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PubMed1980

Dipeptidyl peptidase II of bovine dental pulp. Initial demonstration and characterization as a fibroblastic, lysosomal peptidase of the serine class active on collagen-related peptides.

Dipeptidyl peptidase II (dipeptidylpeptide hydrolase, EC 3.4.14.2), previously known as dipeptidyl aminopeptidase II, was shown to be present in relatively high concentrations in bovine dental pulp. Based on the DNA content of tissue homogenates, the fibroblasts of this connective tissue appeared to contain more dipeptidyl peptidase II than the cells of lysosome-rich tissues such as bovine spleen and rat liver. The newly-recognized properties of dipeptidyl peptidase II, from both pulp and pituitary sources, included a marked propensity for hydrolyzing prolyl bonds at acidic pH. Lys-Pro-2-NNap and Lys-Pro-2(4-methoxy)naphthylamide (designed for cytochemical application) were hydrolyzed at rates equal to that of Lys-Ala-2-NNap. The impure pulp enzyme and the authentic pituitary enzyme showed comparable relative rates of hydrolysis on a variety of fluorogenic substrates with the general structure X-Pro-2-NNap (X = Lys, Arg, Phe, Ala or Gly), and on a variety of collagen-related tripeptides represented by X-Pro-Ala (X = Gly, Ala or Lys). The highest rates wee obtained on Lys-Ala-Ala and Lys-Ala-Pro. The pH optima for the hydrolysis of the 2-naphthylamide derivatives varied from 5.0 to 5.7, and for tripeptides from 4.2 to 5.3. In all cases the N-terminal dipeptide was released intact. Although previously unrecognized as a serine protease, dipeptidyl peptidase II (of pulp and pituitary origin) was strongly inhibited by (1 mM) diisopropyl phosphorofluoridate, p-nitrophenyl-p'-guanidinobenzoate, and phenylmethylsulfonyl fluoride. The enzyme, from both sources, was fully inhibited by 0.1 mM Lys-Ala chloromethyl ketone, a newly-designed, active-site-directed inhibitor. The numerous properties shared by the putative dipeptidyl peptidase II of bovine dental pulp and an authentic preparation of the bovine pituitary enzyme provided strong support for their having a common identity.

Animals↗

PubMed2001

Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum--a new proline-specific peptidase.

The study of a new proline-specific peptidase from bovine serum is presented. The enzyme readily cleaves the prolyl oligopeptidase (PO) substrate Z-Gly-Pro-MCA, liberating the fluorophore MCA, thus allowing quantification of enzyme activity. Unlike PO, however, this peptidase is completely insensitive to the PO-specific inhibitor Z-Pro-prolinal and has been designated Z-Pro-prolinal-insensitive Z-Gly-Pro-MCA-hydrolyzing peptidase (ZIP). The two peptidases were successfully separated from each other by phenyl Sepharose hydrophobic interaction chromatography and the subsequent purification focused on the isolation of ZIP from bovine serum. In addition to phenyl Sepharose, calcium phosphate cellulose and DEAE anion-exchange chromatography were employed in the purification, with an overall enzyme yield of 33% and a purification factor of 4023. SDS-PAGE and size-exclusion chromatography indicated a dimeric structure with a relative molecular mass of 174 kDa. The enzyme was stable over the pH range 2.5-10.0. Optimal activity was detected in the pH range 7.4-8.0. Isoelectric focusing revealed a pI of 5.68. Inhibition by AEBSF suggests the peptidase may be a serine protease and ZIP possibly contains a cysteine residue near the active site. alpha(2)M failed to inhibit activity, suggesting oligopeptidase specificity. HPLC analysis revealed a broad substrate specificity for proline-containing peptides. Kinetic analysis indicated that ZIP had a high affinity for Z-Gly-Pro-MCA with a K(m) of 54 microM deduced. Bovine serum ZIP exhibits biophysical characteristics both similar to and different from those of PO isolated from a number of sources and may serve an important physiological function in the degradation of bioactive oligopeptides.

Animals↗

PubMed2004

Regulation of signal peptidase by phospholipids in membrane: characterization of phospholipid bilayer incorporated Escherichia coli signal peptidase.

Prokaryotic signal peptidases are membrane-bound enzymes. They cleave signal peptides from precursors of secretary proteins. To study the enzyme in its natural environment, which is phospholipid bilayers, we developed a method that allows us effectively to incorporate full-length Escherichia coli signal peptidase I into phospholipid vesicles. The membrane-bound signal peptidase showed high activity on a designed substrate. The autolysis site of the enzyme is separated from its catalytic site in vesicles by the lipid bilayer, resulting in a dramatic decrease of the autolysis rate. Phosphotidylethanolamine, which is the most abundant lipid in Escherichia coli inner membrane, is required to maintain activity of the membrane-incorporated signal peptidase. The maximal activity is achieved at about 55% phosphotidylethanolamine. Negatively charged lipids, which are also abundant in Escherichia coli inner membrane, enhances the activity of the enzyme too. Its mechanism, however, cannot be fully explained by its ability to increase the affinity of the substrate to the membrane. A reaction mechanism was developed based on the observation that cleavage only takes place when the enzyme and the substrate are bound to the same vesicle. Accordingly, a kinetic analysis is presented to explain some of the unique features of phospholipid vesicles incorporated signal peptidase, including the effect of lipid concentration and substrate-vesicle interaction.

