Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.     

Presence of dipeptidyl peptidase II, dipeptidyl peptidase IV, and prolyl endopeptidase in effusion from patients with serous otitis media

We have measured for the first time, using specific substrates and specific fluorometric analyses, activities of three pathophysiologically important peptidases, i.e., dipeptidyl peptidase II, dipeptidyl peptidase IV, and prolyl endopeptidase in effusions from 45 patients with chronic otitis media with effusion. In 20 patients, DPP II and DPP IV were assayed simultaneously in effusions and sera. Activity of PEP was also estimated in effusions and sera from 25 patients. The mean values (+/- SD) of DPP II and DPP IV (n = 45) and PEP (n = 25) in effusion from patients with OME were 0.020 +/- 0.007, 0.66 +/- 0.04, and 0.040 +/- 0.006 nmole/min/mg protein, and 0.21 +/- 0.01, 16.2 +/- 1.87, and 1.90 +/- 0.23 nmole/min/ml of effusion, respectively. The mean values (+/- SD) for DPP II, DPP IV, and PEP in sera were 2.82 +/- 0.18, 54.8 +/- 1.23, and 3.73 +/- 0.33 nmole/min/ml of serum, respectively, which were similar to our previously reported values. Activities of DPP II, DPP IV, and PEP of serous effusions were comparable to those in serum. However, there was no significant correlation between their activities in serum and effusion. This may suggest that the major source of these enzymes in effusions may not be serum but the cells in the middle ear.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.

We examined the activities of peptidases in the synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Dipeptidyl peptidase II (DPP II), prolyl endopeptidase (PEP), and collagenase-like peptidase (CLP) activities were higher in knee joint synovial membrane from patients with RA than in that from patients with OA. DPP II and PEP activities in knee joint synovial membrane of patients with RA increased in parallel with the increase in joint fluid volume, whereas DPP IV activity decreased in parallel with the increase in joint fluid volume. These results suggest that these peptidases in the synovial membrane may play some role in immunological disturbances in the joints of patients with RA. Measurement of these peptidases in synovial membrane may be useful in the diagnosis of the severity of local joint inflammation.     

Effects of hydrosaline treatments on prolyl endopeptidase activity in rat tissues

Enzymatic cleavage of some peptide hormones, neurotransmitters and neuromodulators could be implicated in the regulation of extra- and intracellular fluid volume and osmolality. Prolyl endopeptidase is known to hydrolyze several peptides, which act on hydromineral balance, such as angiotensins, bradykinin, vasopressin, oxytocin, thyrotropin-releasing hormone, neurotensin and opioids. In this work, we analyzed the effects of certain volume and/or osmotic changes in the activity of the soluble and membrane-bound prolyl endopeptidase in several brain areas, heart, lungs, kidney and adrenal and pituitary glands of the rat. Soluble prolyl endopeptidase activity was higher in the renal cortex of the chronic salt-loaded rats than in the control rats. In the water-deprived and polyethylene glycol-treated rats, heart particulate prolyl endopeptidase was lower than in the control rats. Particulate prolyl endopeptidase was also lower in the adrenal gland of the acute salt-loaded rats and in the brain cortex of the water-loaded rats than in the control rats. Data suggest that tissue-dependent peptide hydrolysis evoked by prolyl endopeptidase activity is involved in the water-electrolyte homeostasis.     

Regulation of prolyl endopeptidase activity by the intracellular redox state

The activity of prolyl endopeptidase was markedly decreased during incubation of intact murine erythroleukemia cells at 45 degrees C, but not during incubation of sonicated cells or during incubation at 42 degrees C. The thermal inactivation of prolyl endopeptidase in situ required neither the synthesis of proteins and polynucleotides nor the synergistic activation of inhibitors. Moreover, inhibition of lysosomal proteinases and calpains or depletion of ATP did not affect the thermal inactivation of prolyl endopeptidase. This specific inactivation of prolyl endopeptidase was also observed following the addition to the culture medium of menadione or diamide, compounds known to increase intracellular oxidized glutathione levels. The activity of prolyl endopeptidase in the cell lysate was also dose-dependently decreased by the addition of glutathione disulfide and the decrease of the activity was prevented by coexistence of reduced glutathione. Furthermore, the level of intracellular oxidized glutathione was increased during incubation at 45 degrees C for 15 min, but not at 42 degrees C for 30 min. These results strongly suggest that the activity of prolyl endopeptidase is regulated by changes in the intracellular redox potential.     

Human erythrocyte prolyl endopeptidase II hydrolysing teprotid, an inhibitor of peptidyl peptidase from snake venom

The paper is concerned with the action of 1200-fold purified prolylendopeptidase II (PE-E) from human erythrocytes and the action of highly purified prolyl-D-L-alanine peptidyl hydrolase (PE-A) from bovine adenohypophysis on teprotide (BPP9a, SQ 20881), a nonapeptide from venom of the snake Bothrops Jararaca--an inhibitor of peptidyl dipeptidase A (carboxycathepsin). Both the purified preparation PE-E and highly purified preparation PE-A split teprotide at the bonds Pro3-Arg4 and Pro5-Gln6. The Pro8-Pro9-OH bond was not split by the two enzymes. The comparative characteristics of the properties of PE-E and PE-A are presented and the possible physiological role of these enzymes is discussed.     

