Soluble CD26 / dipeptidyl peptidase IV enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding.

Human CD26 has dipeptidyl peptidase-4 (DPP IV) enzyme activity and binds to adenosine deaminase (ADA). CD26 is costimulatory for lymphocytes and has a circulating soluble form (sCD26). DPP IV enzyme inhibition is a new successful type 2 diabetes therapy. We examined whether the ADA binding and catalytic functions of sCD26 contribute to its effects on T-cell proliferation. Wildtype soluble recombinant human CD26 (srhCD26), an enzyme inactive mutant (srhCD26E-) and an ADA non-binding mutant (srhCD26A-) were co-incubated in in vitro T-cell proliferation assays with peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA), muromonab-CD3 or Herpes simplex virus antigen (HSV Ag). Both srhCD26 and srhCD26E- enhanced PHA-induced T-cell proliferation dose-dependently in all six subjects tested. srhCD26 and srhCD26A- had no overall effect on anti-CD3-stimulated PBMC proliferation in four of five subjects. srhCD26, srhCD26E- and srhCD26A- enhanced HSV Ag induced PBMC proliferation in low responders to HSV Ag, but had no effect or inhibited proliferation in HSV-high responders. Thus, effects of soluble human CD26 on human T-cell proliferation are mechanistically independent of both the enzyme activity and the ADA-binding capability of sCD26.

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

The main neutral aminopeptidase activity of human lymphoid tumour cell lines does not originate from the aminopeptidase N-(APN; CD13) gene.

Lymphocytes and related cell lines are predominantly CD13-negative, however, there are reports describing neutral aminopeptidase activity in or on these cells. The aim of this study was to answer the question, whether this activity originates from APN-gene expression. The total cellular activities (Ala-pNA hydrolysis) of lymphoid cell lines are up to 15 times higher than that of normal lymphocytes. Despite weak or lacking CD13 surface expression all lymphoid cell lines tested contain APNmRNA as quantified by competitive RT-PCR as well as low enzymatic activity in their particulate fractions. By isoelectric focusing two enzyme species with isoelectric points of 5.4 or between 3.5 to 4.8, respectively, were detected. To investigate whether these activities result from APN-gene we established transfectants lacking cellular APN expression of the CD13-positive histiocytic cell line U937 and the CD13-negative T-cell line H9. Studies on these transfectants proved (I) that the main neutral aminopeptidase activity expressed in lymphoid cells is definitively not related to APN and (II) that APN is also expressed in lymphoid cells, although on a low level only.

Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV).

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3-116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1-116), releasing the N-terminal dipeptide Ala-Pro. We hypothesize that PCT(3-116) is the result of the hydrolysis of PCT(1-116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.

Non-radioactive labelling and immunoprecipitation analysis of leukocyte surface proteins using different methods of protein biotinylation.

The biotinylation of surface proteins and the detection of immunoprecipitated protein(s) after transfer to nitrocellulose using chemiluminescence methods is a highly sensitive alternative to hazardous radioactive labelling procedures. Ligation of proteins using the common biotin-NHS-ester (N-hydroxysuccinimido-biotin) is often associated with a decrease in immunoreactivity. Here a new non-radioactive method of leukocyte surface glycoprotein labelling using biotin-LC-hydrazide is described. This technique is based on the labelling of glycoproteins after mild oxidation of carbohydrate hydroxyl groups to reactive aldehydes. Flow cytometric and immunoprecipitation analyses of selected leukocyte markers such as CD3, CD26 and CD65 indicated that the alteration in immunoreactivity achieved by NHS-mediated biotin ligation was different from that obtained with hydrazide-mediated biotin ligation. CD3 and CD26 immunoreactivity was diminished using NHS biotinylation but preserved by biotin-LC-hydrazide, whereas CD65 binding to monoclonal antibodies was completely abolished after treatment with biotin-hydrazide. However, the immunoreactivity of CD13 was found to be totally unaffected by both NHS and hydrazide biotinylation. The combination of different biotinylation methods for surface protein labelling offers a viable alternative to radioiodination in the biochemical analysis of membrane proteins.

