Tripeptidyl Peptidases Research Articles

Cellular locations of proteinases and association with vesicles in porphyromonas gingivalis

We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.

Lysosomal Protease Expression in Mature Enamel

The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

Sedolisins, a New Class of Secreted Proteases fromAspergillus fumigatuswith Endoprotease or Tripeptidyl-Peptidase Activity at Acidic pHs

The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17- to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

Novel Prolyl Tri/Tetra-Peptidyl Aminopeptidase from Streptomyces mobaraensis: Substrate Specificity and Enzyme Gene Cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.

Peptidases affecting recombinant protein production by Streptomyces lividans

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.

Peptidases affecting recombinant protein production by <i>Streptomyces lividans</i>

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.

The occurrence of a tripeptidyl peptidase I in trophozoites of Entamoeba histolytica.

Tripeptidyl aminopeptidases are exopeptidases involved in intracellular protein degradation downstream of endopeptidases [for review, see (1)]. Mammalian tripeptide-releasing enzymes identified to date have been designated as tripeptidyl peptidases I (TPP I), which are localized in lysosomes, and tripeptidyl peptidases II (TPP II), which are cytoplasmatic. Additionally, evolutionarily unrelated TPPs have been identified in bacteria (1). Tripeptidases II and homologues belong to the subtilisin family of serine proteases and, like the proteasome, seem to have an ubiquitous distribution in eukaryotes, suggesting that they cooperate with the proteasome and with dipeptidases in degrading substrate proteins the entire way down into amino acids. Lysosomal TPP I, which further splits peptides generated by cathepsins, may also be a serine-type peptidase. Monomers of the enzyme, which generally are glycosylated, have a molecular mass between 48 kDa (human) and 107 kDa ( Dictyostelium ), and can form aggregates exhibiting molecular masses from 200–700 kDa (2). In addition to its role in lysosomal protein degradation, tripeptidase I has been suggested to be involved in more specific functions, such as generating signaling peptides or proteins for developmental processes (2). Entamoeba histolytica contains vesicular endopeptidases, the genes of which have been well characterized and have been shown to be cysteine proteinases (EhCP1, EhCP2, EhCP5, histolysain, and amebapain). Their activities contribute to lysosomal protein digestion and, extracellularly, to the attack of host structural proteins (3). The cytoplasm contains proteasomes that probably function in the endopeptidolytic cleavage of substrate proteins (4). By contrast, nothing is known about exopeptidases acting downstream of either cathepsins or of the proteasome in this parasite. Here, we report first data on a lysosomal/extracellular tripeptidyl peptidase I, with activity against AAF-X or AFP-X (X being a peptidolytically released fluorophor), in E. histolytica.

Prolyl Tripeptidyl Peptidase from Porphyromonas gingivalis: A NOVEL ENZYME WITH POSSIBLE PATHOLOGICAL IMPLICATIONS FOR THE DEVELOPMENT OF PERIODONTITIS

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.

Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.

Lysosomal heterogeneity of dipeptidyl peptidase II active on collagen-related peptides.

The subcellular distribution of dipeptidyl peptidase II (DPP II) in the rat kidney cortex, as determined by subfractionation of the mitochondrial/lysosomal fraction by rate sedimentation, indicated that this enzyme is mainly associated with the large, fast sedimenting lysosomes (protein droplets). The small lysosomes, on the other hand, displayed considerable size heterogeneity as indicated by the broad distribution of DPP II; cathepsin B, and a tripeptidyl peptidase active on Gly-Pro-Met-2-naphthylamide at pH 4 (TPP 4). Cathepsin D and N-acetyl-beta-D-glucosaminidase were limited primarily to the slower-sedimenting, small lysosomes. Equilibrium banding in sucrose gradients of the two main DPP II-containing lysosomal populations showed that the large lysosomes banded at a density of 1.235-1.24 g/ml while small lysosomes banded at three densities: 1.11-1.15 g/ml (lysosomal fragments), 1.20 g/ml (light lysosomes), and 1.235 g/ml (dense lysosomes). Identical distribution pattern were obtained for DPP II using either Lys-Ala-7-(4-methyl)coumarylamide or Gly-Pro-2-naphthylamide as the substrate at pH 5.5 and 5.0, respectively. Notably, DPP II and TPP 4, and cathepsin B as well, gave banding densities and distributions that were consistent with a lysosomal localization. Since triplets of the Gly-Pro-X-type released by the TPP 4 are ideal substrates for DPP II, the integrated action of tripeptidyl and dipeptidyl peptidases could make a novel contribution to the renal depolymerization and reabsorption of polypeptides, in particular the proline-rich, collagen-derived sequences that possess repeating-triplet primary structures.