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PubMed2002

Tripeptidyl-peptidase II expression and activity are increased in skeletal muscle during sepsis.

Ubiquitin-proteasome-dependent protein degradation plays a central role in sepsis-induced muscle wasting. Because the proteasome degrades proteins into small peptides rather than free amino acids, it is likely that additional mechanisms downstream of the proteasome are involved in sepsis-induced muscle proteolysis. Recent studies suggest that the extralysosomal peptidase tripeptidyl-peptidase II (TPP II) degrades peptides generated by the proteasome. We hypothesized that TPP II expression and activity are increased in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. TPP II activity was determined by using the specific substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-AMC). TPP II protein and gene expression were determined by Western blot and real-time PCR, respectively. Sepsis resulted in increased activity and protein and gene expression of TPP II in extensor digitorum longus muscles. This result was blunted by the glucocorticoid receptor antagonist RU 38486, indicating that glucocorticoids participate in the upregulation of TPP II in skeletal muscle during sepsis. The results suggest that proteolytic mechanisms downstream of the proteasome may be important for the complete degradation of muscle proteins during sepsis.

Aminopeptidases↗
PubMed1980

Rapid chromatographic purification of dipeptidyl peptidase IV in human submaxillary gland.

Pure dipeptidyl peptidase IV (X-prolyl dipeptidyl aminopeptidase), which did not contain aminopeptidase activity at all, was rapidly prepared from the human submaxillary gland by chromatography with concanavalin A-Sepharose and Gly-Pro-NH-(CH2)6-NH-Sepharose. The entire purification took only 3 days. Aminopeptidase, which was very difficult to separate from dipeptidyl peptidase IV by various chromatographic procedures, could be completely removed by chromatography with Gly-Pro-NH-(CH2)6-NH-Sepharose. On SDS gel electrophoresis the purified enzyme gave a single band with a molecular weight of 116,000. The apparent molecular weight of the enzyme was estimated to be 225,000 by gel filtration. Therefore, the enzyme consists of two identical subunits. It did not hydrolyze Ala p-nitroanilide at all, but the hydrolysis of the p-nitroanilides of Gyl-Pro, Lys-Pro and Arg-Pro at pH 8.0 was nearly specific.

Chromatography, Gel↗
PubMed2003

Isolation and properties of a tripeptidyl peptidase from a periodontal pathogen Prevotella nigrescens.

Prolyltripeptidyl amino peptidase activity was found in a crude extract of Prevotella nigrescens and this enzyme was purified by procedures including concentration with ammonium sulfate, ion exchange chromatography, gel filtration, and isoelectric focusing. This peptidase hydrolyzed Ala-Ala-Pro-p-nitroanilide as well as Ala-Phe-Pro-p-nitroanilide. Furthermore, several p-nitroanilide derivatives of dipeptides with a proline residue in the second position from the amino-terminal end (Xaa-Pro) were also cleaved detectably. The molecular mass of this tripeptidase was calculated as 56 kDa and its isoelectric point was 5.8. The enzyme was inactivated completely by heating at 60 degrees C for 5 min and inhibited significantly by specific serine enzyme inhibitors.

Aminopeptidases↗
PubMed2002

Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis.

Tripeptidyl-peptidase II (TPP II) is a 138-kDa subtilisin-like serine peptidase forming high molecular mass oligomers of >1000 kDa. The enzyme participates in general protein turnover and apoptotic pathways, and also has specific substrates such as neuropeptides. Here we report the site-directed mutagenesis of amino acids predicted to be involved in catalysis. The amino acids forming the putative catalytic triad (Asp-44, His-264, Ser-449) as well as the conserved Asn-362, potentially stabilizing the transition state, were replaced by alanine and the mutated cDNAs were transfected into human embryonic kidney (HEK) 293 cells. In clones stably expressing the mutant proteins, TPP II activity did not exceed the endogenous activity, thus confirming the essential role of the above amino acids in catalysis. Mutant and wild-type TPP II subunits co-eluted from a gel filtration column, suggesting that the subunits associate and that the native subunit conformation was retained in the mutants. Interestingly, the S449A and a H264A mutant enzyme affected the quaternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of >10,000 kDa.

Amino Acid Sequence↗
PubMed1989

Expression of enzymatically active rat dipeptidyl peptidase IV in Chinese hamster ovary cells after transfection.

