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Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis.

Nishimata H, Ohara-Nemoto Y, Baba TT, Hoshino T, Fujiwara T, Shimoyama Y, Kimura S, Nemoto TK - PLoS ONE (2014)

Bottom Line: Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99.In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis.The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; Department of Pediatric Dentistry, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

ABSTRACT
Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 µM-1·sec-1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM-1·sec-1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

No MeSH data available.


Cell-associated peptidase activity profiles of P. endodontalis and eight strains of P. gingivalis.(A) Aliquots (5 µL) of bacterial cell suspensions (A600 = 2.0) of P. gingivalis strains and P. endodontalis ATCC 35406 were incubated in a reaction mixture containing 20 µM Boc-Phe-Ser-Arg- or Z-His-Glu-Lys-MCA. (B) DPP activities toward Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA were measured. Activity values are shown as the mean±S.D. (n = 3).
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pone-0114221-g001: Cell-associated peptidase activity profiles of P. endodontalis and eight strains of P. gingivalis.(A) Aliquots (5 µL) of bacterial cell suspensions (A600 = 2.0) of P. gingivalis strains and P. endodontalis ATCC 35406 were incubated in a reaction mixture containing 20 µM Boc-Phe-Ser-Arg- or Z-His-Glu-Lys-MCA. (B) DPP activities toward Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA were measured. Activity values are shown as the mean±S.D. (n = 3).

Mentions: Hydrolysis of benzyloxycarbonyl (Z)-His-Glu-Lys-MCA that was characteristic of Lys-gingipain (Kgp) and t-butyloxycarbonyl (Boc)-Phe-Ser-Arg-MCA of Arg-gingipain (Rgp) was commonly demonstrated in the eight strains of P. gingivalis (ATCC 33277, 16-1, HW24D1, HG1690, ATCC 49417, W83, W50, HNA99), though some variations of these activities were observed. Strains HNA99, HG1690, ATCC 49417, and 16-1 showed relatively high activities for both gingipains, while strain W50 showed the lowest level of hydrolysis (Fig. 1). The absence of gingipain-like activities was obvious in P. endodontalis ATCC 35406, in agreement with previous reports [2], [9]. The DPP activities of these Porphyromonas strains were examined using four different fluorescent synthetic dipeptidyl substrates, Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA, which are specific or preferential substrates of DPP4, DPP5, DPP7, and DPP11, respectively [18]. They were hydrolyzed within a few variations of efficiency by the P. gingivalis strains (Fig. 1). Interestingly, the hydrolyzing activities for Leu-Asp-, Gly-Pro-, and Met-Leu-MCA were higher in P. endodontalis than those respective ones for all of the P. gingivalis strains, with the hydrolysis of Lys-Ala-MCA in particular the most prominent in P. endodontalis.


Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis.

Nishimata H, Ohara-Nemoto Y, Baba TT, Hoshino T, Fujiwara T, Shimoyama Y, Kimura S, Nemoto TK - PLoS ONE (2014)

Cell-associated peptidase activity profiles of P. endodontalis and eight strains of P. gingivalis.(A) Aliquots (5 µL) of bacterial cell suspensions (A600 = 2.0) of P. gingivalis strains and P. endodontalis ATCC 35406 were incubated in a reaction mixture containing 20 µM Boc-Phe-Ser-Arg- or Z-His-Glu-Lys-MCA. (B) DPP activities toward Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA were measured. Activity values are shown as the mean±S.D. (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262410&req=5

pone-0114221-g001: Cell-associated peptidase activity profiles of P. endodontalis and eight strains of P. gingivalis.(A) Aliquots (5 µL) of bacterial cell suspensions (A600 = 2.0) of P. gingivalis strains and P. endodontalis ATCC 35406 were incubated in a reaction mixture containing 20 µM Boc-Phe-Ser-Arg- or Z-His-Glu-Lys-MCA. (B) DPP activities toward Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA were measured. Activity values are shown as the mean±S.D. (n = 3).
Mentions: Hydrolysis of benzyloxycarbonyl (Z)-His-Glu-Lys-MCA that was characteristic of Lys-gingipain (Kgp) and t-butyloxycarbonyl (Boc)-Phe-Ser-Arg-MCA of Arg-gingipain (Rgp) was commonly demonstrated in the eight strains of P. gingivalis (ATCC 33277, 16-1, HW24D1, HG1690, ATCC 49417, W83, W50, HNA99), though some variations of these activities were observed. Strains HNA99, HG1690, ATCC 49417, and 16-1 showed relatively high activities for both gingipains, while strain W50 showed the lowest level of hydrolysis (Fig. 1). The absence of gingipain-like activities was obvious in P. endodontalis ATCC 35406, in agreement with previous reports [2], [9]. The DPP activities of these Porphyromonas strains were examined using four different fluorescent synthetic dipeptidyl substrates, Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA, which are specific or preferential substrates of DPP4, DPP5, DPP7, and DPP11, respectively [18]. They were hydrolyzed within a few variations of efficiency by the P. gingivalis strains (Fig. 1). Interestingly, the hydrolyzing activities for Leu-Asp-, Gly-Pro-, and Met-Leu-MCA were higher in P. endodontalis than those respective ones for all of the P. gingivalis strains, with the hydrolysis of Lys-Ala-MCA in particular the most prominent in P. endodontalis.

Bottom Line: Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99.In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis.The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; Department of Pediatric Dentistry, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

ABSTRACT
Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 µM-1·sec-1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM-1·sec-1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

No MeSH data available.