Enzymatic activity of DPIV/CD26 is involved in PMA-induced hyperphosphorylation of p56lck.

The T-cell activation antigen CD26 (dipeptidyl peptidase IV, DPIV) is a proline specific protease thought to be involved in regulation of the immune response. Several former results characterized this ectoenzyme as a possible accessory molecule of the T-cell surface. The molecular events of lymphocyte activation mediated by this enzyme, as well as the physiological ligands of dipeptidyl peptidase, are only partly established. Here we provide evidence for a direct involvement of DPIV/CD26 in early phosphorylation mechanisms which were known to be essential in the signal transduction cascade of human T lymphocytes. Considering a possible functional linkage between CD26 and the tyrosine kinase p56lck, we have investigated the action of DPIV-specific inhibitors (Lys[Z[NO2)]-thiazolidide and -piperidide) on the PMA-induced hyperphosphorylation of p56lck in human T cells. Interestingly, this hyperphosphorylation of p56lck was strongly suppressed by both inhibitors in a dose-dependent manner. Removal of these inhibitors totally restored the hyperphosphorylation. Therefore, this effect could be considered as reversible. Free thiazolidine and piperidine, used in control experiments, neither inhibit DPIV enzyme activity nor PMA-induced hyperphosphorylation. The data presented here provide evidence that DPIV/CD26 is directly involved in early processes of T-cell activation. Furthermore, these findings strongly support the assumption that the signaling function of CD26 requires its enzymatic activity.

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) specifically suppress proliferation and modulate cytokine production of strongly CD26 expressing U937 cells.

Various studies from different laboratories have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) expressed in T and NK cells is involved in the regulation of DNA synthesis and cytokine production. In this paper, we performed a biochemical and functional characterization of dipeptidyl peptidase IV on the human histiocytic lymphoma cell line U937. Using U937 clones expressing low to high levels of membrane localized CD26, we found that the synthetic reversible inhibitors of DP IV, Lys-[Z(NO2)]-thiazolidide and Lys-[Z(NO2)]-piperidide, have different effects on all functions. In U937-H cells that strongly express high levels of CD26, DP IV inhibitors were shown to suppress DNA synthesis and production of IL-1 beta, but stimulate the secretion of the IL-1 receptor antagonist (IL-1RA) and of TNF-alpha. In contrast, both inhibitors did not influence the cytokine production and DNA synthesis in U937-L cells exhibiting low level CD26 expression. These data support the hypothesis that CD26 plays a crucial role in proliferation and cytokine production, not only in T cells, but also in other cell systems, and that enzymatic activity is essential for its function.

Inhibition of dipeptidyl peptidase IV (DP IV) by anti-DP IV antibodies and non-substrate X-X-Pro- oligopeptides ascertained by capillary electrophoresis.

Dipeptidyl peptidase IV (DP IV)-catalyzed hydrolysis of the NH2-X-Pro-containing N-terminal dodecapeptide of IL-2 was studied using free zone capillary electrophoresis as an alternative peptidase assay. In contrast to the conventional DP IV substrate glycyl-prolyl-p-nitroanilide (Gly-Pro-pNA), the hydrolysis of this peptide by DP IV was found to be significantly inhibited by anti-DP IV antibodies. Inhibition of DP IV was also observed with a number of non-substrate oligopeptides containing an N-terminal X-X-Pro- structure, including the HIV Tat protein. For Met-IL-2(1-6), we determined a competitive inhibition with an inhibition constant of ca. 100 microM.

Dipeptidyl peptidase IV: a cell surface peptidase involved in regulating T cell growth (review).

