Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp(2)-Tat(1-9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1-9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1-9) induces the production of the immunosuppressive cytokine transforming growth factor-beta1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.     

Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., St?ckel-Maschek, A., Lorey, S., K?hne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.     

1H NMR conformational study on n-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure–activity relationships

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1–9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, ¹H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1–9), I⁵-Tat(1–9) and L⁶-Tat(1–9) and to examine the influence of amino acid exchanges on structural features of these peptides. The defined structures revealed differences in the conformations what might be the reason for different interactions of these Tat(1–9) analogues with certain amino acids of the active site of DP IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Determination of the Solution Conformation of HIV-1 Tat(1–9) Peptides by Means of Molecular Dynamics Simulations Considering NMR Data and Docking Studies into an Active Site Model of DP IV

The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro⁶ with Leu and Asp⁵ with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional ¹H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L⁶-Tat(1–9) and I⁵-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089480040200     

Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris. A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme

Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DP IV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-1(7-36) resulted in significantly different binding constants for the human and the porcine enzyme (Kd = 153.0 +/- 17.0 microM and Kd = 33.4 +/- 2.2 microM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd = 2.15 +/- 0.18 nM and Kd = 7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.     

Metabolism of glucagon by dipeptidyl peptidase IV (CD26)

Glucagon is a 29-amino acid polypeptide released from pancreatic islet alpha-cells that acts to maintain euglycemia by stimulating hepatic glycogenolysis and gluconeogenesis. Despite its importance, there remains controversy about the mechanisms responsible for glucagon clearance in the body. In the current study, enzymatic metabolism of glucagon was assessed using sensitive mass spectrometric techniques to identify the molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) yielded sequential production of glucagon(3-29) and glucagon(5-29). In human serum, degradation to glucagon(3-29) was rapidly followed by N-terminal cyclization of glucagon, preventing further DP IV-mediated hydrolysis. Bioassay of glucagon, following incubation with purified DP IV or normal rat serum demonstrated a significant loss of hyperglycemic activity, while a similar incubation in DP IV-deficient rat serum did not show any loss of glucagon bioactivity. Degradation, monitored by mass spectrometry and bioassay, was blocked by the specific DP IV inhibitor, isoleucyl thiazolidine. These results identify DP IV as a primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for the determination of glucagon levels in human plasma.     

Stromal cell-derived factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8

N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.     

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.     

Isoforms of dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24: role of the signal sequence and overexpression in Escherichia coli

The complete nucleotide sequence of dipeptidyl aminopeptidase IV (DAP IV) from Pseudomonas sp. WO24 was determined. Nucleotide sequence analysis revealed an open reading frame of 2238bp, which was assigned to dap4 by N-terminal and internal amino acid sequences previously reported. The predicted amino acid sequence of DAP IV contains a serine protease Gly-X-Ser-X-Gly-Gly consensus motif and displays extensive homology to DAP IVs and the homologous proteins from eukaryotes and bacteria, belonging to the prolyl oligopeptidase family S9. In Pseudomonas sp. WO24, DAP IV is expressed as 82 and 84-kDa isoforms, having two Met, Met-1 and Met-12, in its N-terminal sequence. Met-1 of DAP IV was mutated to Gly and Met-12 was mutated to Ile, and we overexpressed the two mutated genes in Escherichia coli and obtained the recombinant 82 and 84-kDa proteins from the periplasm and the cytoplasm, respectively, suggesting that the 82 and 84-kDa isoforms are derived from the same gene and localize to different compartments in the cell. We developed purification steps for activting a large amount of 84-kDa isoform protein that will be useful for producing protein for crystallographic studies.     

Isolation and characterization of cardiac amyloid in familial amyloid polyneuropathy type IV (Finnish): relation of the amyloid protein to variant gelsolin

Amyloid subunit protein was isolated from familial amyloid polyneuropathy type IV (Finnish type) cardiac tissue and purified to homogeneity. N-terminal amino acid sequence analysis shows that the amyloid protein is a fragment of the inner region of human gelsolin. When compared with the predicted sequence of human plasma gelsolin, the amyloid protein contains an asparagine-for-aspartic acid substitution at position 15 corresponding to residue 187 of the secreted protein. Antibodies raised against the amyloidogenic region of gelsolin specifically stained the amyloid deposited in tissues in familial amyloidosis type IV. The results show that the subunit amyloid protein in familial amyloid polyneuropathy type IV represents a unique type of amyloid derived from a variant (Asn-187) gelsolin molecule by limited proteolysis.     

