Acylpeptidyl Oligopeptidase: Porphyromonas Gingivalis Periplasmic Novel Exopeptidase Releases N-acylated Di-and Tri-peptides from Oligopeptides* Were Used as Expression Vectors. Restriction Enzymes and Dna-modifying Enzymes Were Purchased From

Exopeptidases including dipeptidyland tripeptidyl-peptidase are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as diand tri-peptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser and encoded by PGN_1349 in P. gingivalis ATCC 33277. AOP is currently listed as an unassigned S9-family peptidase or prolyl oligopeptidase. Recombinant AOP did not hydrolyze a Pro-Xaa bond. In addition, though sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, as AOP scarcely released an N-acyl-amino acid as compared to diand tri-peptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-LysMet-4-methycoumaryl-7-amide, the most potential substrate, was 123.3 ± 17.3 μMsec, optimal pH was 7-8.5, and the activity was decreased with increased NaCl concentrations. AOP existed predominantly in the periplasmic fraction as a monomer, while equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met-Glu). The three dimensional modeling revealed the three domain structures: residues Met-Ala, which has no similar homologue with known structure, residues Leu-Met (β-propeller domain) and residues Ala-Phe (α /β hydrolase domain), and further indicated the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference. AOP orthologues are widely distributed in bacteria, archaea, and eukaryotes, suggesting its importance for processing of nutritional and/or bioactive oligopeptides. Porphyromonas gingivalis, a Gram-negative obligate anaerobe, has been implicated as the causative agent of chronic periodontal disease (1, 2), which is a major reason for permanent tooth loss (3-5). Recently, much attention has been paid to this bacterium due to its association with systemic diseases, such as cardiovascular disorders (6), decreased kidney function (7), and rheumatoid arthritis (8). P. gingivalis does not ferment carbohydrates, but rather metabolizes amino acids to produce ATP by a putative respiratory chain of fumarate respiration without oxygen (9). In addition, it incorporates nutritional Acylpeptidyl oligopeptidase in P. gingivalis 2 amino acids not as single amino acids, but preferentially as diand tri-peptides and short oligopeptides, possibly via inner-membrane-associated oligopeptide transporters (9, 10). In P. gingivalis, extracellular nutritional proteins are initially digested to oligopeptides by potent cysteine endopeptidases, such as Arg-gingipain (Rgp) (C25.001 in MEROPS classification) (11) and Lys-gingipain (Kgp) (C25.002) (12), and subsequently degraded into diand tri-peptides by exopeptidases, i.e., dipeptidyl-peptidases (DPPs) (13-16) and prolyl tripeptidylpeptidase A (PTP-A) (17), respectively. Therefore, the topology of the sub-cellular localization of these peptidases, i.e., extracellular and outer membrane-bound Rgp/Kgp, and DPPs/PTP-A in periplasmic space, appears to be suitable for an ordered degradation of proteinaceous substrates and their incorporation into cells. Through amino acid metabolism, the organism excretes sulfide, ammonia, butyrate (18, 19), and methyl mercaptan (20) as end-products, which have been suggested to cause host tissue damage (21-23). Accordingly, DPPs, PTP-A, and gingipains are considered to play crucial roles not only in cell growth but also bacterial pathogenicity. Thus far, four DPPs, i.e., DPP4, DPP5, DPP7, and DPP11, have been identified in P. gingivalis. These are serine peptidases belonging to either the S9 or S46 family, with DPP4 (S09.003) showing a preference for Pro at P1 (13, 14) and DPP5 (S09.012), the first entity identified in bacteria, showing a preference for hydrophobic P1 residues and no specificity at the P2 position (24). Furthermore, DPP7 (S46.001) has a hydrophobic preference for the P1 (15, 25) as well as P2 (26) positions, while we discovered that DPP11 (S46.002) is a unique DPP specific for acidic P1 residues (Asp and Glu) (27). In addition to these DPPs, P. gingivalis possesses a metallopeptidase encoded by the gene PGN_1645, which was identified as DPP3 (M49.001) and specific for P1 Arg. However, DPP3 appears to be localized in the cytosol, while the Arg-specific DPP activity of Rgp plays a role in extracellular substrate processing (24). We also found Lys-specific DPP activity in Kgp (24). DPPs do not cleave polypeptides with Pro at the third position from the N-terminus, while PTP-A (S09.017) is able to release the N-terminal tripeptide Xaa-Xaa-Pro (16, 17). Therefore, most extracellular polypeptides, at least those without N-terminal modification, should be sequentially and completely degraded into dior tri-peptides in P. gingivalis by the cooperative activities of the four DPPs, PTP-A, and gingipains (24). On the other hand, our previous