Specific inhibitors of aminopeptidase P. Peptides and pseudopeptides of 2-hydroxy-3-amino acids.
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Amino Acids↗
Biomedical subjects
K Neubert
Publications and source records attributed to K Neubert.
Induction of the membrane alanyl aminopeptidase gene and surface expression in human T-cells by mitogenic activation.
The metal-dependent membrane alanyl aminopeptidase (amino-peptidase N, APN, CD13; EC 3.4.11.2) is a well-established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. CD13-specific monoclonal antibodies do not bind to the surface of normal B lymphocytes, and APN mRNA was not detectable by Northern analysis in normal lymphocytes or in T-cell lines. Recently the expression of the APN gene in T-cell lines as well as the ability of these cells to cleave chromogenic substrates preferred by APN have been demonstrated [Lendeckel, Wex, Kähne, Frank, Reinhold and Ansorge (1994) Cell. Immunol. 153, 214-226]. Here, by means of dot-blot hybridization and RNase protection assay, evidence is provided that human peripheral T-cells as well as derived cell lines contain significant amounts of APN mRNA, comparable to that in the promyeloic cell line U937, and that mitogenic activation of peripheral human T-cells leads to a more than 4-fold increase in their APN mRNA content. In the course of activation, T-cells increase their total alanine p-nitroanilide-hydrolysing activity to approx. 7-fold that of resting cells. Furthermore these cells become immunoreactive towards CD13 to a significant extent (up to 51%) as shown by surface staining and confirmed by activity staining and immunostaining after isoelectric focusing (pI of T-cell APN = 4.6). In addition it is demonstrated by fluorescence microscopy that viable, activated T-cells effectively cleave the fluorogenic aminopeptidase substrate bis-glycyl-rhodamine 110 and that the corresponding aminopeptidase activity is associated with the cell surface. We show that specific inhibitors of APN, probestin and actinonin, strongly decrease DNA synthesis in phytohaemagglutinin (PHA)-stimulated T-cells. In summary, evidence is presented that in the course of mitogenic activation human peripheral T-cells increase the expression of APN both at the transcriptional level and at the cell surface. This has been demonstrated both at the APN mRNA level and at the protein level with respect to aminopeptidase enzymic activity and CD13 immunoreactivity.
CD13 Antigens↗
Dipeptidyl-peptidase IV-beta, a novel form of cell-surface-expressed protein with dipeptidyl-peptidase IV activity.
The T-cell activation antigen CD26, is a type II membrane glycoprotein with intrinsic dipeptidyl-peptidase IV (DPP IV) activity, characterized by its capacity to cleave off N-terminal dipeptides containing proline as the penultimate residue. Independent of its catalytic activity, CD26 has also been characterized as adenosine deaminase binding protein. By using CD26 negative human C8166 cells, here we describe the existence of another cell-surface protein which manifests CD26-like DPP IV activity. For convenience, this protein will be referred to as DPP IV-beta. Consistent with the cell-surface expression of DPP IV-beta, intact C8166 cells manifested a high level of DPP IV, whereas, they manifested poor activity against substrates of DPP II known to have an intracellular localization. A partially purified preparation of CD26 from human MOLT4 cells, and the DPP IV-beta expressed on intact cells were found to possess similar catalytic activity and pH optimum. In addition, cell-surface CD26 and DPP IV-beta on intact MOLT4 and C8166 cells, respectively, resisted digestion by proteolytic enzymes such as trypsin and proteinase K. However, adenosine deaminase activity was not detectable on the surface of C8166 cells in contrast to CD26 positive MOLT4 cells. In accord with this, 125I-labeled adenosine deaminase which binds CD26 was found not to bind DPP IV-beta. Gel-filtration experiments using 0.5% Triton X-100 extracts from C8166 and MOLT4 cells, revealed that the apparent molecular mass of DPP IV-beta is 82 kDa, whereas that of CD26 is 110 kDa as expected. Taken together, our results suggest that DPP IV-beta is a CD26-like protein which could be characterized by distinct properties.
