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Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis.

ABSTRACT: Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 ?M and 11.02 ?M(-1) s(-1), respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.

SUBMITTER: Ohara-Nemoto Y

PROVIDER: S-EPMC3937620 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis.

Ohara-Nemoto Yuko Y Rouf Shakh M A SM Naito Mariko M Yanase Amie A Tetsuo Fumi F Ono Toshio T Kobayakawa Takeshi T Shimoyama Yu Y Kimura Shigenobu S Nakayama Koji K Saiki Keitarou K Konishi Kiyoshi K Nemoto Takayuki K TK

The Journal of biological chemistry 20140107 9

Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7 ...[more]

PMID: 24398682

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Project description:Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.

Project description:Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0-2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.

Project description:MicroRNAs (miRNAs) are short, noncoding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert any innate immune response. In this study we analyzed, using microarray analysis, the bacterial modulation of miRNAs in bone marrow-derived macrophages (BMMs) in which activity was induced by infection with Porphyromonas gingivalis The expression of several miRNAs was modulated 3 h postinfection (at a multiplicity of infection of 25). A bioinformatic analysis was performed to further identify pathways related to the innate immune host response under the influence of selected miRNAs. To assess the effects of the miRNAs identified on cytokine secretion (tumor necrosis factor alpha [TNF-α] and interleukin-10 [IL-10]), BMMs were transfected with selected miRNA mimics and inhibitors. Transfection with mmu-miR-155 and mmu-miR-2137 did not modify TNF-α secretion, while their inhibitors increased it. Inhibitors of mmu-miR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory factor IL-10. In P. gingivalis-infected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo, in a mouse model of P. gingivalis-induced calvarial bone resorption, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatory cell infiltration, osteoclast activity, and bone loss. Bioinformatic analysis demonstrated that pathways related to cytokine- and chemokine-related pathways but also osteoclast differentiation may be involved in the effects observed. This study contributes further to our understanding of P. gingivalis-induced modulation of miRNAs and their physiological effects. It highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by P. gingivalis infection.

Dataset Information

Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis.

Publications

Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from Porphyromonas gingivalis.

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