The CD26 antigen is identical with the cell surface ectopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5). The post proline cleaving substrate specificity makes DP IV relatively unique among other proteases. Numerous cytokines, chemokines and other bioactive peptides are potential substrates of DP IV, but knowledge about the real in vivo substrates is still very limited. CD26 represents an accessory surface molecule playing an important role in the process of activation and proliferation of human lymphocytes. The molecular events mediated by this ectoenzyme are only partly established and the necessity of DP IV enzymatic activity for its signalling capacity has been controversial. This review out-lines evidence for an involvement of DP IV in the regulation of immune response and focuses on the putative role of the catalytic domain of this peptidase. Inhibition of the catalytic activity can provoke many cellular effects, including induction of tyrosine phosphorylations and p38 MAP kinase activation as well as suppression of DNA synthesis and reduced production of various cytokines. TGF-beta1, the production and secretion of which is increased after DP IV inhibition, supposedly mediates the observed suppressive effects by maintaining p27kip expression levels which leads to a cell cycle arrest in G1. Moreover, anti-CD3-induced signalling pathways can be strongly affected by DP IV inhibition. Thus, the enzymatic activity or at least the interaction of effectors with the catalytic domain of CD26 seem to be important for crucial functions of this cell surface antigen.

CD26/dipeptidyl peptidase IV in lymphocyte growth regulation.

DP IV/CD26 is involved in regulation of DNA synthesis and proliferation as well as production of cytokines of hematopoietic cells under various conditions. Inhibition of DNA synthesis in T lymphocytes, B lymphocytes, NK cells and myelomonocytic cells as well as of the production of IL-2, IL-6 TNF alpha, IL-1, IL-10, IL-12, IL-13, IFN-gamma, GM-CSF are not due to apoptosis of these cells. DP IV/CD26 inhibitors induce TGF-beta 1 mRNA synthesis and latent protein release demonstrating a crucial role of TGF-beta 1 in mediating CD26 function. X-X-Pro peptides as HIV-Tat protein strongly inhibit DP IV enzymatic activity and suppress DNA synthesis. This group of peptides may represent a class of natural DP IV/CD26 ligands and effectors, respectively. Hyperphosphorylation of p56lck as well as protein tyrosine phosphorylation of a number of proteins in T lymphocytes can be modulated by DP IV inhibitors. These data suggest that enzymatic activity or, at least in part, the active site of DP IV are both essential for its regulatory function in lymphocytes. Further work is required to determine the natural ligands, i.e. substrates and effectors, which are play the central role in DP IV/CD26 action in T cell growth and to understand the molecular mechanism of the early steps of this fundamental process.

Signal transduction events induced or affected by inhibition of the catalytic activity of dipeptidyl peptidase IV (DP IV, CD26).

DP IV (CD26) represents an accessory surface molecule playing an important role in the process of activation and proliferation of human lymphocytes. The molecular events mediated by this ectoenzyme are only partly established and the necessity of DP IV enzymatic activity for its signalling capacity has been discussed controversial. Focusing on the putative role of the catalytic domain of this peptidase, it could be shown that inhibition of the catalytic activity can provoke many cellular effects, including induction of tyrosine phosphorylations and p38 MAP kinase activation as well as suppression of DNA synthesis and reduced production of various cytokines. TGF-beta 1, the production and secretion of which is increased after DP IV inhibition, supposedly mediates the observed suppressive effects by maintaining p27kip expression levels which leads to a cell cycle arrest in G1. Moreover, anti-CD3-induced signalling pathways, including Ca2+ mobilisation, MEK1-, Erk1/2- and PKB-activation, can be strongly affected by DP IV inhibition. Thus, the enzymatic activity or at least the interaction of effectors with the catalytic domain of CD26 seems to be important for crucial functions of this cell surface antigen.

Relating structure to function in the beta-propeller domain of dipeptidyl peptidase IV. Point mutations that influence adenosine deaminase binding, antibody binding and enzyme activity.

Point mutations in human CD26/DP IV were analysed for adenosine deaminase (ADA) binding, monoclonal antibody (mAb) binding and DP IV enzyme activity. Point mutations at either Leu294 or Val341 ablated ADA binding. Binding by mAbs that inhibit ADA binding was found to involve both Leu340 to Arg343 and Thr440/Lys441. Glu205 and Glu206 were found to be essential for enzyme activity. All residues of interest were mapped onto a model of the beta-propeller domain of DP IV. These data led us to suggest that in DP IV and related peptidases ligand and antibody binding sites are non-linear and that enzyme activity depends on charged sidechains that surround the entrance to the central tunnel of the beta-propeller.

Dipeptidyl peptidase IV (CD26): role in T cell activation and autoimmune disease.

