CD26: an expanding role in immune regulation and cancer

In this review, we highlight major aspects of the biology of CD26, a dipeptidyl peptidase IV (DPPIV)-containing surface glycoprotein with multiple functions. In particular, we discuss findings demonstrating that CD26/DPPIV has an essential role in immune regulation as a T cell activation molecule and a regulator of chemokine function. We also review recent studies that identify key cellular molecules that physically associate with CD26 and the potential consequences of their interaction, including those with clinically-related implications. Furthermore, we present work suggesting a role for CD26 in the pathogenesis and behavior of selected human cancers, both solid tumors and hematological malignancies. We present recent studies that investigate the potential role of CD26 as a molecular target for novel treatment modalities for T cell lymphoid malignancies and possibly other hematological malignancies, with work involving the use of anti-CD26 monoclonal antibody, CD26-transfected cells as well as soluble CD26 molecules.     

Dipeptidyl peptidase IV activity and/or structure homologs: Contributing factors in the pathogenesis of rheumatoid arthritis?

Several of the proinflammatory peptides involved in rheumatoid arthritis pathogenesis, including peptides induced downstream of tumor necrosis factor-α as well as the monocyte/T cell-attracting chemokines RANTES and stromal cell-derived factor (SDF)-1α and the neuropeptides vasoactive intestinal peptide (VIP) and substance P, have their biological half-lives controlled by dipeptidyl peptidase IV (DPPIV). Proteolysis by DPPIV regulates not only the half-life but also receptor preference and downstream signaling. In this article, we examine the role of DPPIV homologs, including CD26, the canonical DPPIV, and their substrates in the pathogenesis of rheumatoid arthritis. The differing specific activities of the DPPIV family members and their differential inhibitor response provide new insights into therapeutic design.     

Rapid assay to detect possible natural substrates of proteases in living cells

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.     

Expression,purification, and characterization of human dipeptidyl peptidase IV/CD26 in Sf9 insect cells

The human dipeptidyl peptidase IV/CD26 (DPPIV/CD26) is a multifunctional type-II membrane bound glycoprotein. As a receptor of collagen I and fibronectin it mediates cell-cell and cell-matrix adhesion, and by interacting with extracellular adenosine deaminase and CD45 it is involved in regulatory and costimulatory events in the immune system. DPPIV/CD26 has a very distinct substrate specificity, and is potentially capable of truncating many cytokines, chemokines, and peptide hormones. In this study, we describe the overexpression, purification, and characterization of human DPPIV/CD26 in Spodoptera frugiperda (Sf9) cells, using the baculovirus system. Overexpression of DPPIV/CD26 was confirmed by measurement of its peptidase specificity, SDS-PAGE, and Western blot analyses. Expression rates were between 6.4 and 17.6 mg protein per liter suspension culture (1.5 x 10(9)cells). The N-linked oligosaccharide composition was examined and compared with that of mammalian cell-expressed DPPIV/CD26. Two-step purification by immunoaffinity chromatography and size-exclusion fast protein liquid chromatography (SE-FPLC) led to highly stable protein with significant peptidase activity. A subsequent gel filtration step on a Superdex 200 column yielded 2mg homogeneous dimeric DPPIV/CD26 (per liter insect cell culture) for crystallographic studies. Protein homogeneity was confirmed by silver staining of non-denaturating PAGE gels and by MALDI-TOF analysis of tryptic peptides.     

The multifunctional or moonlighting protein CD26/DPPIV

CD26/DPPIV can be considered a moonlighting protein because it is a multifunctional protein that exerts its different functions depending on cell type and intra- or extracellular conditions in which it is expressed. In the present review, we summarize all its known functions in relation to physiological and pathophysiological conditions. The protein is a proteolytic enzyme, receptor, costimulatory protein, and is involved in adhesion and apoptosis. The CD26/DPPIV protein plays a major role in immune response. Abnormal expression is found in the case of autoimmune diseases, HIV-related diseases and cancer. Natural substrates for CD26/DPPIV are involved in immunomodulation, psycho/neuronal modulation and physiological processes in general. Therefore, targeting of CD26/ DPPIV and especially its proteolytic activity has many therapeutic potentials. On the other hand, there are homologous proteins with overlapping proteolytic activity, which thus may prevent specific modulation of CD26/DPPIV. In conclusion, CD26/DPPIV is a protein present both in various cellular compartments and extracellularly where it exerts different functions and thus is a true moonlighting protein.     