Dipeptidyl peptidase IV (DPPIV) is a cell surface membrane glycoprotein expressed in many tissues. We have subcloned the coding region of a full-length cDNA for DPPIV into the inducible eukaryotic expression vector pMSG. The resulting construct was used to transfect Chinese hamster ovary (CHO) cells. Stable transformants were found to express DPPIV, and the expression is enhanced by dexamethasone. Metabolic labeling of the transfected cells with [35S]Met followed by immunoprecipitation revealed the presence of two specific products of apparent Mr 100,000 (100-kDa form) and 110,000 (110-kDa form), respectively. Pulse-chase experiments demonstrated that the 100-kDa form can be chased into the 110-kDa form, suggesting the 100-kDa form is the precursor of the 110-kDa form. Further studies with endo H treatment demonstrated that the carbohydrate structures are of the high-mannose type, and of the complex type for the 100- and 110-kDa forms, respectively. The 110-kDa form is present at the cell surface as shown by its accessibility to cell surface iodination. The DPPIV expressed on the cell surface is resistant to digestion by relatively high concentrations of trypsin. Studies also demonstrated that the surface DPPIV is fairly stable with a half-life for turnover of about 40 h. Furthermore, the DPPIV produced in the transfected cells displays specific dipeptidyl peptidase activity. The stably transfected cells that express enzymatically active DPPIV in an inducible manner will provide an excellent system for further biochemical, functional, and cell biological characterizations of DPPIV.

Animals↗
PubMedDate not supplied

Rapid parallel synthesis of dipeptide diphenyl phosphonate esters as inhibitors of dipeptidyl peptidases.

In this paper, we present a parallel synthesis of several series of dipeptide diphenyl phosphonates that are known to be irreversible inhibitors of serine proteases. Polymer-assisted solution-phase synthesis (PASP) is used for the rapid and clean coupling between various alpha-aminoalkyl diphenyl phosphonate ester building blocks and commercially available or easily accessible amino acids. These compounds were used for the rapid profiling of dipeptidyl peptidase II (DPP II) and the closely related dipeptidyl peptidase IV (DPP IV). A highly selective DPP II inhibitor was identified, N-cyclopentylglycyl-NHCH(C(6)H(5))PO(OPh)(2) (9.35), that will be useful to discriminate between DPP II and DPP IV in biological systems in order to further elucidate the biological function of DPP II.

Alanine↗
PubMed2001

Cloning and functional expression of rat kidney dipeptidyl peptidase II.

Dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from rat kidney was purified to a specific activity of 65.4 micromol/min per mg of protein for Lys-Ala-beta-naphthylamide. The N-terminal and partial amino acid sequences of the enzyme were determined. The peptide sequences were used to identify expressed sequence tag (EST) clones. By using the cDNA fragment of one of the EST clones as a probe, we isolated a cDNA clone with 1710 bp encoding DPP II from a rat kidney cDNA library. The cDNA of rat DPP II contained an open reading frame of 1500 bp, coding for a protein of 500 amino acids. The first 10 residues of the purified enzyme matched the deduced protein sequence starting with residue 37, suggesting the presence of a signal peptide. The mature enzyme (464 residues) had a calculated molecular mass of 51400 Da, which was lower than the value (about 60000 Da) determined by SDS/PAGE; and the deduced amino acid sequence showed six potential N-glycosylation sites. The deduced amino acid sequence of rat DPP II shared high similarity with quiescent-cell proline dipeptidase (78% identity) and prolyl carboxypeptidase (38% identity) and bore the putative catalytic triad (Ser, Asp, His) conserved in serine peptidase families. We transiently transfected COS-7 cells with pcDNA3.1 containing the cloned cDNA and obtained the overexpression of an immunoreactive protein (of about 60000 Da). The transfected cells showed Lys-Ala-methylcoumarinamide-hydrolysing activity that was 50 times higher than the control cells.

Amino Acid Sequence↗
PubMed1987

Tripeptidyl peptidase II in haemolysates and liver homogenates of various species.

Rabbit antibodies against purified tripeptidyl peptidase II (TPP II) of human erythrocytes have been prepared and used in immunoblot analysis. Antibodies affinity-purified against the denatured 135,000-Mr subunit of the human peptidase were shown to cross-react with purified rat liver TPP II, as well as a polypeptide of Mr 135,000 in haemolysates and liver homogenates from several other species. TPP II activity in haemolysates from monkey, hog, horse and rabbit corresponded to the levels found in human erythrocytes, whereas this activity was absent or low in erythrocytes from calf and hen. On immunoblot analysis of the calf haemolysates, the 135,000-Mr polypeptide could not be detected. In contrast, analysis of liver homogenates from these species revealed both TPP activity and immunoreactive polypeptides. Immunoreactive polypeptides with Mr about 105,000 and 84,000 of variable occurrence were also detected. In addition, extracts of Escherichia coli showed no TPP II activity under the conditions of the standard assay, although polypeptides of Mr 135,000 and 40,000 were revealed on immunoblot analysis of this species.