Global changes in protein composition of N2-fixing-Azoarcus sp. strain BH72 upon diazosome formation.

The strictly respiratory, diazotrophic bacterium Azoarcus sp. strain BH72 fixes nitrogen under microaerobic conditions. In empirically optimized batch cultures at nanomolar O2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N2 fixation are formed. Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, Kallar grass. To initiate studies on function of diazosomes and regulation of their formation, diazosome-containing bacteria were compared with respect to composition or total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions. In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions. N-terminal amino acid sequence analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P(II), an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism. According to its electrophoretic mobility, it might be uridylylated in diazosome-free cells but not in diazosome-containing cells, or it may represent a second, not identical P(II) protein. Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp. strain BH72 in Southern hybridizations.     

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. Sch?n, 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 (Sch?n, 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.     

The mouse type IV c-abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization

The subcellular localization of the mouse type IV c-abl protein was determined by indirect immunofluorescence of nontransformed NIH 3T3 fibroblasts that overexpress the protein. Unlike the viral transforming protein p160gag/v-abl, which has cytoplasmic and plasma membrane localization, a large fraction of the c-abl (IV) protein is nuclear, with the remainder in the cytoplasm and plasma membrane. Deletion of a small N-terminal regulatory region of the c-abl (IV) protein, sufficient to activate its transforming potential fully, changes the distribution of the protein from the nucleus to the cytoplasm. Mapping of an amino acid sequence responsible for the nuclear localization of the c-abl (IV) protein reveals a nuclear localization signal similar to that of SV40 large T antigen.     

Subunit IV of human cytochrome c oxidase, polymorphism and a putative isoform.

As part of our study of isoenzyme forms of human cytochrome c oxidase, we purified subunit IV from human heart and skeletal muscle with reversed-phase HPLC and determined the N-terminal amino acid sequences and the electrophoretic mobility. The N-terminus of human heart subunit IV proved to be ragged with 30% of the protein lacking the first three residues. Also a Tyr/Phe polymorphism was observed at residue 16. No differences in N-terminal sequence and electrophoretic mobility were observed between subunit IV of cytochrome c oxidase from human heart and skeletal muscle. Therefore, our results suggest that identical subunits IV are present in cytochrome c oxidase from human heart and skeletal muscle. A putative isoform of subunit IV with a blocked N-terminus was purified from human heart cytochrome c oxidase, which proved to have a different retention time on a reversed-phase column and also a slightly higher electrophoretic mobility on an SDS-polyacrylamide gel compared to the native subunit IV. We could not demonstrate the existence of isoforms of subunit IV in human skeletal muscle.     

Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26)

Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.     

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.     

Hydrolysis of proteins using dipeptidyl aminopeptidases: analysis of the N-terminal portion of spinach plastocyanin

The exopeptidases dipeptidyl aminopeptidases I and IV were used to hydrolyze the N-terminal portion of spinach plastocyanin to dipeptides. The enzymes were used individually as well as in a mixture and the dipeptides were analyzed by combined gas chromatography-mass spectrometry. Data are presented for native plastocyanin and the S-methylated protein. Of the 98 residues which make up this protein, the first 44 were released in the form of 22 dipeptides by the combined action of DAP I and DAP IV. These dipeptides were aligned by homology to other plastocyanins of known sequence. The results demonstrate the versatility of the two enzymes in hydrolyzing proteins to obtain information on their primary sequence.     

Does human attractin have DP4 activity?

Mutations in the mouse ATRN gene, which encodes attractin, offer links between this protein and pigmentation, metabolism, immune status and neurodegeneration. However, the mechanisms of attractin action are not understood. The protein was first identified in humans in a circulating form in serum. A protease activity was postulated similar to the membrane-bound ectoenzyme DP4/CD26. In the last decade, both DP4/CD26 and attractin were controversially described to be the major source of human serum DP4 activity. We purified attractin from human plasma, and found that the DP4-like activity of the preparation shows nearly identical kinetic properties to that of recombinant human DP4. In contrast, the native electrophoretic behavior of this activity is clearly different from human and porcine DP4, but co-migrates with the protein band identified as attractin by Western blotting and N-terminal sequencing. Nevertheless, a DP4 impurity could be demonstrated in purified plasma attractin and the activity could be removed by ADA affinity chromatography, resulting in a homogenous attractin preparation without DP4 activity. These results are substantiated by expression of different attractin isoforms, in which no DP4 activity was found either. This indicates that the multidomain protein attractin acts as a receptor or adhesion protein rather than a protease.