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• W2038423623 abstract "A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis.This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of theStaphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, andP. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium. A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis.This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of theStaphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, andP. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium. dipeptidylpeptidase 4-morpholineethanesulfonic acid 3-(cyclohexylamino)propanesulfonic acid p-nitroanilide succinyl- benzyloxycarbonyl group of overlapping clones open reading frame Porphyromonas gingivalis, an oral anaerobic bacterium, has been implicated as a causative agent of adult type periodontitis. As an asaccharolytic organism, P. gingivalis is totally dependent on external sources of peptides, which are necessary for its growth and proliferation. To fulfill such a fastidious nutritional requirement, this bacterium evolved a complex system of proteolytic enzymes, which are now recognized as important virulence factors in the development of periodontal disease (1Travis J. Banbula A. Potempa J. Adv. Exp. Med. Biol. 2000; 477: 455-465Crossref PubMed Google Scholar). The best known and well characterized enzymes of this system are gingipains R and K, arginine- and lysine-specific cysteine proteinases (2Curtis M.A. Kuramitsu H.K. Lantz M. Macrina F.L. Nakayama K. Potempa J. Reynolds E.C. Aduse-Opoku J. J. Periodontal Res. 1999; 34: 464-472Crossref PubMed Scopus (175) Google Scholar). Working in concert with the proteinases periodontain (3Nelson D. Potempa J. Kordula T. Travis J. J. Biol. Chem. 1999; 274: 12245-12251Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), collagenases/gelatinases (4Birkedal-Hansen H. Taylor R.E. Zambon J.J. Barwa P.K. Neiders M.E. J. Periodontal Res. 1988; 23: 258-264Crossref PubMed Scopus (97) Google Scholar, 5Lawson D.A. Meyer T.F. Infect. Immun. 1992; 60: 1524-1529Crossref PubMed Google Scholar, 6Kato T. Takahashi N. Kuramitsu H.K. J. Bacteriol. 1992; 174: 3889-3895Crossref PubMed Scopus (116) Google Scholar), prtT (7Otogoto J. Kuramitsu H.K. Infect. Immun. 1993; 61: 117-123Crossref PubMed Google Scholar), and Tpr (8Bourgeau G. Lapointe H. Peloquin P. Mayrand D. Infect. Immun. 1992; 60: 3186-3192Crossref PubMed Google Scholar) as well as host proteinases, this array of enzymes has the potential to degrade proteins from both the periodontal ligamentum and surrounding tissues. Their concerted action leads to the formation of a large pool of oligopeptides, which can be further utilized byP. gingivalis and other oral bacteria. However, P. gingivalis cannot transport poly- and oligopeptides into the cell, although it has the ability to thrive on dipeptides as a sole source of carbon. For this reason, we have focused our attention on a specialized group of P. gingivalis peptidases capable of hydrolyzing oligopeptides to di- and tripeptides, which can be subsequently metabolized by this periodontopathogen. In our previous report (9Banbula A. Mak P. Bugno M. Silberring J. Dubin A. Nelson D. Travis J. Potempa J. J. Biol. Chem. 1999; 274: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), we presented the purification, characterization, and cloning of prolyl tripeptidylpeptidase A, an enzyme that liberates tripeptides from the N-terminal regions of substrates containing proline residues in the third position. DPP1IV, an enzyme with similar specificity but only dipeptidylpeptidase activity, has also been cloned (10Kiyama M. Hayakawa M. Shiroza T. Nakamura S. Takeuchi A. Masamoto Y. Abiko Y. Biochim. Biophys. Acta. 1998; 1396: 39-46Crossref PubMed Scopus (37) Google Scholar), purified, and characterized (11Kumagai Y. Konishi K. Gomi T. Yagishita H. Yajima A. Yoshikawa M. Infect. Immun. 