Aminopeptidases↗
PubMed1987

Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity.

Tripeptidyl peptidase II is an extralysosomal serine peptidase of an unusually large size, i.e. Mr greater than 10(6) for the native enzyme and Mr 135000 for the subunit. The enzyme from human erythrocytes was studied by electron microscopy on samples negatively stained by ammonium molybdate. Two different structural representations of the purified enzyme were obtained, both with a length of about 50 nm, and consisting of repetitive substructures. Upon dialysis of the enzyme against a Tris/HCl buffer, the activity was gradually decreased. This decrease was shown to parallel the dissociation of the large enzyme structures into smaller ones, the smallest measuring 3 nm by 10 nm and apparently corresponding to the repetitive substructures. The results indicate that a large polymeric form of the enzyme is a prerequisite for full activity.

Aminopeptidases↗
PubMed1994

Characterization of cDNA for murine tripeptidyl-peptidase II reveals alternative splicing.

Tripeptidyl-peptidase II (TPP II) is a cytosolic high-M(r) exopeptidase with an active site of the subtilisin type. This paper describes cloning of cDNA encoding murine TPP II. Four clones were isolated from a murine mastocytoma cDNA library and the 5'-end was isolated by use of 5'-RACE (rapid amplification of cDNA ends). A total of 4611 bp were isolated, including the complete coding region. The deduced amino acid sequence shows a 96% overall identity when compared with the previously cloned human TPP II. The remarkably high identity indicates that not only the catalytic domain, but almost the entire subunit, must be of functional importance. Alignment with subtilisin-like serine peptidases identified Asp44, His264 and Ser449 as the catalytic triad, thus defining an extra domain of approximately 200 amino acids between the catalytic Asp and His in TPP II as compared with other subtilases. In addition, it was demonstrated that different polyadenylation signals can be utilized, since two different clones with untranslated 3'-ends of 155 bp and 781 bp respectively have been isolated. Finally, one of the isolated clones contains an extra 39 bp insert encoding 13 amino acids, which implies alternative splicing of the mRNA.

Alternative Splicing↗
PubMed1999

Increased collagenase and dipeptidyl peptidase I activity in leucocytes from healthy elderly people.

The incidence of infectious diseases increases with ageing. The enzymatic activity of leucocytes may have a relevant role in the morbidity and mortality due to infections in the elderly. In this study we have compared the activity of enzymes involved in the inflammatory response in leucocytes from young and elderly women. A total of 35 healthy females was studied, 20 volunteers aged 78-98 years (mean 89.1 years) and 15 young controls aged 19-34 years (mean 26 years). All of them were in good clinical condition, without any acute or chronic disease. Intracellular enzyme activity was analysed by flow cytometry in leucocytes from young and elderly women. The enzyme substrates employed were for oxidative burst, L-aminopeptidase, collagenase, cathepsin B, C, D and, G and dipeptidyl peptidase I. The intracellular enzyme activity assessed by flow cytometry in leucocytes from young and elderly women was similar, as far as oxidative burst, L-aminopeptidase, cathepsin B, C, D and G are concerned. An increased collagenase activity was detected in granulocytes from elders. The mean fluorescence channels for this enzyme corresponded to 86 +/- 23 and 60 +/- 15 in cells from elders and controls, respectively (P = 0.01224). An increased dipeptidyl peptidase I activity was detected in lymphocytes from elderly women. The corresponding values for this enzyme in elders and the young were 65.9 +/- 43.3 and 17.3 +/- 5, respectively (P = 0. 0036). The proper functional activity of intracellular enzymes involved in inflammatory responses is likely to be determinant for successful ageing.

Adult↗
PubMed2005

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II.

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Aminopeptidases↗
PubMed1998

Characterization and cloning of tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster.

We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.

Amino Acid Sequence↗
PubMed1994

Activation of thrombin-inactivated single-chain urokinase-type plasminogen activator by dipeptidyl peptidase I (cathepsin C).

Single-chain urokinase-type plasminogen activator (scu-PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), we found that the lysosomal aminopeptidase dipeptidyl-peptidase I or cathepsin C efficiently activates tcu-PA/T. Cathepsin C was as active towards tcu-PA/T as the bacterial proteinase thermolysin and about 300-times more active than plasmin. The activation by cathepsin C proceeded in a concentration-dependent and time-dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu-PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu-PA/T occurs by cleavage between Lys158-Ile159 and removal of the two N-terminal amino acid residues (Phe157-Lys158) of the B chain of tcu-PA/T. We conclude that both thrombin and dipeptidyl-peptidases like cathepsin C might play a regulatory role in the plasminogen-plasmin system by inactivating scu-PA and activating tcu-PA/T, respectively.