observations of P. gingivalis NDP212, a dpp4-5-7-11-knockout strain, suggested the existence of an unidentified DPP responsible for Met-Leu-MCA hydrolysis, since the activity was markedly elevated in the mutant strain as compared to a dpp4-5-7-knockout strain (24). In order to define this entity, we focused on and studied the remaining uncharacterized three putative S9-family proteins, i.e., PGN_1349, PGN_1542, and PGN_1878, of P. gingivalis in the present study. We found peptidase activity exclusively in PGN_1349 and, interestingly, most exopeptidase activities remaining in NDP212 were explained by the activity of PGN_1349. The observed peptidase properties of PGN_1349 indicated it as a novel oligopeptidase, designated acylpeptidyl oligopeptidase (AOP), with a preference for hydrophobic amino acids at the P1 position of its substrates, especially those with N-terminal modification. EXPERIMENTAL PROCEDURES Materials—pQE60 (Qiagen Inc., Chatsworth, CA) and pTrcHis2-TOPO (Invitrogen, Carlsbad, CA) were used as expression vectors. Restriction enzymes and DNA-modifying enzymes were purchased from Takara Bio (Tokyo, Japan) and New England Biolabs (Ipswich, MA), respectively. Quick Taq HS DyeMix and KOD-Plus-Neo DNA polymerase came from Toyobo (Tokyo, Japan). Met-Leu-4-methycoumaryl-7-amide (MCA) was from Bachem (Bubendorf, Switzerland), and Leu-Asp-, Leu-Glu-, Lys-Met-, benzyloxycarbonyl-(Z-)Lys-Met-, and Z-AVKM-MCA were synthesized by Thermo Fisher Scientific (Ulm, Germany) and Scrum (Tokyo, Japan). Other MCA peptides were purchased from the Peptide Institute (Osaka, Japan). Oligonucleotide primers were synthesized by FASMAC (Atsugi, Japan). Low-molecular-weight markers, full-range rainbow molecular weight markers, rabbit muscle aldolase, egg white ovalbumin, and a Sephacryl S-200 High Resolution and Superdex 200 Increase 10/300 columns were from GE Healthcare (Little Chalfont, UK). Bovine liver Acylpeptidyl oligopeptidase in P. gingivalis 3 catalase and bovine milk α-lactalbumin were obtained from Sigma-Aldrich (St. Louis, MS). Lysozyme from egg white and formyl cellulofine were from Seikagaku Biobusiness Corp. (Tokyo, Japan). Culture Conditions—P. gingivalis strains ATCC 33277, KDP136 (28), NDP212 (24), and NDP600 were grown anaerobically (80% N2, 10% CO2, 10% H2) in enriched brain heart infusion broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 5 μg/ml of hemin and 0.5 μg/ml of menadione. Ampicillin (10 μg/ml) for NDP600 and appropriate antibiotics (ampicillin, erythromycin, tetracycline, chloramphenicol) were added to the cultures of KDP136 and NDP212, as previously described (24, 27). Bacterial cells were suspended in phosphate-buffered saline (PBS) at pH 7.4, then centrifuged at 6000 x g for 15 min at 4 ̊C. The cell pellet was washed once with PBS, re-suspended in PBS to adjust absorbance to 2.0 at 600 nm, and then used in the following experiments. Expression and Purification of Recombinant Proteins—Oligonucleotides and plasmids used in this study are listed in Table 1. DNA fragments encoding the mature forms (Met-Lys) of PGN_1349 (29) (GenBank Accession Number 6329856, MEROPS code MER034614) and Leu-Glu of PGN_1542 (MER110015) were amplified by PCR using sets of primers (5PGN1349-M16 and 3PGN1349-K759B, and 5PGN1542-L6Bam and 3PGN1542-E279Bam, respectively), with genomic DNA of P. gingivalis ATCC 33277 utilized as a template. The PCR products were ligated with pTrcHis2-TOPO according to the manufacturer’s protocol, resulting in production of pTrcHis2-PGN1349 and -PGN1542, respectively. A DNA fragment encoding Ile-Leu of PGN_1878 (MER109588) was PCR amplified using a set of primers (5PGN1878-I6Bgl and 3PGN1878-L473Bgl). After restriction cleavage by BglII, the fragment was inserted into the BamHI site of pQE60, resulting in production of pQE60-PGN1878. A deletion mutation of Met-Gly (designated pTrcHis2-PGN1349-N102) was constructed using a PCR-based technique with primers 5PGN1349-N102 and 3pTrcHisTopo-L-1, with the substitution of Ser by Ala, (designated pTrcHis2-PGN1349-S615A and pTrcHis2-PGN1349-Ν102-S615A) constructed from pTrcHis2-PGN1349 and pTrcHis-PGN1349-N102 using the primers 5PGN1349-S615A and 3PGN1349-A614, respectively. Mutations were confirmed by DNA sequencing. Escherichia coli XL-1 Blue cells carrying expression plasmids were cultured in Luria-Bertani broth supplemented with 75 μg/ml ampicillin at 37 ̊C. Recombinant proteins were induced with 0.2 mM isopropyl-thiogalactopyranoside at 30 ̊C for 4 h, then purified using Talon affinity chromatography as previously reported (27). Disruption of P. gingivalis Genes—P. gingivalis NDP212, with deletion of four DPP genes (dpp4, dpp5, dpp7, dpp11) (24), and KDP136, with deletions of kgp, rgpA, and rgpB (28), were previously reported. To construct the PGN_1349 gene deletion mutant, DNA fragments of each 5’and 3’-part of the PGN_1349 gene were PCR