The ectoenzyme dipeptidyl peptidase IV (DP IV; EC 3.4.14.5; CD26) has been shown to play a crucial role in T cell activation. In the present study, we show by flow cytometry and by enzymatic DP IV assay that myelin basic protein (MBP)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis (MS) express high levels of DP IV/CD26. The enzymatic activity of resting TCC was found to be three to fourfold higher than on resting peripheral blood T cells and close to that of T cells 48 hours after PHA stimulation. The DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide suppress in a dose-dependent manner DNA synthesis and IFN-gamma, IL-4, and TNF-alpha production of the antigen-stimulated TCC. These data suggest that CD26 plays a role in regulating activation of autoreactive TCC. Further in vivo investigations will clarify, whether the inhibition of the enzymatic activity of DP IV could be a useful tool for therapeutic interventions in MS and/or other autoimmune diseases.

Cathepsin protease activity modulates amyloid load in extracerebral amyloidosis.

In cerebral amyloidoses, such as Alzheimer's disease, proteolytic processing of the precursor protein is a fundamental mechanism of the disease, since it generates the amyloid protein. However, the putative significance of proteases in extracerebral amyloidoses is less well defined. In this study, we investigated the biological significance of cathepsin (Cath) B, CathK, and CathL in the pathology and pathogenesis of extracerebral amyloidoses by using the murine model of reactive or secondary AA amyloidosis with three different cathepsin-deficient mouse strains. Extracerebral AA amyloid was induced by injecting amyloid-enhancing factor and silver nitrate into CathB(-/-), CathK(-/-), and CathL(-/-) mice. Wild-type mice served as a control. CathK(-/-) mice deposited over 90% more amyloid and CathL(-/-) mice 60% less amyloid than the control (p < 0.0001). The amyloid load in CathB(-/-) mice did not differ from that in wild-type mice. In vitro degradation experiments with recombinant human and murine serum amyloid A (SAA) 1.1 and CathK and CathL showed that CathL generates a large number of differently sized SAA cleavage products. One of these fragments spans the heparin/heparan sulphate binding site and the neutral cholesterol ester hydrolase activating region of SAA. CathK showed only endoproteolytic activity and did not generate any AA amyloid-like peptides. This study provides unequivocal evidence that proteases modulate amyloid load in extracerebral amyloidosis. CathL was identified as an amyloid-promoting and CathK as an amyloid-retarding cysteine protease. CathB may only modulate the primary structure of the amyloid peptide without affecting amyloid load.

Proteolysis of serum amyloid A and AA amyloid proteins by cysteine proteases: cathepsin B generates AA amyloid proteins and cathepsin L may prevent their formation.

BACKGROUND: AA amyloidosis develops in patients with chronic inflammatory diseases. The AA amyloid proteins are proteolytic fragments obtained from serum amyloid A (SAA). Previous studies have provided evidence that endosomes or lysosomes might be involved in the processing of SAA, and contribute to the pathology of AA amyloidosis. OBJECTIVE: To investigate the anatomical distribution of cathepsin (Cath) B and CathL in AA amyloidosis and their ability to process SAA and AA amyloid proteins. METHODS: and results: CathB and CathL were found immunohistochemically in every patient with AA amyloidosis and displayed a spatial relationship with amyloid in all the cases studied. Both degraded SAA and AA amyloid proteins in vitro. With the help of mass spectrometry 27 fragments were identified after incubation of SAA with CathB, nine of which resembled AA amyloid proteins, and seven fragments after incubation with CathL. CathL did not generate AA amyloid-like peptides. When native human AA amyloid proteins were used as a substrate 26 fragments were identified after incubation with CathB and 18 after incubation with CathL. CONCLUSION: The two most abundant and ubiquitously expressed lysosomal proteases can cleave SAA and AA amyloid proteins. CathB generates nine AA amyloid-like proteins by its carboxypeptidase activity, whereas CathL may prevent the formation of AA amyloid proteins by endoproteolytic activity within the N-terminal region of SAA. This is particularly interesting, because AA amyloidosis is a systemic disease affecting many organs and tissue types, almost all of which express CathB and CathL.