2000; 68: 716-724Crossref PubMed Scopus (57) Google Scholar, 12Banbula A. Bugno M. Goldstein J. Yen J. Nelson D. Travis J. Potempa J. Infect. Immun. 2000; 68: 1176-1182Crossref PubMed Scopus (54) Google Scholar). Together with a recently described angiotensinogen-converting enzyme analogue (13Awano S. Ansai T. Mochizuki H., Yu, W. Tanzawa K. Turner A.J. Takehara T. FEBS Lett. 1999; 460: 139-144Crossref PubMed Scopus (40) Google Scholar), all of these proteases can hydrolyze peptide bonds containing proline residues. In addition, the P. gingivalis genome contains three further putative genes encoding proteinases homologous with dipeptidyl peptidase IV, although their activities have not yet been identified (9Banbula A. Mak P. Bugno M. Silberring J. Dubin A. Nelson D. Travis J. Potempa J. J. Biol. Chem. 1999; 274: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).In the present study, we show purification, biochemical characterization, and the gene sequence of a new cell surface-associated serine protease with dipeptidylpeptidase activity. This enzyme liberates dipeptides from the free amino terminus and has a broad specificity for both aliphatic and aromatic residues in the penultimate position.RESULTSA 76-kDa dipeptidylpeptidase associated with P. gingivalis membranes was solubilized by mild detergent treatment. This procedure released more than 90%of the amidolytic activity against H-Ala-Phe-pNA into the medium. After acetone precipitation and subsequent chromatography steps including the use of hydroxyapatite, phenyl-Sepharose, and MonoS columns (Fig.1), a pure enzyme preparation was obtained. The homogeneity of the preparation and molecular mass of the protein were checked both by SDS-PAGE (Fig.2) and gel filtration on a TSK G3000 SW column (data not shown).Figure 2SDS-PAGE of fractions obtained during the purification of P. gingivalis DPP-7. Lane a, molecular mass markers (phosphorylase b, 97 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; α-lactalbumin, 14 kDa);lane b, Triton X-100 extract of P. gingivalis; lane c, acetone precipitate from Triton X-100 extract of P. gingivalis; lane d, hydroxyapatite column eluate;lane e, phenyl-Sepharose column eluate; lane f, MonoS column eluate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition ProfileBased on the inhibition studies (TableI) DPP-7 was classified as a serine protease, being inactivated by diisopropylfluorophosphate, Pefablock, and 3,4-dichloisocoumarin but not by typical cysteine class inhibitors such as E-64 or iododoacetic acid. Metal chelators including EDTA and 1,10-orthophenanthroline, as well as reducing agents did not influence its activity. The enzyme was not sensitive to inactivation by either detergents (0.5%SDS, 1%Triton X-100) or heavy metal ions including Zn2+, Co2+, and Ni2+. Human plasma inhibitors, such as α1-proteinase inhibitor, α1-antichymotrypsin, and α2-macroglobulin, did not affect enzyme activity, nor were they cleaved by DPP-7 (data not shown).Table IEffect of different compounds on P. gingivalis DPP-7 activityInhibitorConcentrationPercentage of residual activity%Diisopropylfluorophosphate10 mm34Pefablock4 mg/ml13,4-Dichloroisocoumarin2 mm0E-641 μm96Iodoacetic acid0.1 mm102EDTA10 mm901,10-Orthophenanthroline1 mm98Leupeptin0.1 mm107Aprotinin0.5 mg/ml128Pepstatin0.5 mg/ml127Cysteine10 mm90Gly-Ala100 mm102Arg-Phe100 mm69Ala-Gly100 mm96Arg-Gly10 mm84Lys-Gly10 mm96Ni2+1 mm95Zn2+1 mm95Co2+1 mm116SDS0.5%65SDS1%0Triton X-1000.1%144Triton X-1000.5%103Triton X-1001%94 Open table in a new tab pH Optimum and StabilityPurified DPP-7 was active against H-Ala-Phe-pNA over a broad pH range, from neutral to basic pH (6.5–9.0) (Fig. 3). This activity also changed with the ionic strength of the buffer, reaching 200%at 0.5 m NaCl concentration in 100 mm HEPES, pH 8.0. DPP-7 was stable in 0.2 m HEPES, pH 8.0, for 1 week at 4 °C. The protease showed no appreciable loss of activity when kept frozen at −80 °C for 1 month. After a 3-h incubation at either room temperature or 37 °C, activity was reduced to 62 and 20%, respectively. The optimum temperature for the hydrolysis of H-Ala-Phe-pNA was determined to be 43 °C.Figure 3pH optimum of the DPP-7 activity against Ala-Phe-pNA. Enzyme activity was tested on Ala-Phe-pNA substrate in different buffers including HEPES (●), PIPES (■), potassium phosphate (▪), Tris (○), and MES (▴).