-) during the development of and recovery from DSS-colitis. However, DPIV(-/-) mice still possessed significant levels of plasma DPIV-like activity. We conclude that loss of DPIV activity does not increase resistance to experimental colitis and hypothesize that other DPIV family members may also be involved in the cleavage of GLP-2.
Title: Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity Ajami K, Abbott CA, McCaughan GW, Gorrell MD Ref: Biochimica & Biophysica Acta, 1679:18, 2004 : PubMed
Dipeptidyl peptidase (DP) IV has a distinct substrate specificity in hydrolyzing a post-proline bond. Here we present novel data on the sizes and tissue distribution of human and rat gene products and the peptidase activity of the DPIV-related gene DP9. A short cDNA of 2589 bp and a long cDNA of 3006 bp of DP9 were cloned. A ubiquitous predominant DP9 mRNA transcript at 4.4 kb represented the short form, whereas a less abundant 5.0-kb transcript present predominantly in muscle represented the long form. Both forms of DP9 have no transmembrane domain and two potential N-linked glycosylation sites. DP9 exhibited post-proline dipeptidyl aminopeptidase activity and was a cytoplasmic, 110-kDa monomer. Thus, the six DPIV gene family members have diverse characteristics: only DP9 and DP8 have exclusively cytoplasmic localization and only DP9, DP8, fibroblast activation protein (FAP) and DPIV have peptidase activity.
Dipeptidyl peptidase IV (DP-IV/CD26), fibroblast activation protein (FAP), DP-like 1 (DPL1), DP8, DP9, and DPL2 comprise the CD26 gene family. CD26/DP-IV has roles in liver disease, T cell costimulation, chemokine biology, type II diabetes, and tumor biology. DPIV substrates include the glucagonlike peptides, neuropeptide Y, and the chemokines CCL3, CCL5, CCL11, CCL22, and CXCL12. We have proposed that the extracellular region of CD26 is analogous to prolyl oligopeptidase in consisting of an alpha/beta hydrolase domain contributed by both N- and C-terminal portions of the polypeptide and a seven-blade beta-propeller domain. Replacing the C-terminal portion of the predicted alpha/beta hydrolase domain of CD26 (residues 501-766) with the homologous portion of DP8 or DP9 produced intact proteins. However, these chimeric proteins lacked dimerization and peptidase activity, suggesting that CD26 dimerization requires the C-terminal portion of the alpha/beta hydrolase domain. Deleting some N-terminal residues of the alpha/beta hydrolase domain of CD26 ablated peptidase activity and greatly diminished cell surface expression. Together with previous data that CD26 peptidase activity requires the C-terminal 20 residues, this suggests that peptidase activity requires the entire alpha/beta hydrolase domain. The catalytic triad of DP8 was shown to be Ser(739)-Asp (817)-His(849). Glu(259) of DP8, a residue distant from the catalytic triad yet greatly conserved in the CD26 gene family, was shown to be required for peptidase activity. These data concord with our predicted CD26 structure, indicate that biosynthesis of a functional fragment of CD26 is difficult, and confirm the functional homology of DP8 with CD26.
We have identified three novel members of the DPIV gene family using database mining approaches. Recombinant DP8 shares a post-proline dipeptidyl aminopeptidase activity with the closely related enzymes DPIV and FAP. The similarities between DP8, DP9 and DPIV in tissue expression pattern suggest a potential role for DP8 and DP9 in liver disease, T cell activation and immune function. The role of the two novel enzymes DP8 and DP9 and the other non-enzyme member DPL2 in human disease will be the focus of further studies.
Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.
Title: Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity Abbott CA, McCaughan GW, Gorrell MD Ref: FEBS Letters, 458:278, 1999 : PubMed
Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.