amplified with a set of primers (1349-5F1 and cep-comp-1349-5R for the 5’ part, cep3F-1349-3F1 and 1349-3Rcomp for the 3’ part). A cepA fragment was amplified using primers (1349-5F1-cepF, 1349-3Fcomp-cep3R-comp) and pCR4-TOPO as a template. Nested PCR was performed with a mixture of these three fragments using primers (1349-5F2, 1349-3R2comp), then the obtained DNA fragment (3,440 bp) was introduced into P. gingivalis by electroporation, resulting in NDP600 (pgn1349::cepA). Measurement of Peptidase Activity—Peptidase activity was measured using peptidyl-MCA as previously reported (24, 27). Briefly, the reaction was started by addition of recombinant proteins (5-100 ng), a periplasmic cell fraction (1-5 μl), or P. gingivalis cell suspensions (1-5 μl) in a reaction mixture (200 μl) composed of 50 mM sodium phosphate (pH 7.5), 5 mM EDTA, and 20 μM peptidyl MCA. After 30 min at 37 ̊C, fluorescence intensity was measured with excitation at 380 nm and emission at 460 nm. In some experiments, pH values varied from 5.5-9.5 with 50 mM phosphate (pH 5.5-8.5) or Tris-HCl (pH 7.0-9.5), and NaCl concentrations varied from 0-1.6 M. To determine enzymatic parameters, recombinant proteins were incubated with various concentrations of peptidyl MCA. Obtained data were analyzed using a nonlinear regression curve fitted to the Michaelis-Menten equation with the GraphPad Prism software program (San Diego, CA). Values are shown as the average ± S.D. and calculated from 4 independent measurements. Subcellular Fractionation—All procedures Acylpeptidyl oligopeptidase in P. gingivalis 4 were carried out at 4 ̊C according to a previously reported method (30), with a sight modification. Briefly, a 20-ml culture of P. gingivalis in the log-phase was centrifuged at 6000 x g for 15 min, then the extracellular fraction was obtained from the supernatant by filtration with a 0.20-μm membrane filter. Bacterial cells were washed with ice-cold PBS, resuspended in 4 ml of 0.25 M sucrose in 5 mM Tris-HCl (pH 7.5), and then left on ice for 10 min. Cells were precipitated at 12,500 x g for 15 min, resuspended in 4 ml of 5 mM Tris-HCl, and mixed gently for 10 min to disrupt the outer membrane. The supernatant was obtained by centrifugation at 6000 x g for 15 min and collected as the periplasmic fraction containing the outer membrane. The obtained spheroplasts as precipitate were resuspended in PBS and then disrupted in an ice-water bath by sonication pulse 10 times for 10 seconds each with 2-second intervals. The cytosol and inner membrane fractions were separately prepared by ultracentrifugation, as previously described (24). Size Exclusion HPLC—Recombinant proteins and the periplasmic fraction were subjected to size exclusion HPLC using ÄKTA explorer 10S (GE Healthcare) with a Superdex200 increase 10/300 column (1.5 X 30 cm) equilibrated with 20 mM Tris-HCl (pH 8.0) containing 1 mM EDTA. They were separated with the identical buffer at a rate of 0.75 ml/min at room temperature, then 0.5-ml fractions were collected. Aliquots of the fractions were subjected to a peptidase assay with Z-VKM-MCA, as well as SDS-PAGE or native PAGE. Immunoblotting Analysis—Recombinant PGN_1349 (Met-Lys) was purified by use of the Talon affinity chromatography and further subjected to a Sephacryl S200 HR column equilibrated with 20 mM ammonium bicarbonate (pH 8.5). Rabbit anti-PGN_1349 (AOP) antiserum was prepared as previously reported (24, 27). For immunoblotting, separated proteins on polyvinylidene difluoride membranes (Millipore) were incubated with anti-AOP antiserum (10-fold dilution), followed by alkaline phosphatase-conjugated anti-rabbit IgG. Finally, specific bands were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Similarly, immunoblotting against P. gingivalis DPP5 and DPP7 was performed as previously reported (24, 25). N-Terminal sequencing of proteins—Proteins separated on SDS-PAGE and transferred to a Sequi-Blot membrane (Bio-Rad) were stained with Coomassie Brilliant Blue, and subjected to the protein sequencing by use of a model Procise 49XcLC protein sequencer (Applied Biosystems). 3D Homology Modeling—In order to search for an ideal template for homology modeling, P. gingivalis AOP sequence was submitted to Psipred (31) and GenThreader (32) server for domain and fold assignment. Following, a Blast search (33) against the Protein Data Bank was performed to identify the closest homologue to P. gingivalis AOP with known 3D structure. The model was generated via the server Phyre2 (Protein Homology/Analogy Recognition Engine) (34), using the tool “one-to-one threading”, in the expert mode. The coordinates of Pyrococcus horikoshii acylaminoacyl peptidase (AAP) (35) (PDB code: 4HXE) and P. gingivalis AOP sequence were submitted to structural alignment and further model building. The final model covered around 85% of the sequence, sharing 20% identity with the template.