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Substrate SpecificityAmong the several chromogenic substrates tested, only those with aliphatic or aromatic side chains residues in the second, penultimate position were rapidly hydrolyzed by DPP-7 (Table II). To further confirm specificity, several synthetic peptides were also tested as substrates for this enzyme. Again, only those with an aliphatic or aromatic residue in the second position from the amino-terminal end were cleaved (Table III), with glycine, proline, or charged amino acids being not acceptable in the P1 position. The protease did not show any endopeptidase activity on gelatin, insulin β-chain, carboxymethylated lysozyme, and azocasein or type I collagen (data not shown). Purified DPP-7 was devoid of any aminopeptidase activity and did not cleave model substrates with blocked amino termini.Table IIKinetic analysis for para-nitroanalide cleavage by DPP-7SubstrateKmVmaxmmH-Ala-Ala-pNA0.313129.65H-Ala-Phe-pNA0.441170.06H-Gly-Phe-pNA0.25654.54Several other substrates, including H-Ala-Pro-pNA, H-Ala-pNA, H-Gly-pNA, H-Ile-pNA, H-Leu-pNA, H-Lys-pNA, H-Phe-pNA, H-Gly-Arg-pNA, H-Gly-Glu-pNA, H-Gly-Lys-pNA, H-Ala-Gly-pNA, H-Gly-Gly-pNA, H-Ala-Ala-Phe-pNA, H-Ala-Gly-Arg-pNA, H-Leu-Thr-Arg-pNA, H-Ala-Phe-Pro-pNA,N α-benzoyl-dl-arginine-pNA,N-Met-Ala-Pro-Val-pNA,N-Suc-Ala-Ala-pNA,N-Suc-Ala-Ala-Pro-Glu-pNA,N-Suc-Ala-Ala-Pro-Leu-pNA,N-Suc-Ala-Ala-Val-Ala-pNA, Z-Ala-Ala-pNA, Z-Lys-pNA, Z-Arg-pNA, Z-Glu-Glu-pNA, Z-Leu-Leu-Gly-pNA, Z-Lys-Arg-pNA, Z-Phe-Arg-pNA, Z-Phe-Val-Arg-pNA, Z-Tyr-Lys-Arg-pNA were tested, but none of these was hydrolyzed by DPP-7. Open table in a new tab Table IIISpecificity of P. gingivalis DPP-7 on synthetic peptidesPeptides cleavedPeptides not cleavedTrp-Ala-↓-Gly-Gly-Asp-Ala-Ser-Gly-GluTrp-His-Trp-Leu-Glu-Leu-Lys-Pro-Gly-Glu-Pro-Met-TyrIle-Ala-↓-Arg-Arg-His-Pro-Tyr-Phe-LeuSer-Pro-Tyr-Ser-Ser-Glu-Thr-ThrLys-Ile-↓-Ala-Gly-Tyr-His-Leu-Glu-LeuAla-Pro-Val-Arg-Ser-LeuPhe-Leu-↓-Arg-Glu-Pro-Val-Ile-Phe-LeuGln-Lys-Gln-Met-Ser-Asp-Arg-Arg-Glu Open table in a new tab DPP-7 Sequence AnalysisPurified DPP-7 was resolved on SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. It had an amino-terminal sequence ADKGMMWLLNELNQENLDRMRELGFT. After proteolytic in-gel digestion of the enzyme, additional internal sequences were obtained, including DNKPYK, EMTYL, FAQFAN, VLPAML, SVVPY, and LFFAGL. All of this sequence data allowed us to identify theP. gingivalis genomic contig gln[vert]TIGR[vert]P. gingivalis_ in the Unfinished Microbial Genomes data base, the Institute of Genomic Research. An ORF corresponding to the DPP-7 amino acid sequence was found, as indicated by the fact, that all sequences of the DPP-7-derived peptides obtained by the enzyme polypeptide fragmentation by trypsin were present in the protein primary structure inferred from the nucleotide sequence of the ORF as shown in Fig.4. The entire ORF corresponds to a 675-amino acid polypeptide with a calculated mass of 76247.4 Da. Interestingly, the DPP-7 ORF contains the consensus sequence for the active-site serine residue of serine type proteases, TGGNSGSPVF. As indicated in Fig.5, the DPP-7 carboxyl terminus exhibits a high degree of identity to that of the V8 serine protease, particularly around the putative active site serine residue. This is surprising, since the P. gingivalis DPP-7 is a dipeptidyl peptidase specific for small aliphatic and aromatic residues, whereasStaphylococcus