Biomedical subjects

K Neubert

Publications and source records attributed to K Neubert.

At least 19 recordsLinked to original sources

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1993

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↗

PubMed1993

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↗

PubMed1993

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↗

PubMed1991

Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?

Dipeptidyl peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) are inhibitors for dipeptidyl peptidase IV. We could show that both compounds as well as other tripeptides with a penultimate proline residue are substrates for dipeptidyl peptidase IV. An apparent competitive inhibition by those compounds is a kinetic artifact due to the substrate-like structure of such tripeptides.

Amino Acid Sequence↗

PubMed1991

Cyclization studies with tetra- and pentapeptide sequences corresponding to beta-casomorphins.

The tetrapeptide Boc-D-Orn-Phe-D-Pro-Gly-OH and the pentapeptide sequence Boc-Tyr(tBu)-D-Orn-Phe-D-Pro-Gly-OH were used to study the influence of different coupling reagents on the yield and purity of these model peptides. The simple structure prevented racemization and cyclodimerization and facilitated the ring formation. The most favorable effects on yield and purity were obtained in both reactions using diphenyl-phosphoryl azide (DPPA) and norborn-5-ene-2,3-dicarboximidodiphenylphosphate (NDPP), while the cyclizations with the powerful activating reagents benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) with the exception of the cyclopentapeptide reaction with HBTU/4-dimethylaminopyridine gave unsatisfactory results.

Amino Acid Sequence↗

PubMed1991

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Schön, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Schön, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.

Binding Sites↗

PubMed1991

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.

Animals↗

PubMed1991

Structure-activity studies of novel casomorphin analogues: binding profiles towards mu 1-, mu 2- and delta -opioid receptors.

The beta -casomorphin-5 sequence was systematically modified by substitution of the naturally occurring amino acids. The derivatives are compared on the basis of their affinities towards mu 1-, mu 2 and delta -opioid binding sites estimated by means of binding studies with [3H]dihydromorphine and [3H]D-Ala2-Leu5-enkephalin as labels and computer curve fitting. A C-terminal amide group which is known to increase mu-site affinity of beta -CM-4 and beta -CM-5 mainly enhances the mu 2-site affinity. Furthermore, the Pro4-amide structure, which seems to be responsible for the affinity enhancement can be substituted by the pyrrolidide ring structure. Modifications in position 2, 3 and 4 can lead to an increase in mu 1-, mu 2- or delta -site affinity and/or selectivity. The specificity of these effects might be dependent on the respective changes in the charge or hydrophobicity due to the introduction of other amino acids. The results suggest firstly that the alternating proline residues in the beta -CM-5 molecule are not absolutely necessary for its mu-site affinities, and secondly that both opioid receptor subtype affinity and selectivity may be modified by changing charge and/or hydrophobicity in the middle part of the beta -casomorphin molecule.

Amino Acid Sequence↗

PubMed1990

[3H]naloxone as an opioid receptor label: analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues.

The nonselective antagonist [3H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opioid ligands as well as various beta-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between mu 1- and mu 2-binding sites. The affinities to the mu-sites obtained with [3H]naloxone as label are in a good agreement with those from experiments with the mu-selective radioligand [3H]DAGO. The mu 1-site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity mu 1-subtype.

Affinity Labels↗

PubMed1989

Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV.

Specific inhibitors of the membrane-bound dipeptidyl peptidase IV (DP IV) and polyclonal antibodies against this enzyme were used to investigate the relationships between DP IV activity and the production and action of T cell-derived lymphokines. Production of interleukin 2 (IL-2) and gamma interferon by mitogen plus phorbol ester-stimulated mononuclear cells from human blood was found to be reduced in the presence of N-Ala-Pro-O-(nitrobenzoyl-)-hydroxylamine, epsilon-(4'-nitro) benzoxycarbonyl-Lys-Pro, and anti-(DP IV) immunoglobulin in a dose-dependent manner. Moreover, the proliferative response of mitogen-stimulated mononuclear cells to IL-2 is impaired in the presence of DP IV inhibitors. Therefore it is suggested that the membrane peptidase DP IV is involved in the induction and activation of cytokines controlling lymphocyte proliferation.

Cells, Cultured↗

PubMed1989

[Application of dipeptidylpeptidase IV in formation of the peptide bond].

According to the increasing importance of enzyme catalysed peptide synthesis the capability of the protease dipeptidyl peptidase IV is shown to form the peptide bond. To our best knowledge, it's the first time that has been used successfully proline as one of the amino acids. The formation of the peptide bond is kinetically controlled. Free amino acids have been used as nucleophiles. The reaction media contain 50 vol.-% glycerol. A first approach to a kinetic treatment is shown.

Amino Acid Sequence↗