Bacteriolysis↗

PubMed1984

Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.

Binding Sites↗

PubMed1999

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases.

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.

Amino Acid Sequence↗

PubMed1986

Histochemical localization of cathepsin B, dipeptidyl peptidase I, and dipeptidyl peptidase II in rat bone.

The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues.

Animals↗

PubMed2000

Molecular characterization of dipeptidyl peptidase activity in serum: soluble CD26/dipeptidyl peptidase IV is responsible for the release of X-Pro dipeptides.

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV-like activity in serum. Using ADA-affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA-binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N-terminal sequence, glycosylation type, CD-spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.

Amino Acid Sequence↗

PubMed1990

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.

Adult↗

PubMed1992

The activity of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice immunized with type II collagen.

We investigated the activity of peptidases in the serum of mice with experimental polyarthritis that was induced by the injection of type II collagen, an experimental model of human rheumatoid arthritis. The activity of dipeptidyl peptidase II (DPP II) was increased and that of dipeptidyl peptidase IV (DPP IV) was decreased resulting in the significant increase of the serum DPP II/DPP IV ratio in the polyarthritic mice compared with that of controls. These results indicate that the DPP II/DPP IV ratio is a novel index of disease activity in mice with collagen-induced polyarthritis and may be useful in assessing the activity of rheumatoid arthritis in humans.

Animals↗

PubMed2004

Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity.

Dipeptidyl peptidase (DP) IV has a distinct substrate specificity in hydrolyzing a post-proline bond. Here we present novel data on the sizes and tissue distribution of human and rat gene products and the peptidase activity of the DPIV-related gene DP9. A short cDNA of 2589 bp and a long cDNA of 3006 bp of DP9 were cloned. A ubiquitous predominant DP9 mRNA transcript at 4.4 kb represented the short form, whereas a less abundant 5.0-kb transcript present predominantly in muscle represented the long form. Both forms of DP9 have no transmembrane domain and two potential N-linked glycosylation sites. DP9 exhibited post-proline dipeptidyl aminopeptidase activity and was a cytoplasmic, 110-kDa monomer. Thus, the six DPIV gene family members have diverse characteristics: only DP9 and DP8 have exclusively cytoplasmic localization and only DP9, DP8, fibroblast activation protein (FAP) and DPIV have peptidase activity.

Adenosine Deaminase↗

PubMed2000

Tripeptidyl-peptidase I deficiency in classical late-infantile neuronal ceroid lipofuscinosis brain tissue. Evidence for defective peptidase rather than proteinase activity.

Brain tissue from patients with classical late-infantile neuronal ceroid lipofuscinosis (LINCL, an infantile form of Batten disease) is deficient in the lysosomal enzyme tripeptidyl-peptidase I (EC 3.4.14.9). The activities of other lysosomal enzymes are either increased or decreased. Tripeptidyl-peptidase I is a pepstatin-insensitive exo-tripeptidase, with little or no endo-proteolytic activity, that is active on small peptides but not on large proteins. Using haemoglobin and casein as substrates for proteolytic activity, we were unable to demonstrate any significant defect in pepstatin-sensitive or pepstatin-insensitive proteinase activity in brain tissue or cultured skin fibroblasts of LINCL patients. These observations suggest that the lysosomal storage of undegraded, small peptides in LINCL results from the absence of peptidase rather than proteinase activity.

Adult↗

PubMed2005

Kinetic investigation of human dipeptidyl peptidase II (DPPII)-mediated hydrolysis of dipeptide derivatives and its identification as quiescent cell proline dipeptidase (QPP)/dipeptidyl peptidase 7 (DPP7).