aureus V8 endopeptidase is specific toward substrates containing glutamic and aspartic acid residues in the P1 position. The similarity search performed using the NCBI TBLASTN tool against GenBankTM, EMBL, DDBJ, and PDB data bases showed no significant similarity of DPP-7 to any other known dipeptidyl peptidases, indicating that this enzyme could be regarded as a member of a new family of proteases. Additional searches against data bases containing unfinished and finished microbial genomes allowed us to identify more genes coding for similar proteases with consensus active site sequence TGGNSGSPV (Fig. 6). A gene of related protein has been found in the P. gingivalis W83 unfinished fragment of the complete genome between positions 1,360,759 and 1,362,718. The inferred primary structure of this putative proteinase shows significant similarity to DPP-7 (267/691 identities). Another organism, Shewanella putrefaciens, possesses two related genes (gnl [vert]TIGR_24[vert]sputre 6401 and gnl [vert]TIGR_24[vert]sputre 6410), while a plant pathogen,Xylella fastidiosa, contains one gene coding for similar proteinase (gb[vert]AE004008.1[vert]).Figure 4ORF for the gene coding P. gingivalis DPP-7. Underlined are sequences obtained from the Edman degradation of the trypsin fragmented DPP-7 polypeptide chain. The putative active site serine residue is marked by the black background.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Comparison of the C-terminal regions of theP. gingivalis DPP-7 (residues 664–695) and S. aureus V8 endopeptidase (residues 704–863).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Multiple sequence alignment of P. gingivalis DPP-7 and its putative homologues. Sequences of DPP-7-related proteinases were obtained from the conceptual translation of the following ORFs retrieved from unfinished and finished genome data bases: S1, S. putrefaciensgnl [vert]TIGR_24[vert]sputre 6401; S2, S. putrefaciens gnl [vert]TIGR_24[vert]sputre 6410; X,X. fastidiosa gb[vert]AE004008.1[vert]; P1,P. gingivalis gnl [vert]TIGR[vert]P. gingivalis_CPG.con; P2, P. gingivalis DPP-7 gnl [vert]TIGR[vert]P. gingivalis_CPG.con. The sequences were subsequently aligned using the ClustalW multiple sequence alignment tool.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In addition, the computer-assisted search for sequential motifs characteristic for transmembrane domains revealed the presence of two such putative regions within the amino-terminal sequence of DPP-7, with residues 7–24 and 62–78 most likely folded into hydrophobic α-helices responsible for membrane anchoring of this enzyme.DISCUSSIONSeveral studies indicate that the outer membrane of P. gingivalis contains a complex proteolytic machinery, which serves multiple physiological functions. In this study, we have described the identification of a novel proteinase localized on the bacterial surface.The purified enzyme migrated as a single band with a molecular mass of 76 kDa on SDS-PAGE, and its amino-terminal sequence was located within the primary structure of the translated product of the dpp-7gene. Apparently, the enzyme is truncated at the amino terminus due to the action of a lysine-specific proteinase, most likely gingipain K. Taking into account that the N terminus of DPP-7 contains membrane anchorage domains, it is likely that the N-terminal truncation noted here occurred during the isolation procedure and may not represent its true membrane form.The inhibition by typical serine protease inhibitors like diisopropyl fluorophosphate, Pefablock, and phenylmethylsulfonyl fluoride, as well as resistance to sulfhydryl group blocking reagents and chelating agents, allowed us to classify this enzyme as a serine protease. However, the P. gingivalis DPP-7 does not belong to any of the six previously described types of dipeptidylpeptidases (20Barrett A.J. Rawlings N.D. Woessner J. Handbook of Proteolytic Enzymes. Academic Press, London1998Google Scholar). DPP I is a member of a cysteine class of peptidases and possesses a broad specificity with an exclusion for basic amino acid and proline residues in the P1 site of the scissile peptide bond (21McGuire M.J. Lipsky P.E. Thiele D.L. Arch. Biochem. Biophys. 