Screening of crude drug extracts for prolyl endopeptidase inhibitory activity.

Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes. Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition. Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var. trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity. In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition.     

Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy

We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide. Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity. The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo. While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability. A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer. Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan. (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase [specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C]. The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan. The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis. The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications.     

Porcine muscle prolyl endopeptidase and its endogenous substrates

Prolyl endopeptidase [EC 3.4.21.26] was purified 4,675-fold with a yield of 26.3% from porcine muscle. The purified enzyme was shown to be very similar to the liver enzyme with respect to its molecular weight (72,000-74,000), antigenicity, substrate specificity, and susceptibility to protease inhibitors. Among several bioactive peptides, angiotensins I, II, and III had the lowest Km of 0.6 to 3 microM with the lowest kcat of 0.19 to 0.85 s-1, while thyrotropin-releasing hormone had the highest Km of 98 microM with the highest kcat of 14.4 s-1. Interestingly, mastoparan was hydrolyzed at alanyl bonds, but insulin was only slightly hydrolyzed and glucagon was not hydrolyzed although the latter two peptides contain prolyl and/or alanyl bonds. Muscle prolyl endopeptidase failed to hydrolyze proteins with high molecular weight such as albumin, immunoglobulin G, elastin, collagen, and muscle soluble and insoluble proteins. However, 8 of 14 peptides with molecular weights lower than 3,000, which were isolated from muscle extract, were digested by this enzyme, and they were proved to contain prolyl and/or alanyl residues in their molecules. The data suggest that they are probable endogenous substrates for prolyl endopeptidase.     

Purification and characterization of prolyl endopeptidase from pig brain

The prolyl endopeptidase from pig brain was purified to homogeneity according to SDS-gel electrophoresis and visualization with the silver staining procedure. The molecular weight of prolyl endopeptidase was estimated as 70 kDa, and the isoelectric point as 4.9. The molecular properties of prolyl endopeptidase from pig brain are therefore similar to those of prolyl endopeptidases from other mammalian tissues. Diisopropylfluorophosphate, diethylpyrocarbonate and p-chloromercuribenzoic acid are strong irreversible inhibitors of prolyl endopeptidase from pig brain. We showed that diisopropylfluorophosphate und diethylpyrocarbonate act as competitive inhibitors with respect to substrate. Therefore it is assumed that at least one serine and one histidine residue are located at the active site of this enzyme. This result supports the assumption that the prolyl endopeptidase from pig brain is a typical serine protease. Substance P, thyreoliberin, beta-casomorphin-5 and morphiceptin are hydrolysed by prolyl endopeptidase in vitro.     

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.     

Porcine muscle prolyl endopeptidase: limited proteolysis of tryptic peptides from hemoglobin beta-chains at prolyl and alanyl bonds

Tryptic peptides from hemoglobin (Hb) beta-chains were used as model substrates for limited proteolysis by prolyl endopeptidase (EC 3.4.21.26) from porcine muscle. From the physicochemical and enzymatic properties of prolyl endopeptidase the conditions for routine digestion were established as follows: the molar ratio of enzyme to substrate was 1 to 100, and the reaction was carried out in sodium phosphate buffer (pH 6.4) at 37 degrees C for 4 h. Under these conditions the peptide bonds on the carboxyl terminal sides of proline and alanine residues in the tryptic peptides from Hb beta-chains (with Mr values of less than 2100) were hydrolyzed by the enzyme with the exception of the amino terminal alanyl bond and aminoacyl alanyl bond. In addition, one of five seryl bonds was cleaved by the enzyme. However, the Hb beta-chain itself, Mr 16,600, and its two CNBr-peptides with Mr 10,200 and Mr 6400, respectively, were not hydrolyzed. Under the same conditions a prolyl bond in oxidized B-chains of insulin, Mr 3400, was partially digested, and an alanyl bond was not hydrolyzed. The data indicate that the prolyl endopeptidase is useful for the limited proteolysis of peptides with relative masses of less than 3000 at both prolyl and alanyl bonds.     

A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase

The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1' position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1' position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.     

No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population

Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.     

Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time-dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation-induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.     

Automated parallel synthesis of a tetrahydroisoquinolin-based library: potential prolyl endopeptidase inhibitors

Solution-phase automated parallel synthesis of a Tic-based library is described. This library comprising 2560 members, was obtained from the combination of 80 carboxylic acids and 32 amines and was screened against Tc80 protease, a parasitic prolyl endopeptidase secreted by Trypanosoma cruzi. Pyrrolidine derivatives proved the most potent inhibitors with IC50 values found in the low nanomolar range.