Adenosine Deaminase↗
The N-terminal X-X-Pro sequence of the HIV-1 Tat protein is important for the inhibition of dipeptidyl peptidase IV (DP IV/CD26) and the suppression of mitogen-induced proliferation of human T cells.
Recent data in the literature suggest that the HIV-1 Tat(1-86) protein exhibits immunosuppressive effects. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV), which is identical to the T cell activation marker CD26. Here we show that the N-terminal amino acid sequence of Tat is essential for the inhibition of DP IV-catalyzed IL-2(1-12) degradation. N-terminal modification of Tat with rhodamine prevented inhibition of enzymatic activity of DP IV as well as suppression of DNA synthesis of mitogen-stimulated human T cells. Moreover, natural peptides containing the X-X-Pro N-terminal motif of Tat also inhibited DP IV activity. These data suggest the existence of endogenous immunomodulatory oligopeptides which influence immune cell proliferation and differentiation via DP IV as does HIV-1 Tat.
Amino Acid Sequence↗
Proton NMR conformational analysis of cyclic beta-casomorphin analogues of the type Tyr-cyclo[N omega-D-Orn-Xaa-Yaa-Gly-].
A conformational study of the cyclic beta-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] (1) (mu-selective agonist; 2-Nal = 2 naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] (2) (mixed mu agonist/delta antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] (3) (highly potent mu and delta agonist) has been carried out using 1H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal3 and Pro4, and a minor one (10%) showing cis amide bonds both between D-Orn2 and 2-Nal3 and between 2-Nal3 and Pro4. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive gamma turns around the D-Pro4 and D-Orn2 residues, respectively, and by a D-Orn2-CO<--HN delta-D-Orn2 hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro4 residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for mu opioid receptor binding compounds.
Amino Acid Sequence↗
CD26 mediates the action of HIV-1 Tat protein on DNA synthesis and cytokine production in U937 cells.
The human immunodeficiency virus 1 (HIV-1) Tat protein is known to be capable of suppressing antigen- and CD3-induced activation of human T cells. Previously, it was shown that Tat can bind to the dipeptidyl peptidase IV (DP IV, CD26) and inhibit the degradation of the chromogenic substrate Gly-Pro-p-nitroanilide. Using the method of free zone capillary electrophoresis, here we have shown that the DP IV-catalyzed hydrolysis of the NH2-X-Pro-containing cytokine peptides IL-2(1-12), IL-1 beta(1-6), and IL-6(1-12) was also significantly inhibited by the Tat protein. Moreover, HIV-1 Tat at a concentration of 10 micrograms/ml was found to have a strong suppressive effect on DNA synthesis and IL-1 beta production, but stimulates secretion of IL-1 receptor antagonist (IL-1RA) and TNF-alpha of CD26-expressing U937-H cells. It did not impair neither DNA synthesis nor cytokine production of low CD26-expressing U937-L cells. Similar results have been found with synthetic DP IV/CD26 inhibitors (Immunobiol., 1994, vol. 192, pp. 121-136). These data strongly suggest that Tat protein is a potent "natural" inhibitor of DP IV/CD26, and they support the hypothesis that DPIV plays a role in Tat's immunosuppressive activity.
Cytokines↗
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.
Amino Acid Sequence↗
Functional role of CD26 on human B lymphocytes.
CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0-5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.
B-Lymphocytes↗
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.
Dipeptidyl Peptidase 4↗
Synthesis of dipeptide 4-nitroanilides containing non-proteinogenic amino acids.
A series of tert-butyloxycarbonyl amino acid 4-nitroanilides, including N-alkylated amino acids and (R)-thiazolidine-4-carboxylic acid, (S)-oxazolidine-4-carboxylic acid. (4S,5S)-5-methyloxazolidine-4-carboxylic acid, (4S,5R)-5-methyloxazolidine-4-carboxylic acid, (S)-azetidine-2-carboxylic acid, (S)-pipecolic acid and (S)-3,4-dehydroproline, were prepared conveniently by the isocyanate method or the mixed anhydride procedure. The resulting amino acid 4-nitroanilides were extended to corresponding dipeptide 4-nitroanilides with tert-butyloxycarbonyl-(S)-alanine. In the case of sterically hindered amino acid 4-nitroanilides the mixed anhydride procedure with diphenylphosphinyl chloride was successful.