1992; 295: 280-288Crossref PubMed Scopus (93) Google Scholar). DPP VI is another representative of the cysteine proteinase family with dipeptidylpeptidase activity toward the broad spectrum of substrates (22Vacheron M.J. Guinand M. Francon A. Michel G. Eur. J. Biochem. 1979; 100: 189-196Crossref PubMed Scopus (24) Google Scholar). DPP II, DPP IV, and DPP V belong to the S9 family of the serine proteases (20Barrett A.J. Rawlings N.D. Woessner J. Handbook of Proteolytic Enzymes. Academic Press, London1998Google Scholar). Both DPP II and DPP IV share similar specificity directed against Pro and Ala residues in the penultimate position, whereas DPP V is an enzyme secreted by Aspergillus fumigatuswith a unique substrate specificity limited to X-Ala, His-Ser, and Ser-Tyr dipeptides (23Beauvais A. Monod M. Debeaupuis J.P. Diaquin M. Kobayashi H. Latge J.P. J. Biol. Chem. 1997; 272: 6238-6244Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). DPP III is also classified as a serine peptidase, with its action being restricted to the Arg residue in the P1 position (24Ellis S. Nuenke J.M. J. Biol. Chem. 1967; 242: 4623-4629Abstract Full Text PDF PubMed Google Scholar). In terms of biochemical features, DPP-7 resembles a dipeptidyl aminopeptidase (DAP BII), which was isolated from Pseudomonas sp. strain WO24, but the gene sequence of that enzyme remains unknown and does not allow a sequence comparison of these proteins (25Ogasawara W. Kobayashi G. Okada H. Morikawa Y. J. Bacteriol. 1996; 178: 6288-6295Crossref PubMed Google Scholar). Because P. gingivalisdipeptidylpeptidase does not exhibit any significant homology to any of the dipeptidyl peptidases described so far, we have named this enzyme DPP-7.Interestingly, the P. gingivalis DPP-7 displays the consensus sequence characteristic for the catalytic site of the V-8 like proteases, a group of endopeptidases cleaving after glutamic or aspartic acid residues (26Carmona C. Gray G.L. Nucleic Acids Res. 1987; 15: 6757Crossref PubMed Scopus (61) Google Scholar). This region of significant sequence similarity is specifically located only at the C-terminal region of both proteases and includes the putative active site serine residue. Interestingly, we identified more genes coding for putative, DPP-7-related proteases in P. gingivalis, X. fastidiosa and S. putrefaciens. Based on the enzymological and gene sequence data presented above, we conclude thatP. gingivalis DPP-7 does not belong to any of the peptidase families previously reported and should, therefore, be regarded as a prototype enzyme that defines a new family of dipeptidylpeptidases. Porphyromonas gingivalis, an oral anaerobic bacterium, has been implicated as a causative agent of adult type periodontitis. As an asaccharolytic organism, P. gingivalis is totally dependent on external sources of peptides, which are necessary for its growth and proliferation. To fulfill such a fastidious nutritional requirement, this bacterium evolved a complex system of proteolytic enzymes, which are now recognized as important virulence factors in the development of periodontal disease (1Travis J. Banbula A. Potempa J. Adv. Exp. Med. Biol. 2000; 477: 455-465Crossref PubMed Google Scholar). The best known and well characterized enzymes of this system are gingipains R and K, arginine- and lysine-specific cysteine proteinases (2Curtis M.A. Kuramitsu H.K. Lantz M. Macrina F.L. Nakayama K. Potempa J. Reynolds E.C. Aduse-Opoku J. J. Periodontal Res. 1999; 34: 464-472Crossref PubMed Scopus (175) Google Scholar). Working in concert with the proteinases periodontain (3Nelson D. Potempa J. Kordula T. Travis J. J. Biol. Chem. 1999; 274: 12245-12251Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), collagenases/gelatinases (4Birkedal-Hansen H. Taylor R.E. Zambon J.J. Barwa P.K. Neiders M.E. J. Periodontal Res. 1988; 23: 258-264Crossref PubMed Scopus (97) Google Scholar, 5Lawson D.A. Meyer T.F. Infect. Immun. 