The presence of DPPII (dipeptidyl peptidase II; E.C. 3.4.14.2) has been demonstrated in various mammalian tissues. However, a profound molecular and catalytic characterization, including substrate selectivity, kinetics and pH-dependence, has not been conducted. In the present study, DPPII was purified from human seminal plasma to apparent homogeneity with a high yield (40%) purification scheme, including an inhibitor-based affinity chromatographic step. The inhibitor lysyl-piperidide (K(i) approximately 0.9 microM at pH 5.5) was chosen, as it provided a favourable affinity/recovery ratio. The human enzyme appeared as a 120 kDa homodimer. Mass spectrometric analysis after tryptic digestion together with a kinetic comparison indicate strongly its identity with QPP (quiescent cell proline dipeptidase), also called dipeptidyl peptidase 7. pH profiles of both kcat and kcat/K(m) clearly demonstrated that DPPII/QPP possesses an acidic and not a neutral optimum as was reported for QPP. Kinetic parameters of the human natural DPPII for dipeptide-derived chromogenic [pNA (p-nitroanilide)] and fluorogenic [4Me2NA (4-methoxy-2-naphthylamide)] substrates were determined under different assay conditions. DPPII preferred the chromogenic pNA-derived substrates over the fluorogenic 4Me2NA-derived substrates. Natural human DPPII showed high efficiency towards synthetic substrates containing proline at the P1 position and lysine at P2. The importance of the P1' group for P2 and P1 selectivity was revealed, explaining many discrepancies in the literature. Furthermore, substrate preferences of human DPPII and dipeptidyl peptidase IV were compared based on their selectivity constants (kcat/K(m)). Lys-Pro-pNA (k(cat)/K(m) 4.1x10(6) s(-1) x M(-1)) and Ala-Pro-pNA (kcat/K(m) 2.6x10(6) s(-1) x M(-1)) were found to be the most sensitive chromogenic substrates for human DPPII, but were less selective than Lys-Ala-pNA (kcat/K(m) 0.4x10(6) s(-1) x M(-1)).

Amino Acid Sequence↗

PubMed1987

Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis.

We examined the activities of peptidases in plasma and tissues of the New Zealand Black (NZB) mouse as an animal model of human systemic lupus erythematosus, and also in serum from patients with rheumatoid arthritis and systemic lupus erythematosus. Activities of dipeptidyl peptidase II (DAP II) and post-proline cleaving enzyme (PPCE) were increased, and dipeptidyl peptidase IV (DAP IV) activity was decreased in plasma and spleen of NZB mice, as compared with the control BALB/c mice. Likewise, the activity of DAP II was increased and that of DAP IV was decreased in serum of patients with rheumatoid arthritis and systemic lupus erythematosus. These results indicate the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.

Animals↗

PubMed2006

Compensatory effect of the minor Streptomyces lividans type I signal peptidases on the SipY major signal peptidase deficiency as determined by extracellular proteome analysis.

The developmentally complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of protein and possesses four different type I signal peptidase genes (sipW, sipX, sipY and sipZ) that are unusually clustered in its chromosome. 2-DE and subsequent MS of extracellular proteins showed that proteins with typical export signals for type I and type II signal peptidases are the main components of the S. lividans secretome. Secretion of extracellular proteins is severely reduced in a strain deficient in the major type I signal peptidase (SipY). This deficiency was efficiently compensated by complementation with any of the other three signal peptidases as deduced from a comparison of the corresponding 2-D PAGE patterns with that of the wild-type strain.

Amino Acid Sequence↗

PubMed2005

Mitochondrial signal peptidases of yeast: the rhomboid peptidase Pcp1 and its substrate cytochrome C peroxidase.

The rhomboid peptidase Pcp1 of yeast is the first mitochondrial enzyme of this new class of serine peptidases. Pcp1 is an integral part of the inner membrane and was identified by its signal peptidase activity responsible for processing of the intermediate of cytochrome c peroxidase (iCcp1) to the mature enzyme. Here we describe studies on the expression of the PCP1 gene. Proteolytic processing of Pcp1 itself was found. The precursor and the intermediate of Ccp1 were localized to the inner membrane. The results confirm our previous report on a two-step processing pathway of cytochrome c peroxidase and the identification of the signal peptidases involved.

Cytochrome-c Peroxidase↗

PubMed2006

Discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 by a single mutation.

It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is approximately 7 times higher than the peptidase activity with Ac-Leu-p-nitroanilide as substrate. However, with the same substrates, this difference was increased to approximately 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on k(cat) and K(m) of the enzyme were discussed based on molecular dynamics simulation studies.

Aeropyrum↗

PubMed1984

Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas.

Signal peptidase has been enriched extensively from microsomal membranes of porcine pancreas. Microsomal membranes were washed with 1 M KCl and Brij 35, and then solubilized with 1% Nonidet P-40. The solubilized signal peptidase was purified by DEAE-cellulose chromatography and Sepharose CL-6B filtration. Cleavage of pre-human placental lactogen with the partially purified enzyme gave the mature form, whose NH2-terminus was identified as valine. The signal peptidase is heat-labile and approximately 90% of the enzymatic activity was lost at 60 degrees C within 1 min. The pH optimum of the activity was 7 to 8. Chymostatin and o-phenanthroline at concentrations of 2.5 mM inhibited the signal peptidase activity by 62% and 30%, respectively.

Amino Acid Sequence↗

PubMed2000

Peptidase E, a peptidase specific for N-terminal aspartic dipeptides, is a serine hydrolase.

Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (10(4)-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available.

Amino Acid Sequence↗