Expanded substrate specificity supported by P1' and P2' residues enables bacterial dipeptidyl-peptidase 7 to degrade bioactive peptides.

Dipeptide production from extracellular proteins is crucial for Porphyromonas gingivalis, a pathogen related to chronic periodontitis, because its energy production is entirely dependent on the metabolism of amino acids predominantly incorporated as dipeptides. These dipeptides are produced by periplasmic dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11. Although the substrate specificities of these four DPPs cover most amino acids at the penultimate position from the N terminus (P1), no DPP is known to cleave penultimate Gly, Ser, Thr, or His. Here, we report an expanded substrate preference of bacterial DPP7 that covers those residues. MALDI-TOF mass spectrometry analysis demonstrated that DPP7 efficiently degraded incretins and other gastrointestinal peptides, which were successively cleaved at every second residue, including Ala, Gly, Ser, and Gln, as well as authentic hydrophobic residues. Intravenous injection of DPP7 into mice orally administered glucose caused declines in plasma glucagon-like peptide-1 and insulin, accompanied by increased blood glucose levels. A newly developed coupled enzyme reaction system that uses synthetic fluorogenic peptides revealed that the P1' and P2' residues of substrates significantly elevated kcat values, providing an expanded substrate preference. This activity enhancement was most effective toward the substrates with nonfavorable but nonrepulsive P1 residues in DPP7. Enhancement of kcat by prime-side residues was also observed in DPP11 but not DPP4 and DPP5. Based on this expanded substrate specificity, we demonstrate that a combination of DPPs enables proteolytic liberation of all types of N-terminal dipeptides and ensures P. gingivalis growth and pathogenicity.

Immunoelectron Microscopic Analysis of Dipeptidyl-Peptidases and Dipeptide Transporter Involved in Nutrient Acquisition in Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic, Gram-negative, anaerobic bacterium representing a keystone pathogen in chronic periodontitis. The bacterium's energy production depends on the metabolism of amino acids, which are predominantly incorporated as dipeptides via the proton-dependent oligopeptide transporter (Pot). In this study, the localization of dipeptidyl-peptidases (DPPs) and Pot was investigated for the first time in P. gingivalis using immunoelectron microscopy with specific antibodies for the bacterial molecules and gold-conjugated secondary antibodies on ultrathin sections. High-temperature protein G and hemin-binding protein 35 were used as controls, and the cytoplasmic localization of the former and outer membrane localization of the latter were confirmed. P. gingivalis DPP4, DPP5, DPP7, and DPP11, which are considered sufficient for complete dipeptide production, were detected in the periplasmic space. In contrast, DPP3 was localized in the cytoplasmic space in accord with the absence of a signal sequence. The inner membrane localization of Pot was confirmed. Thus, spatial integration of the nutrient acquisition system exists in P. gingivalis, in which where dipeptides are produced in the periplasmic space by DPPs and readily transported across the inner membrane via Pot.

Characterization of substrate specificity and novel autoprocessing mechanism of dipeptidase A from Prevotella intermedia.

Prevotella intermedia, a Gram-negative anaerobic rod, is frequently observed in subgingival polymicrobial biofilms from adults with chronic periodontitis. Peptidases in periodontopathic bacteria are considered to function as etiological reagents. Prevotella intermedia OMA14 cells abundantly express an unidentified cysteine peptidase specific for Arg-4-methycoumaryl-7-amide (MCA). BAU17746 (locus tag, PIOMA14_I_1238) and BAU18827 (locus tag, PIOMA14_II_0322) emerged as candidates of this peptidase from the substrate specificity and sequence similarity with C69-family Streptococcus gordonii Arg-aminopeptidase. The recombinant form of the former solely exhibited hydrolyzing activity toward Arg-MCA, and BAU17746 possesses a 26.6% amino acid identity with the C69-family Lactobacillus helveticus dipeptidase A. It was found that BAU17746 as well as L. helveticus dipeptidase A was a P1-position Arg-specific dipeptidase A, although the L. helveticus entity, a representative of the C69 family, had been reported to be specific for Leu and Phe. The full-length form of BAU17746 was intramolecularly processed to a mature form carrying the N-terminus of Cys15. In conclusion, the marked Arg-MCA-hydrolyzing activity in Pre. intermedia was mediated by BAU17746 belonging to the C69-family dipeptidase A, in which the mature form carries an essential cysteine at the N-terminus.

Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor.

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.

Establishment of potent and specific synthetic substrate for dipeptidyl-peptidase 7.

Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40 ±â€¯0.83 µM-1 s-1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06-3.77 µM-1 s-1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7.

A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides.

Exopeptidases, including dipeptidyl- and tripeptidylpeptidase, are crucial for the growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium that incorporates amino acids mainly as di- and tripeptides. In this study, we identified a novel exopeptidase, designated acylpeptidyl oligopeptidase (AOP), composed of 759 amino acid residues with active Ser(615) 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, although sequence similarities to human and archaea-type acylaminoacyl peptidase sequences were observed, its enzymatic properties were apparently distinct from those, because AOP scarcely released an N-acyl-amino acid as compared with di- and tripeptides, especially with N-terminal modification. The kcat/Km value against benzyloxycarbonyl-Val-Lys-Met-4-methycoumaryl-7-amide, the most potent substrate, was 123.3 ± 17.3 µm(-1) s(-1), 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, whereas equilibrium between monomers and oligomers was observed with a recombinant molecule, suggesting a tendency of oligomerization mediated by the N-terminal region (Met(16)-Glu(101)). Three-dimensional modeling revealed the three domain structures (residues Met(16)-Ala(126), which has no similar homologue with known structure; residues Leu(127)-Met(495) (ß-propeller domain); and residues Ala(496)-Phe(736) (α/ß-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.

Degradation of Incretins and Modulation of Blood Glucose Levels by Periodontopathic Bacterial Dipeptidyl Peptidase 4.

Severe periodontitis is known to aggravate diabetes mellitus, though molecular events related to that link have not been fully elucidated. Porphyromonas gingivalis, a major pathogen of periodontitis, expresses dipeptidyl peptidase 4 (DPP4), which is involved in regulation of blood glucose levels by cleaving incretins in humans. We examined the enzymatic characteristics of DPP4 from P. gingivalis as well as two other periodontopathic bacteria, Tannerella forsythia and Prevotella intermedia, and determined whether it is capable of regulating blood glucose levels. Cell-associated DPP4 activity was found in those microorganisms, which was effectively suppressed by inhibitors of human DPP4, and molecules sized 73 kDa in P. gingivalis, and 71 kDa in T. forsythia and P. intermedia were immunologically detected. The kcat/Km values of recombinant DPP4s ranged from 721 ± 55 to 1,283 ± 23 µM-1s-1 toward Gly-Pro-4-methylcoumaryl-7-amide (MCA), while those were much lower for His-Ala-MCA. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis showed His/Tyr-Ala dipeptide release from the N termini of incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide, respectively, with the action of microbial DPP4. Moreover, intravenous injection of DPP4 into mice decreased plasma active GLP-1 and insulin levels, accompanied by a substantial elevation in blood glucose over the control after oral glucose administration. These results are the first to show that periodontopathic bacterial DPP4 is capable of modulating blood glucose levels the same as mammalian DPP4; thus, the incidence of periodontopathic bacteremia may exacerbate diabetes mellitus via molecular events of bacterial DPP4 activities.

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

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 µM and 11.02 µM(-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.

A bone substitute with high affinity for vitamin D-binding protein--relationship with niche of osteoclasts.

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism.

Potential elevation of exopeptidase activity of Glu-specific endopeptidase I/GluV8 mediated by hydrophobic P1'-position amino acid residue.

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in kcat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2'-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1' and P2' residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1'-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased kcat/KM primarily through a maximum 500-fold elevation of kcat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1' residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1' amino acid residues.

Asp- and Glu-specific novel dipeptidyl peptidase 11 of Porphyromonas gingivalis ensures utilization of proteinaceous energy sources.

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp(22). A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the k(cat)/K(m) value was higher for Asp than Glu. Those activities were lost by substitution of Ser(652) in P. endodontalis and Ser(655) in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg(670) is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg(670) interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods.

Dipeptidyl-peptidases: Key enzymes producing entry forms of extracellular proteins in asaccharolytic periodontopathic bacterium Porphyromonas gingivalis.