1992; 60: 1524-1529Crossref PubMed Google Scholar, 6Kato T. Takahashi N. Kuramitsu H.K. J. Bacteriol. 1992; 174: 3889-3895Crossref PubMed Scopus (116) Google Scholar), prtT (7Otogoto J. Kuramitsu H.K. Infect. Immun. 1993; 61: 117-123Crossref PubMed Google Scholar), and Tpr (8Bourgeau G. Lapointe H. Peloquin P. Mayrand D. Infect. Immun. 1992; 60: 3186-3192Crossref PubMed Google Scholar) as well as host proteinases, this array of enzymes has the potential to degrade proteins from both the periodontal ligamentum and surrounding tissues. Their concerted action leads to the formation of a large pool of oligopeptides, which can be further utilized byP. gingivalis and other oral bacteria. However, P. gingivalis cannot transport poly- and oligopeptides into the cell, although it has the ability to thrive on dipeptides as a sole source of carbon. For this reason, we have focused our attention on a specialized group of P. gingivalis peptidases capable of hydrolyzing oligopeptides to di- and tripeptides, which can be subsequently metabolized by this periodontopathogen. In our previous report (9Banbula A. Mak P. Bugno M. Silberring J. Dubin A. Nelson D. Travis J. Potempa J. J. Biol. Chem. 1999; 274: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), we presented the purification, characterization, and cloning of prolyl tripeptidylpeptidase A, an enzyme that liberates tripeptides from the N-terminal regions of substrates containing proline residues in the third position. DPP1IV, an enzyme with similar specificity but only dipeptidylpeptidase activity, has also been cloned (10Kiyama M. Hayakawa M. Shiroza T. Nakamura S. Takeuchi A. Masamoto Y. Abiko Y. Biochim. Biophys. Acta. 1998; 1396: 39-46Crossref PubMed Scopus (37) Google Scholar), purified, and characterized (11Kumagai Y. Konishi K. Gomi T. Yagishita H. Yajima A. Yoshikawa M. Infect. Immun. 2000; 68: 716-724Crossref PubMed Scopus (57) Google Scholar, 12Banbula A. Bugno M. Goldstein J. Yen J. Nelson D. Travis J. Potempa J. Infect. Immun. 2000; 68: 1176-1182Crossref PubMed Scopus (54) Google Scholar). Together with a recently described angiotensinogen-converting enzyme analogue (13Awano S. Ansai T. Mochizuki H., Yu, W. Tanzawa K. Turner A.J. Takehara T. FEBS Lett. 1999; 460: 139-144Crossref PubMed Scopus (40) Google Scholar), all of these proteases can hydrolyze peptide bonds containing proline residues. In addition, the P. gingivalis genome contains three further putative genes encoding proteinases homologous with dipeptidyl peptidase IV, although their activities have not yet been identified (9Banbula A. Mak P. Bugno M. Silberring J. Dubin A. Nelson D. Travis J. Potempa J. J. Biol. Chem. 1999; 274: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In the present study, we show purification, biochemical characterization, and the gene sequence of a new cell surface-associated serine protease with dipeptidylpeptidase activity. This enzyme liberates dipeptides from the free amino terminus and has a broad specificity for both aliphatic and aromatic residues in the penultimate position. RESULTSA 76-kDa dipeptidylpeptidase associated with P. gingivalis membranes was solubilized by mild detergent treatment. This procedure released more than 90%of the amidolytic activity against H-Ala-Phe-pNA into the medium. After acetone precipitation and subsequent chromatography steps including the use of hydroxyapatite, phenyl-Sepharose, and MonoS columns (Fig.1), a pure enzyme preparation was obtained. The homogeneity of the preparation and molecular mass of the protein were checked both by SDS-PAGE (Fig.2) and gel