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PubMed2004

The metal-binding motif of dipeptidyl peptidase III directly influences the enzyme activity in the copper derivative of dipeptidyl peptidase III.

The zinc-binding motif (HELLGH) of dipeptidyl peptidase III (DPP III) is different from the common zinc-binding motif (HExxH) of metallopeptidases. To clarify the importance of the zinc-binding motif part of DPP III for enzymatic activity, we measured the recovery of the enzyme activity of apo-Leu(453)-deleted dipeptidyl peptidase III (apo-Leu(453)-del-DPP III), which has a motif (HELGH) like that of the common peptidase (HExxH), in the presence of various metal ions. The enzyme activity of apo-Leu(453)-deleted DPP III could not be recovered by the addition of cupric ions, while apo-DPP III could be easily reactivated by the addition of cupric ions. The visible and electron paramagnetic resonance spectra of the isolated Cu(II)-Leu(453)-del DPP III clearly show that the cupric ions of Cu(II)-Leu(453)-del-DPP III bound to the motif part (HELGH) but did not exhibit any enzyme activity. The motif part of DPP III directly influences the expression of the enzyme activity in the copper derivative of DPP III. The competitive inhibitor that is not at all digested by DPP III, Hisprophen (His-Pro-Phe-His-Leu-d-Leu-Val-Tyr), has been determined. The inhibition constant (K(i)) of Hisprophen for DPP III or Cu(II)-DPP III was 4.1x10(-5) or 3.8x10(-5)M, respectively. In the presence of the competitive peptide inhibitor, Hisprophen, the EPR spectra of Cu(II)-DPP III were completely different from that of Cu(II)-DPP III itself. This result clearly indicates that the metal ions of DPP III are located in the active site and directly interact with the substrate.

Amino Acid Motifs↗
PubMed1991

Distribution of peptidases in cultured cells from middle ear mucosa: prolyl endopeptidase is localized in fibroblasts and dipeptidyl peptidase IV and II in epithelial cells.

To examine the distribution of prolyl endopeptidase (PEP), dipeptidyl peptidase IV (DPP IV), and dipeptidyl peptidase II (DPP II) in specific cell types, fibroblasts and epithelial cells were selectively cultured from middle ear mucosal tissues of guinea pigs. In fibroblasts, PEP had the highest activity, 12.28 +/- 4.00 nmole/min/mg protein (mean +/- SD), 45-fold higher than corresponding DPP II levels. In epithelial cells, DPP IV activity was the highest, 6.48 +/- 0.90 nmole/min/mg protein. This communication describes, for the first time, the distribution of the enzyme activities of PEP, DPP IV, and DPP II in fibroblasts and epithelial cells, and the occurrence of PEP in fibroblasts.

Animals↗
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↗
PubMed1998

Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.

Biotinylation↗
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↗
PubMed2003

A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases.

BACKGROUND: Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. RESULTS: We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis). A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. CONCLUSION: CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2.

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↗
PubMed2005

Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell proline dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents.

Animals↗
PubMed1989

Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in synovial fluid from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase IV (DPP IV) activity was lower in synovial fluid from patients with RA, in contrast to the increase of DPP II activity in synovial fluid, as compared with OA. The DPP II/DPP IV ratio for synovial fluid was significantly higher in patients with RA than in patients with OA. A significant correlation was observed between the DPP II/DPP IV ratio for synovial fluid from patients with RA and the amount of C-reactive protein reaction. These results may be useful in the diagnosis of joint effusion of unknown origin.

Arthritis, Rheumatoid↗
PubMed1992

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.

Amino Acid Sequence↗
PubMed1979

Purification and properties of a collagen peptidase (PZ-peptidase) from rabbit serum.

A peptidase cleaving a synthetic substrate for collagen peptidases, 4-phenylazobenzyloxcarbonyl-L-Pro-L-Leu-Gly-L-pro-D-Arg (designated as PZ-peptide) has been purified 1200-fold from rabbit serum and characterized. The enzyme preparation is free of collagenase and unspecific proteinase activity. The natural substrates are denatured collagen and collagen peptides. The peptidase has a molecular weight of 124 000 and an isoelectric point at pH 5.1. The pH dependence curve exhibits two maxima, one at pH 7.1 and the other at pH 7.9. The enzymic reaction is completely inhibited by Zn2+ and to a slower degree by Hg2+, Mn2+ and p-hydroxymercuribenzoate. It is not affected by EDTA and KCN but totally blocked by o-phenanthroline. Phenylmethylsulfonylfluoride is completely inhibitory and points to a serine residue in the active site.