Amino Acids↗
A model of the active site of dipeptidyl peptidase IV predicted by comparative molecular field analysis and molecular modelling simulations.
A molecular model of the active site of the serine protease dipeptidyl peptidase IV (DPP IV or CD26) has been developed on the basis of comparative molecular field analysis (CoMFA) of competitive inhibitors and by force field calculations. By application of CoMFA experimentally obtained inhibition constants Ki have been successfully predicted. The resulting steric and electrostatic coefficients of CoMFA were used for the development of the molecular model. The main assumptions of the model are the recognition of substrates or inhibitors by the side chains of a tyrosine (S1-position) and a tryptophan residue (S2-position). The model helps us to understand a multitude of experimental data regarding the substrate specificity of this enzyme as well as results obtained by genetic engineering experiments by other authors. General conclusions concerning a new family of serine proteases are drawn and discussed.
Binding Sites↗
Cyclic beta-casomorphin analogues with mixed mu agonist/delta antagonist properties: synthesis, pharmacological characterization, and conformational aspects.
Analogues of the potent and moderately mu-opioid-receptor-selective cyclic beta-casomorphin-5 derivative H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] (2) were prepared by conventional solution synthesis. Replacement of the Phe3 residue by 2-naphthylalanine (2-Nal) led to a peptide (4) with high affinity for both mu and delta opioid receptors. This compound turned out to be an agonist in the mu-receptor-representative guinea pig ileum (GPI) assay but a moderately potent antagonist against various delta agonists in the delta-receptor-representative mouse vas deferens (MVD) assay. It thus represents the first known cyclic opioid peptide analogue with mixed mu agonist/delta antagonist properties. Interestingly, replacement of 2-Nal3 in compound 4 with 1-naphthylalanine (1-Nal) resulted in an analogue (5) showing high affinity for mu receptors and a full agonist effect in the MVD assay that was mediated by both mu and delta receptors. Substitution of Trp for Phe3 in 2 (compound 8) was well tolerated at both receptors and led to an analogue with agonist activity in both the GPI and MVD assays. Variation of the peptide ring size in 4 was achieved by substitution of D-Orn2 with D-Lys (compound 6) or D-2,4-diaminobutyric acid (compound 7). Analogue 6 was also a mixed mu agonist/delta antagonist with somewhat lower potency than 4, whereas compound 7 displayed mu agonist and partial delta agonist properties. Further reduction of the peptide ring size, as achieved by deletion of the Gly5 residue, produced a compound (9) which was a full agonist in both bioassays. Conformational analysis of analogues 2, 4, and 5 by 1H NMR spectroscopy and molecular mechanics studies suggested that the overall conformation of parent compound 2 and the 2-Nal-containing peptide 4 was similar, while the side-chain orientation of 1-Nal in peptide 5 was different. These results suggest that the delta antagonist properties of analogue 4 may not be due to a difference in its overall conformation as compared to the agonist 2 but may be a direct effect of the 2-naphthyl moiety per se preventing proper alignment of the peptide for receptor activation.
Amino Acid Sequence↗
Influence on proline-specific enzymes of a substrate containing the thioxoaminoacyl-prolyl peptide bond.