Title: Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins Abbott CA, Mackness MI, Kumar S, Boulton AJ, Durrington PN Ref: Arterioscler Thromb Vasc Biol, 15:1812, 1995 : PubMed
Human serum paraoxonase is physically associated with HDL and has been implicated in the detoxification of organophosphates and possibly in the prevention of LDL lipid peroxidation. We investigated the serum activity and concentration of paraoxonase in 78 patients with type 1 diabetes mellitus, 92 with type 2 diabetes, and 82 nondiabetic control subjects. Paraoxonase activity was generally lower in diabetics than in control subjects. This decrease was unrelated to differences in paraoxonase phenotype distribution or its serum concentration. Rather, the difference in paraoxonase activity was explained by its specific activity, which was lower in diabetics, indicating either the presence of a circulating inhibitor or disturbance of the interaction of paraoxonase with HDL affecting its activity. Paraoxonase specific activity was lowest in patients with peripheral neuropathy, suggesting an association of paraoxonase with neuropathy. In control subjects but not patients with diabetes, paraoxonase correlated with HDL cholesterol and apolipoprotein A-1. Our results indicate that the low paraoxonase activity in diabetes is due to decreased specific activity. In other studies low serum paraoxonase activity has been associated with increased susceptibility to atherosclerosis, and the present results also suggest an association with peripheral neuropathy, which could be due to reduced capacity to detoxify lipid peroxides in diabetes.
Title: Genomic organization, exact localization, and tissue expression of the human CD26 (dipeptidyl peptidase IV) gene Abbott CA, Baker E, Sutherland GR, McCaughan GW Ref: Immunogenetics, 40:331, 1994 : PubMed
CD26 is a lymphocyte cell surface antigen which is increased during T-cell activation and is also expressed in other tissues. It is an atypical serine protease belonging to the prolyl oligopeptidase family. CD26 has been implicated in a variety of biological functions including T-cell activation, cell-to-cell adhesion, and recently in HIV infection. This paper describes, through the isolation and partial sequencing of eight human CD26 genomic clones, the first information on the genomic organization of the prolyl oligopeptidase family. We have established that the human CD26 gene spans approximately 70 kilobases (kb) and contains 26 exons, ranging in size from 45 base pairs (bp) to 1.4 kb. The nucleotides that encode the serine recognition site (G-W-S-Y-G) are split between two exons. This clearly distinguishes the genomic organization of the prolyl oligopeptidase family from that of the classical serine protease family. The 5' flanking domain of the CD26 gene contains neither a TATA box nor a CAAT box, but a 300 bp region extremely rich in C and G (72%) contains potential binding sites for several transcriptional factors. The human CD26 gene encodes two messages sized at about 4.2 and 2.8 kb. These are both expressed at high levels in the placenta and kidney and at moderate levels in the lung and liver. Only the 4.2 kb mRNA was expressed at low levels in skeletal muscle, heart, brain, and pancreas. Fluorescence in situ hybridization on metaphase chromosome spreads located the human CD26 gene to the long arm of chromosome 2(2q24.3).
1. The activity of serum butyrylcholinesterase ('pseudocholinesterase', EC3.1.1.8) was investigated in 56 patients with type 1 diabetes mellitus, 51 patients with type 2 diabetes mellitus and 101 healthy control subjects. 2. Butyrylcholinesterase activity was significantly elevated in both type 1 (8.10 +/- 3.35 units/ml) and type 2 (7.22 +/- 1.95 units/ml) diabetes compared with the control subjects (4.23 +/- 1.89 units/ml) (P < 0.001). 3. In the patients with type 1 and type 2 diabetes, serum butyrylcholinesterase activity was correlated with log serum fasting triacylglycerol concentration (r = 0.41 and r = 0.43, respectively, P < 0.001). In the type 2 population serum butyrylcholinesterase activity was also correlated with insulin sensitivity (r = -0.51, P < 0.001). 4. Serum butyrylcholinesterase activity was unrelated to age, gender, serum gamma-glutamyltranspeptidase activity, body mass index, or treatment for diabetes in both the diabetic populations. 5. In 37 non-diabetic patients with butyrylcholinesterase deficiency serum triacylglycerol levels were in the normal range. 6. These results are consistent with the view that butyrylcholinesterase may have a role in the altered lipoprotein metabolism in hypertriglyceridaemia associated with insulin insensitivity or insulin deficiency in diabetes mellitus.
Human serum paraoxonase is responsible for the hydrolysis of organophosphate anticholinesterases, however, whether the enzyme has a physiological role other than the detoxication of insecticides and nerve gases has remained uncertain. Recently, evidence has begun to accumulate of a relationship between the serum activity of paraoxonase and atherosclerosis. Paraoxonase may a fundamental role in lipoprotein metabolism, preventing oxidative changes to low-density lipoprotein which render the particle atherogenic.