Animals↗
PubMedDate not supplied

Chorionic peptidase inactivates GnRH as a post-proline peptidase.

Recently, we have described a chorionic peptidase (C-ase-1) which inactivates gonadotropin releasing hormone (GnRH), oxytocin, angiotensin II and thyrotropin releasing hormone. Since all these hormones contain a proline residue, we proposed that C-ase-1 may act as a post-proline peptidase. Using HPLC and amino acid analyses, we have defined the products which resulted from enzymatic inactivation of GnRH by C-ase-1. The N-terminal nonapeptide of GnRH was isolated by HPLC and confirmed by amino acid composition analyses. Thus, it was demonstrated that C-ase-1 acts as a post-proline peptidase when inactivating GnRH, yielding the nonapeptide, i.e., des-Gly10-NH2-GnRH, and Gly-NH2. The levels of intrauterine GnRH, angiotensin II, oxytocin and thyrotropin releasing hormone may be affected and integrated by this enzyme. Thus, C-ase-1 may play an important role in the regulation of the paracrine and endocrine function during pregnancy.

Amino Acids↗
PubMed1976

Dipeptidyl peptidase IV, a kidney brush-border serine peptidase.

Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.

Animals↗
PubMed2006

Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase.

Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I-CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.

Animals↗
PubMed2004

Influence of dipeptidyl peptidase inhibitors on growth, peptidase activity, and ammonia production by ruminal microorganisms.

The aim was to investigate known and potential new inhibitiors of dipeptidyl peptidases (DPP) for their effects on ruminal microorganisms. Gly-Phe diazomethylketone (GPD), Ala-Ala chloromethylketone (AAC), benserazide (DL-serine 2-(2,3,4- trihydroxybenzyl) hydrazide), and diprotin A (Ile-Pro-Ile) inhibited DPP activities of Prevotella albensis, P. ruminicola, P. bryantii, P. brevis, and mixed ruminal microorganisms, though incompletely and, except for diprotin A, without absolute specificity for any of the peptidases. Leucine aminopeptidase activity of Streptococcus bovis was also inhibited by GPD and benserazide. The inhibitors had no effect on the growth of the bacteria, except for GPD, which inhibited growth of P. albensis when only peptides were available for growth. Benserazide had some inhibitory effects on the growth of Megasphaera elsdenii and Prevotella spp., even in the absence of peptides. The predatory activity of ciliate protozoa on bacteria was unaffected by DPP inhibitors. Ammonia production from casein by mixed ruminal microorganisms was inhibited significantly (P < 0.05) by AAC (29% inhibition) and benserazide (33%). It was concluded that DPP inhibitors can influence the rate of NH3 production in the rumen and may form the basis for developing protein-sparing feed additives for ruminants.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Tripeptidyl peptidase II is the major peptidase needed to trim long antigenic precursors, but is not required for most MHC class I antigen presentation.

Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14-17 residues) more than short ones (9-12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopepsidase 1) are required for the generation of a subset of epitopes.

Amino Acid Sequence↗
PubMed1999

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.

Adenosine Deaminase↗
PubMed1987

The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro.

Dipeptidyl peptidase IV (DP IV), an ectoenzyme in the cell membrane of T lymphocytes, is an important constituent in the process of lymphocyte activation. This conclusion is drawn from the following observations: (a) Proliferation of peripheral blood lymphocytes induced by mitogenic lectins (phytohemagglutinin, concanavalin A, pokeweed mitogen) is suppressed in the presence of DP IV inhibitors. This effect is specific and applies to an irreversible suicide inhibitor as well as to a competitive one in a dose-dependent fashion. (b) Inhibition of DNA synthesis was also induced by a polyclonal anti-DP IV immunoglobulin. (c) As a consequence of impaired T cell function the production of immunoglobulins by pokeweed mitogen-stimulated lymphocytes is also markedly reduced in the presence of DP IV inhibitors.

Antibody Formation↗