Cathepsin C↗
PubMed2002

Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis.

A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases.

Alanine↗
PubMed2002

Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease.

Cysteine peptidases (CPs) are phylogenetically ubiquitous enzymes that can be classified into clans of evolutionarily independent proteins based on the structural organization of the active site. In mammals, two of the major clans represented in the genome are: the CA clan, whose members share a structure and evolutionary history with papain; and the CD clan, which includes the legumains and caspases. This review focuses on the properties of these enzymes, with an emphasis on their potential roles in the oral cavity. The human genome encodes at least (but possibly no more than) 11 distinct enzymes, called cathepsins, that are members of the papain family C1A. Ten of these are present in rodents, which also carry additional genes encoding other cathepsins and cathepsin-like proteins. Human cathepsins are best known from the ubiquitously expressed lysosomal cathepsins B, H, and L, and dipeptidyl peptidase I (DPP I), which until recently were considered to mediate primarily "housekeeping" functions in the cell. However, mutations in DPP I have now been shown to underlie Papillon-Lefevre syndrome and pre-pubertal periodontitis. Other cathepsins are involved in tissue-specific functions such as bone remodeling, but relatively little is known about the functions of several recently discovered enzymes. Collectively, CPs participate in multiple host systems that are active in health and in disease. They are involved in tissue remodeling and turnover of the extracellular matrix, immune system function, and modulation and alteration of cell function. Intracellularly, CPs function in diverse processes including normal protein turnover, antigen and proprotein processing, and apoptosis. Extracellularly, they can contribute directly to the degradation of foreign proteins and the extracellular matrix. However, CPs can also participate in proteolytic cascades that amplify the degradative capacity, potentially leading to pathological damage, and facilitating the penetration of tissues by cancer cells. We know relatively little regarding the role of human CPs in the oral cavity in health or disease. Most studies to date have focused on the potential use of the lysosomal enzymes as markers for periodontal disease activity. Human saliva contains high levels of cystatins, which are potent CP inhibitors. Although these proteins are presumed to serve a protective function, their in vivo targets are unknown, and it remains to be discovered whether they serve to control any human CP activity.

Aggressive Periodontitis↗
PubMed1983

Subcellular localization of dipeptidyl peptidases II and IV in rat and rabbit alveolar macrophages.

Pulmonary alveolar macrophages, obtained from healthy adult rats and rabbits by lung lavage, were evaluated for the presence of two serine exopeptidases having affinity for penultimate prolyl residues. Dipeptidyl peptidase II was visualized using either lysyl-prolyl or lysyl-alanyl derivatives of 4-methoxy-2-naphthylamine (MNA) as synthetic substrates at pH 5.5 and employing the osmiophilic coupler hexazotized pararosanaline (HXP) for localization. Reaction product was observed in 50-90% of alveolar macrophages and was confined principally to lysosomal structures. Interestingly, not all lysosomes reacted, suggesting that they have a variable but distinct degree of heterogeneity for this protease. Dipeptidyl peptidase IV was localized using glycyl-prolyl-MNA at pH 7.3 and HXP. Reaction product was observed exclusively on the external surface of the plasma membrane of the rat only. Its intensity appeared to be greater along the outer edge of what might be interpreted as the cell's glycocalyx. These proteases, by their localization and known substrate specificity, would be expected to make a significant contribution to the alveolar macrophage's proteolytic capabilities.

Animals↗
PubMed1988

Dipeptidyl peptidase III from human erythrocytes.

Purification procedure for dipeptidyl peptidase III (DPP III) from human erythrocytes cytosol, entailing separations on DEAE-cellulose, hydroxylapatite and Sephacryl S-200 column, which gave homogeneous preparation in 35% yield, is described. The enzyme was shown to be a monomeric acidic protein (Mr approximately 82,000, pI approximately 4.5-4.6), sensitive to freezing and temperatures above 40 degrees C. It was inhibited by metallo-chelators and sulphydryl reagents, the activity being restored by divalent cations and thiol compounds. Co2 and Zn2 at low concentrations activated the enzyme, most probably by binding at the same site. Co2 prevented DPP III inactivation by di(4-pyridyl)disulfide, indicating that it is a metallo-peptidase with essential SH-groups which might be near or at the binding site for the metal. Among various naphthylamides Arg-Arg-2-naphthylamide was the best substrate (Km = 7.7 microM, kcat = 28 s-1) of the enzyme. DPP III from human erythrocytes hydrolysed also tri- to decapeptides of different composition, provided they did not have proline at P1 or P'1 position. A hydrophobic residue at P'1 was preferred. Among substrates were angiotensins and Leu-enkephalin. The enzyme showed particularly high affinity for angiotensin III.

Catalysis↗