Amino-terminal processing of MIP-1beta/CCL4 by CD26/dipeptidyl-peptidase IV

CD26 is a membrane-bound ectopeptidase with dipeptidyl peptidase IV (DPPIV) activity that has diverse functional properties in T cell physiology and in regulation of bioactive peptides. We have previously reported that activated human peripheral lymphocytes (PBL) secrete an amino-terminal truncated form of macrophage inflammatory protein (MIP)-1beta/(3-69) with novel functional specificity for CCR1, 2, and 5. In this report, we show that the full length MIP-1beta is processed by CD26/DPPIV to the truncated form and that cleavage can be blocked by DPPIV inhibitory peptides derived from HIV Tat(1-9) or the thromboxane A2 receptor, TAX2-R(1-9). Addition of Tat(1-9) or TAX2-R(1-9) peptides to PBL cultures partially blocks endogenous MIP-1beta processing. The kinetics of conversion of MIP-1beta from intact to MIP-1beta(3-69) in activated PBLs correlates with cell surface expression of CD26. Our results suggest that NH2-terminal processing of MIP-1beta and possibly other chemokines may depend on the balance between CD26/DPPIV enzymatic activity and cellular and viral proteins that modulate enzyme function.     

Vesicular localization and characterization of a novel post-proline-cleaving aminodipeptidase, quiescent cell proline dipeptidase

A large number of chemokines, cytokines, and signal peptides share a highly conserved X-Pro motif on the N-terminus. The cleavage of this N-terminal X-Pro dipeptide results in functional alterations of chemokines such as RANTES, stroma-derived factor-1, and macrophage-derived chemokine. Until recently, CD26/DPPIV was the only known protease with the ability to cleave N-terminal X-Pro motifs at neutral pH. We have isolated and cloned a novel serine protease, quiescent cell proline dipeptidase (QPP), with substrate specificity similar to that of CD26/DPPIV. In this paper we show that QPP, like CD26/DPPIV, is synthesized with a propeptide and undergoes N:-glycosylation. Interestingly, this glycosylation is required for QPP enzymatic activity, but not for its localization. Unlike the cell surface molecule, CD26/DPPIV, QPP is targeted to intracellular vesicles that are distinct from lysosomes. Proteinase K treatment of intact vesicles indicates that QPP is located within the vesicles. These vesicles appear to have a secretory component, as QPP is secreted in a functionally active form in response to calcium release. The presence of QPP in the vesicular compartment suggests that molecules bearing the N-terminal X-Pro motif can be cleaved at multiple sites within and outside the cell. These results expand the potential site(s) and scope of a process that appears to be an important mechanism of post-translational regulation.     

Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1alpha (SDF-1alpha) at its position two proline. CXCL12/SDF-1alpha induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34(+) hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34(+) hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1alpha lacks the ability to induce the migration of CD34(+) cord blood cells and acts to inhibit normal CXCL12/SDF-1alpha-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34(+) cells enhances the migratory response of these cells to CXCL12/SDF-1alpha. This process of CXCL12/SDF-1alpha cleavage by CD26/DPPIV on a subpopulation of CD34(+) cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.     

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.     

CD26/DPPIV signal transduction function, but not proteolytic activity, is directly related to its expression level on human Th1 and Th2 cell lines as detected with living cell cytochemistry.

CD26/DPPIV is a cell surface glycoprotein that functions both in signal transduction and as a proteolytic enzyme, dipeptidyl peptidase IV (DPPIV). To investigate how two separate functions of one molecule are regulated, we analyzed CD26 protein expression and DPPIV enzyme activity on living human T-helper 1 (Th1) and Th2 cells that express different levels of CD26/DPPIV. DPPIV activity was specifically determined with the synthetic fluorogenic substrate ala-pro-cresyl violet and CD26 protein expression was demonstrated with an FITC-conjugated CD26-specific antibody. Fluorescence of liberated cresyl violet (red) and FITC (green) was detected simultaneously on living T-cells using flow cytometry and spectrofluorometry. Th1 cells expressed three- to sixfold more CD26 protein than Th2 cells. The signal transduction function of the CD26/DPPIV complex, tested by measuring its co-stimulatory potential for proliferation, was directly related to the amount of CD26 protein at the cell surface. However, DPPIV activity was similar in both cell populations at physiological substrate concentrations because of differences in K(m) and V(max) values of DPPIV on Th1 and Th2 cells. Western blotting and zymography of Th1 and Th2 whole-cell lysates demonstrated similar patterns. This study shows that two functions of one molecule can be controlled differentially.     