filtration on a TSK G3000 SW column (data not shown).Inhibition ProfileBased on the inhibition studies (TableI) DPP-7 was classified as a serine protease, being inactivated by diisopropylfluorophosphate, Pefablock, and 3,4-dichloisocoumarin but not by typical cysteine class inhibitors such as E-64 or iododoacetic acid. Metal chelators including EDTA and 1,10-orthophenanthroline, as well as reducing agents did not influence its activity. The enzyme was not sensitive to inactivation by either detergents (0.5%SDS, 1%Triton X-100) or heavy metal ions including Zn2+, Co2+, and Ni2+. Human plasma inhibitors, such as α1-proteinase inhibitor, α1-antichymotrypsin, and α2-macroglobulin, did not affect enzyme activity, nor were they cleaved by DPP-7 (data not shown).Table IEffect of different compounds on P. gingivalis DPP-7 activityInhibitorConcentrationPercentage of residual activity%Diisopropylfluorophosphate10 mm34Pefablock4 mg/ml13,4-Dichloroisocoumarin2 mm0E-641 μm96Iodoacetic acid0.1 mm102EDTA10 mm901,10-Orthophenanthroline1 mm98Leupeptin0.1 mm107Aprotinin0.5 mg/ml128Pepstatin0.5 mg/ml127Cysteine10 mm90Gly-Ala100 mm102Arg-Phe100 mm69Ala-Gly100 mm96Arg-Gly10 mm84Lys-Gly10 mm96Ni2+1 mm95Zn2+1 mm95Co2+1 mm116SDS0.5%65SDS1%0Triton X-1000.1%144Triton X-1000.5%103Triton X-1001%94 Open table in a new tab pH Optimum and StabilityPurified DPP-7 was active against H-Ala-Phe-pNA over a broad pH range, from neutral to basic pH (6.5–9.0) (Fig. 3). This activity also changed with the ionic strength of the buffer, reaching 200%at 0.5 m NaCl concentration in 100 mm HEPES, pH 8.0. DPP-7 was stable in 0.2 m HEPES, pH 8.0, for 1 week at 4 °C. The protease showed no appreciable loss of activity when kept frozen at −80 °C for 1 month. After a 3-h incubation at either room temperature or 37 °C, activity was reduced to 62 and 20%, respectively. The optimum temperature for the hydrolysis of H-Ala-Phe-pNA was determined to be 43 °C.Figure 3pH optimum of the DPP-7 activity against Ala-Phe-pNA. Enzyme activity was tested on Ala-Phe-pNA substrate in different buffers including HEPES (●), PIPES (■), potassium phosphate (▪), Tris (○), and MES (▴).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Substrate SpecificityAmong the several chromogenic substrates tested, only those with aliphatic or aromatic side chains residues in the second, penultimate position were rapidly hydrolyzed by DPP-7 (Table II). To further confirm specificity, several synthetic peptides were also tested as substrates for this enzyme. Again, only those with an aliphatic or aromatic residue in the second position from the amino-terminal end were cleaved (Table III), with glycine, proline, or charged amino acids being not acceptable in the P1 position. The protease did not show any endopeptidase activity on gelatin, insulin β-chain, carboxymethylated lysozyme, and azocasein or type I collagen (data not shown). Purified DPP-7 was devoid of any aminopeptidase activity and did not cleave model substrates with blocked amino termini.Table IIKinetic analysis for para-nitroanalide cleavage by DPP-7SubstrateKmVmaxmmH-Ala-Ala-pNA0.313129.65H-Ala-Phe-pNA0.441170.06H-Gly-Phe-pNA0.25654.54Several other substrates, including H-Ala-Pro-pNA, H-Ala-pNA, H-Gly-pNA, H-Ile-pNA, H-Leu-pNA, H-Lys-pNA, H-Phe-pNA, H-Gly-Arg-pNA, H-Gly-Glu-pNA, H-Gly-Lys-pNA, H-Ala-Gly-pNA, H-Gly-Gly-pNA, H-Ala-Ala-Phe-pNA, H-Ala-Gly-Arg-pNA, H-Leu-Thr-Arg-pNA, H-Ala-Phe-Pro-pNA,N α-benzoyl-dl-arginine-pNA,N-Met-Ala-Pro-Val-pNA,N-Suc-Ala-Ala-pNA,N-Suc-Ala-Ala-Pro-Glu-pNA,N-Suc-Ala-Ala-Pro-Leu-pNA,N-Suc-Ala-Ala-Val-Ala-pNA, Z-Ala-Ala-pNA, Z-Lys-pNA, Z-Arg-pNA, Z-Glu-Glu-pNA, Z-Leu-Leu-Gly-pNA, Z-Lys-Arg-pNA, Z-Phe-Arg-pNA, Z-Phe-Val-Arg-pN" @default.
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• W2038423623 title "Porphyromonas gingivalis DPP-7 Represents a Novel Type of Dipeptidylpeptidase" @default.
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