Porphyromonas gingivalis, a pathogen of chronic periodontitis, is an asaccharolytic microorganism that solely utilizes nutritional amino acids as its energy source and cellular constituents. The bacterium is considered to incorporate proteinaceous nutrients mainly as dipeptides, thus exopeptidases that produce dipeptides from polypeptides are critical for survival and proliferation. We present here an overview of dipeptide production by P. gingivalis mediated by dipeptidyl-peptidases (DPPs), e.g., DPP4, DPP5, DPP7, and DPP11, serine exopeptidases localized in periplasm, which release dipeptides from the N-terminus of polypeptides. Additionally, two other exopeptidases, acylpeptidyl-oligopeptidase (AOP) and prolyl tripeptidyl-peptidase A (PTP-A), which liberate N-terminal acylated di-/tri-peptides and tripeptides with Pro at the third position, respectively, provide polypeptides in an acceptable form for DPPs. Hence, a large fraction of dipeptides is produced from nutritional polypeptides by DPPs with differential specificities in combination with AOP and PTP-A. The resultant dipeptides are then incorporated across the inner membrane mainly via a proton-dependent oligopeptide transporter (POT), a member of the major facilitator superfamily. Recent studies also indicate that DPP4 and DPP7 directly link between periodontal and systemic diseases, such as type 2 diabetes mellitus and coagulation abnormality, respectively. Therefore, these dipeptide-producing and incorporation molecules are considered to be potent targets for prevention and treatment of periodontal and related systemic diseases.

Amino acid residues modulating the activities of staphylococcal glutamyl endopeptidases.

The glutamyl endopeptidase family of enzymes from staphylococci has been shown to be important virulence determinants of pathogenic family members, such as Staphylococcus aureus. Previous studies have identified the N-terminus and residues from positions 185-195 as potentially important regions that determine the activity of three members of the family. Cloning and sequencing of the new family members from Staphylococcus caprae (GluScpr) and Staphylococcus cohnii (GluScoh) revealed that the N-terminal Val residue is maintained in all family members. Mutants of the GluV8 enzyme from S. aureus with altered N-terminal residues, including amino acids with similar properties, were inactive, indicating that the Val residue is specifically required at the N-terminus of this enzyme family in order for them to function correctly. Recombinant GluScpr was found to have peptidase activity intermediate between GluV8 and GluSE from Staphylococcus epidermis and to be somewhat less specific in its substrate requirements than other family members. The 185-195 region was found to contribute to the activity of GluScpr, although other regions of the enzyme must also play a role in defining the activity. Our results strongly indicate the importance of the N-terminal and the 185-195 region in the activity of the glutamyl endopeptidases of staphylococci.

Preferential dipeptide incorporation of Porphyromonas gingivalis mediated by proton-dependent oligopeptide transporter (Pot).

Multiple dipeptidyl-peptidases (DPPs) are present in the periplasmic space of Porphyromonas gingivalis, an asaccharolytic periodontopathic bacterium. Dipeptides produced by DPPs are presumed to be transported into the bacterial cells and metabolized to generate energy and cellular components. The present study aimed to identify a transporter responsible for dipeptide uptake in the bacterium. A real-time metabolic analysis demonstrated that P. gingivalis preferentially incorporated Gly-Xaa dipeptides, and then, single amino acids, tripeptides and longer oligopeptides to lesser extents. Heterologous expression of the P. gingivalis serine/threonine transporter (SstT; PGN_1460), oligopeptide transporter (Opt; PGN_1518) and proton-dependent oligopeptide transporter (Pot; PGN_0135) genes demonstrated that Escherichia coli expressing Pot exclusively incorporated Gly-Gly, while SstT managed Ser uptake and Opt was responsible for Gly-Gly-Gly uptake. Dipeptide uptake was significantly decreased in a P. gingivalis Δpot strain and further suppressed in a Δpot-Δopt double-deficient strain. In addition, the growth of the Δpot strain was markedly attenuated and the Δpot-Δopt strain scarcely grew, whereas the ΔsstT strain grew well almost like wild type. Consequently, these results demonstrate that predominant uptake of dipeptide in P. gingivalis is mostly managed by Pot. We thus propose that Pot is a potential therapeutic target of periodontal disease and P. gingivalis-related systemic diseases.