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).     

Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.     

Fluorogenic substrate [Ala-Pro]2-cresyl violet but not Ala-Pro-rhodamine 110 is cleaved specifically by DPPIV activity: a study in living Jurkat cells and CD26/DPPIV-transfected Jurkat cells.

Fluorogenic substrates [Ala-Pro](2)-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro](2)-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro](2)-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro](2)-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro](2)-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction.     

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.     

Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.     

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.     

Adenosine downregulates DPPIV on HT-29 colon cancer cells by stimulating protein tyrosine phosphatase(s) and reducing ERK1/2 activity via a novel pathway

The multifunctional cell-surface protein dipeptidyl peptidase IV (DPPIV/CD26) is aberrantly expressed in many cancers and plays a key role in tumorigenesis and metastasis. Its diverse cellular roles include modulation of chemokine activity by cleaving dipeptides from the chemokine NH(2)-terminus, perturbation of extracellular nucleoside metabolism by binding the ecto-enzyme adenosine deaminase, and interaction with the extracellular matrix by binding proteins such as collagen and fibronectin. We have recently shown that DPPIV can be downregulated from the cell surface of HT-29 colorectal carcinoma cells by adenosine, which is a metabolite that becomes concentrated in the extracellular fluid of hypoxic solid tumors. Most of the known responses to adenosine are mediated through four different subtypes of G protein-coupled adenosine receptors: A(1), A(2A), A(2B), and A(3). We report here that adenosine downregulation of DPPIV from the surface of HT-29 cells occurs independently of these classic receptor subtypes, and is mediated by a novel cell-surface mechanism that induces an increase in protein tyrosine phosphatase activity. The increase in protein tyrosine phosphatase activity leads to a decrease in the tyrosine phosphorylation of ERK1/2 MAP kinase that in turn links to the decline in DPPIV mRNA and protein. The downregulation of DPPIV occurs independently of changes in the activities of protein kinases A or C, phosphatidylinositol 3-kinase, other serine/threonine phosphatases, or the p38 or JNK MAP kinases. This novel action of adenosine has implications for our ability to manipulate adenosine-dependent events within the solid tumor microenvironment.     

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.     

Clinical Significance of Soluble CD26 in Malignant Pleural Mesothelioma

There is no established single diagnostic marker for malignant pleural mesothelioma (MPM). CD26 is a 110 kDa, multifunctional, membrane-bound glycoprotein that has dipeptidyl peptidase IV (DPPIV) enzyme activity. The aim of this study was to evaluate the clinical significance of soluble CD26 (sCD26) in patients with MPM. The study included 80 MPM patients, 79 subjects with past asbestos exposure (SPE), and 134 patients with other benign pleural diseases (OPD) that were included as a control group. sCD26 levels and DPPIV activity in serum and/or pleural fluid were determined using an ELISA kit. Serum sCD26 levels and DPPIV enzyme activity in patients with MPM were significantly decreased compared with those in the SPE group (P = 0.000). The level of serum sCD26 was significantly decreased in patients with advanced stages of MPM compared with those with earlier stages (P = 0.047). The median OS of patients with MPM who had higher DPPIV enzyme activity was significantly longer than that of those with lower DPPIV enzyme activity (P = 0.032). The sCD26 levels in the pleural fluid of MPM patients with an epithelioid subtype were significantly increased compared with the OPD cohort (P = 0.012). Moreover, DPPIV enzyme activity in the pleural fluid of patients with MPM with an epithelioid subtype were significantly increased compared with those in the OPD cohort (P = 0.009). Patients with MPM who had lower specific DPPIV activity, determined as DPPIV/sCD26, showed significantly prolonged survival compared with those with higher specific DPPIV activity (P = 0.028). Serum sCD26 and DPPIV enzyme activity appear to be useful biomarkers for differentiating patients with MPM from SPE. The sCD26 levels or DPPIV enzyme activity in pleural fluid appear to be biomarkers in patients with an epithelioid subtype of MPM. DPPIV activity in serum or pleural fluid appears to be predictive for the prognosis of patients with MPM.