Dipeptidyl peptidase IV from porcine kidney and aminopeptidase P from Escherichia coli can utilize thioxoalanyl-proline 4-nitroanilide but with decreased kinetic constants compared to the normal substrates. Product analysis showed that exclusively thioxoalanyl-proline was liberated in the case of dipeptidyl peptidase IV catalysis and thioxo-alanine in the case of aminopeptidase-P-mediated thioxo peptide bond hydrolysis. For the proline-specific aminopeptidase P the kcat/Km value for the thioxo peptide is 1100-fold lower than for the corresponding oxo peptide. This difference is entirely due to kcat. Because the rotation about the thioxo amide bond is about 12.5 kJ mol-1 more difficult than rotation about an amide bond, these data support a mechanism involving rate-limiting rotation about the scissile peptide bond. It was found that the specificity rate constant for the reaction of thioxoalanyl-proline 4-nitroanilide and dipeptidyl peptidase IV is 100-1000-fold lower compared to the corresponding rate constant for alanyl-proline 4-nitroanilide. This remarkable effect is interpreted in terms of a distorted binding of the transition state for the thioxo substrate. The hydrolysis of the thioxo substrate by dipeptidyl peptidase IV is isomer-specific. The conformation about the nonscissile P2-P1 thioxo amide bond has to be in trans for successful cleavage of the scissile peptide bond. We can now directly compare the rotational energy barrier of the prolyl peptide bond for the oxo and the thioxo form.
Aminopeptidases↗
High-performance liquid chromatographic separation of cis-trans isomers of proline-containing peptides. II. Fractionation in different cyclodextrin systems.
beta-Cyclodextrin-bonded silica is demonstrated to be a suitable stationary phase for high-performance liquid chromatography of conformational isomers of proline-containing peptides. In contrast to reversed-phase chromatography, the principle of inclusion complexation shows significant selectivities in conformer resolution based on a variety of interactions. New results of inclusion HPLC of biologically active oligopeptides related to beta-casomorphin on stationary phases containing bonded cyclodextrins of different internal diameters indicate a steric discrimination process during the conformer separation. beta-Cyclodextrin used as a mobile phase additive in reversed-phase systems is shown to offer the opportunity to investigate conformational changes using commercially available reversed-phase columns.
Amino Acid Sequence↗
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.
Blotting, Western↗
Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13) catalyzed hydrolysis of cytokines and peptides with N-terminal cytokine sequences.
A number of natural cytokines are characterized as having dipeptidyl peptidase (DP) IV susceptible N-terminal peptide sequences. Here we demonstrate that oligopeptides with sequences analogous to the N-terminal part of human IL-1 beta, IL-2, TNF-beta and murine IL-6 were hydrolyzed by purified DP IV and aminopeptidase N (AP-N). The rate of DP IV-catalyzed hydrolysis of these peptides was negatively correlated with their chain length. In contrast to these results, no degradation was found under our conditions for the intact recombinant cytokines, IL-1 alpha, IL-1 beta, IL-2, G-CSF and for natural IL-2, independent of whether DP IV and AP-N were used separately or in combination.
Amino Acid Sequence↗
Dipeptidyl peptidase IV (CD26) on human lymphocytes. Synthetic inhibitors of and antibodies against dipeptidyl peptidase IV suppress the proliferation of pokeweed mitogen-stimulated peripheral blood mononuclear cells, and IL-2 and IL-6 production.
In the present report, we describe that synthetic inhibitors of and polyclonal and monoclonal antibodies against the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) inhibit the production of IL-2 and IL-6 and, concomitantly, DNA synthesis of pokeweed mitogen-stimulated peripheral blood mononuclear cells (PBMC). The release of IL-1 and TNF-alpha, was not influenced under these conditions. The data support the hypothesis that DP IV, possibly in conjunction with other peptidase, is involved in the regulation of activation and proliferation of T lymphocytes.
Antibodies, Monoclonal↗
Enzymatic activity of CD26 (dipeptidylpeptidase IV) is not required for its signalling function in T cells.
CD26 is a proteolytic enzyme (dipeptidylpeptidase IV) expressed on the T cell surface that defines an alternative activation signal for human T lymphocytes. Crosslinking of CD26 via monoclonal antibodies triggers proliferation and cytotoxicity in preactivated T cells. In this study, we used highly specific competitive and irreversible inhibitors of dipeptidylpeptidase IV to study the role of the enzymatic activity in activation of CD26-transfected T cells as well as of CD26-expressing normal human T cell clones. These inhibitors at concentrations that blocked up to 95% of the enzymatic activity, did not specifically inhibit T cell activation neither via TCR/CD3 nor via CD26 itself. This demonstrates that the enzymatic activity of CD26 is not required for its T cell activating properties.
Animals↗