Single nucleotide polymorphism that accompanies a missense mutation (Gln488His) impedes the dimerization of Hsp90.

A single nucleotide polymorphism (SNP) that causes a missense mutation of highly conserved Gln488 to His of the alpha isoform of the 90-kDa heat shock protein (Hsp90alpha) molecular chaperone is observed in Caucasians. The mutated Hsp90alpha severely reduced the growth of yeast cells. To investigate this molecular mechanism, we examined the domain-domain interactions of human Hsp90alpha by using bacterial 2-hybrid system. Hsp90alpha was expressed as a full-length form, N-terminal domain (residues 1-400), or middle (residues 401-617) plus C-terminal (residues 618-732) domains (MC domain/amino acids 401-732). The Gln488His substitution in MC domain did not affect the intra-molecular interaction with N-terminal domain, whereas the dimeric interaction-mediated by the inter-molecular interaction between MC domains was decreased to 32%. Gln488Ala caused a similar change, whereas Gln488Thr, which exceptionally occurs in mitochondrial Hsp90 paralog, fully maintained the dimeric interaction. Therefore, the SNP causing Gln488His mutation could abrogate the Hsp90 function due to reduced dimerization.

Characterization of bacterial acylpeptidyl-oligopeptidase.

Acylpeptidyl-oligopeptidase (AOP), which has been recently identified as a novel enzyme in a periodontopathic bacterium, Porphyromonas gingivalis, removes di- and tri-peptides from N-terminally acylated polypeptides, with a preference for hydrophobic P1-position amino acid residues. To validate enzymatic properties of AOP, characteristics of two bacterial orthologues from Bacteroides dorei (BdAOP), a Gram-negative intestinal rod, and Lysinibacillus sphaericus (LsAOP), a Gram-positive soil rod, were determined. Like P. gingivalis AOP (PgAOP), two orthologues more efficiently hydrolyzed N-terminal acylated peptidyl substrates than non-acylated ones. Optimal pH was shifted from 7.0 to 8.9 for N-acylated to 8.5-9.5 for non-acylated substrates, indicating preference for non-charged hydrophobic N-terminal residues. Hydrophobic P1- and P2-position preferences were common in the three AOPs, although PgAOP preferred Leu and the others preferred Phe at the P1 position. In vitro mutagenesis demonstrated that Phe647 in PgAOP was responsible for the P1 Leu preference. In addition, bacterial AOPs commonly liberated acetyl-Ser1-Tyr2 from α-melanocyte-stimulating hormone. Taken together, although these three bacterial AOPs exhibited some variations in biochemical properties, the present study demonstrated the existence of a group of exopeptidases that preferentially liberates mainly dipeptides from N-terminally acylated polypeptides with a preference for hydrophobic P1 and P2-position residues.

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli.

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation.

V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis--the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.

Distribution of dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11 in human oral microbiota-potent biomarkers indicating presence of periodontopathic bacteria.

Dipeptidyl peptidase (DPP) 4, DPP5, DPP7 and DPP11, expressed in the periplasmic space, are crucial for energy production for Porphyromonas gingivalis, an asaccharolytic bacterium that causes periodontal disease. Bacterial DPP4 seems to be involved in regulation of blood glucose level via degradation of incretins. The present study aimed to identify four dpp orthologs in oral microbiota by database searches, and their enzymatic activities in periodontopathic and cariogenic bacteria, as well as oral specimens were determined. Search in the databases suggested that 43 species of 772 taxa possess dpp4 and other dpp genes. Most species are in the genera Bacteroides, Capnocytophaga, Porphyromonas, Prevotella and Tannerella, indicating a limited distribution of dpp orthologs in anaerobic periodontopathic rods. In accordance with those results, activities of all four DPPs were demonstrated in P. gingivalis, Porphyromonas endodontalis and Tannerella forsythia, while they were negligible in Treponema denticola, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Furthermore, DPP activities were also detected in subgingival dental plaque at different intensities among individual specimens, while DPP4 activity presumably derived from human entity was solely predominant in saliva samples. These findings demonstrated that DPP activities in dental plaque serve as potent biomarkers to indicate the presence of periodontopathic bacteria.

Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides.

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly673 in DPP7 and Arg673 in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly673 exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg673 was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly673 from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser571-Leu700) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members.