key: cord-1033385-dvq637cs
authors: Nemoto, Takayuki K.; Ohara Nemoto, Yuko
title: Dipeptidyl‐peptidases: Key enzymes producing entry forms of extracellular proteins in asaccharolytic periodontopathic bacterium Porphyromonas gingivalis
date: 2020-10-12
journal: Mol Oral Microbiol
DOI: 10.1111/omi.12317
sha: 4e35f95ff0275b1e53cc65364b15039f114847aa
doc_id: 1033385
cord_uid: dvq637cs

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.

Periodontal disease, a bacterial-associated inflammatory condition that occurs around the gingivae, is a leading cause of tooth loss in adults, with 20%-50% of individuals affected worldwide, which results in a decrement in overall quality of life especially in elderly individuals (Nazir, 2017) . A large number of epidemiological studies have shown a keen association of chronic periodontitis and type 2 diabetes mellitus (Grossi & Genco, 1998; Lalla & Papapanou, 2011; Preshaw et al., 2012) , and recently much attention has been given to this oral disease because of its close relationship to systemic diseases, such as atherosclerotic cardiovascular disorder (Genco & VanDyke, 2010; Tabeta et al., 2014) , decreased kidney function (Kshirsagar et al., 2007) , rheumatoid arthritis (Detert et al., 2010) , and Alzheimer's disease (Dominy et al., 2019; Teixeira et al., 2017) .

Periodontal disease is initiated by a complex of bacterial species that form subgingival biofilm in and around the gingival crevice.

When allowed to progress, inflammatory cytokines and chemokines are released from affected tissues, implying a connection of this oral inflammatory condition to other systemic diseases. In previous studies, Socransky and Haffajee and their colleagues stratified the microbiota into five major complexes, among which the first one, designated "red complex" and comprised of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, is strikingly associated with clinical measures of periodontal disease, and thus those members are considered to be major periodontopathic bacteria implicated in severe forms (Holt & Ebersole, 2005; Socransky & Haffajee, 2002; Socransky et al., 1998) . Whole genome analysis has suggested that P. gingivalis can metabolize several sugars including melibiose and galactose (Nelson et al., 2003) . Nevertheless, P. gingivalis and T. forsythia are in practical terms asaccharolytic and their growth is supported by proteinaceous substrates. Sugar utilization may be not so beneficial for periodontal bacteria, because sugar supply is quite limited in their niche. This feature of utilization of amino acids as the sole carbon and energy source is quite singular among oral microorganisms and seems to be advantageous for settlement of these pathogenic bacteria in subgingival plaque. However, until recently little has been reported regarding how nutritional amino acids are produced from extracellular proteins and become incorporated into cells of those bacteria. Extracellular proteins in P. gingivalis are initially degraded into oligopeptides by the well-characterized cysteine endopeptidases Arg-gingipains (Kadowaki et al., 1994; Pavloff et al., 1995) and Lysgingipain (Pavloff et al., 1997; Scott et al., 1993) , which are localized in the outer membrane and outer membrane vesicles. The resulting oligopeptides are transported into the periplasmic space. As they are still larger than the size incorporated across the inner membrane through the outer membrane, they must be further trimmed by exopeptidases prior to incorporation.

Collateral evidence has indicated that P. gingivalis incorporates dipeptides derived from proteinaceous nutrients. Tang-Larsen et al. (1995) reported that additions of Met-Met and Cys-Cys enhanced the production of methyl mercaptan and hydrogen sulfide, respectively, whereas neither a single Met nor Cys showed increased production. The dominance of dipeptides as nutrients in P. gingivalis was also demonstrated by findings showing significant accumulation of their metabolites, such as ammonia and short-chain fatty acids (Takahashi & Sato, 2001 . These findings strongly suggest that not a single amino acid but dipeptide is the major form incorporated into P. gingivalis cells. In accordance with this notion, a series of dipeptide-producing exopeptidases, dipeptidyl-peptidases (DPPs), have been discovered in P. gingivalis.

In this article, we discuss the roles of DPPs and two additional exopeptidases in dipeptide production, as well as the specific distribution of dpp genes in periodontopathic bacteria. Furthermore, we demonstrate the importance of a proton-dependent oligopeptide transporter (POT) in dipeptide incorporation in P. gingivalis. The relationship of bacterial DPPs with systemic diseases is also discussed.

Gingipains are cysteine proteinases that consist of Lys-specific (Lys-gingipain/Kgp) (Pavloff et al., 1997; Scott et al., 1993) and two Arg-specific (Arg-gingipains/Rgps) proteinases (Pavloff et al., 1995) .

Gingipains degrade various human proteins, including complement system proteins, cytokines, and integrin (O'Brien-Simpson et al., 2001) , and their roles as virulence factors have been reviewed elsewhere (Fitzpatrick et al., 2009; Kadowaki et al., 2007; Potempa et al., 2003) . The triple-gingipain gene null mutant KDP136 was shown unable to grow in a defined medium with human albumin as the sole carbon source (Shi et al., 1999) , implicating that gingipains are not only virulence factors but also essential for bacterial growth.

Following degradation of extracellular proteins by gingipains, the resultant oligopeptides are imported into periplasmic space by likely an import mechanism. Madej et al., 2020) recently reported the molecular structure of RagAB, the cell wall importer for oligopeptides, and showed that the RagAB dimer forms a large internal cavity, which seems adaptive for accepting oligopeptides from 7 to 29 residues in length.

DPPs are exopeptidases that liberate dipeptides from the unblocked N-terminus of oligopeptides and proteins, with eight members classified by amino acid sequence similarity and substrate specificities; DPP1-6 (or I-VI), 7, and 11. Roman numerals have been used for DPPI-DPPVI, though for simplicity and to follow more recent usage, this study uses Arabic numbers for all DPPs. Other DPP members with various designations, i.e., QPP, FAPα (Retting et al., 1994) , DPP6, DPP8, and DPP9 (Olsen & Wagtmann, 2002) , DPP10 (Qi et al., 2003) , and DPP-A and DPP-B are subtypes of eukaryotic DPP4, because of their sequence similarities (Table 1) . The substrate specificity of DPPs is primarily defined based on the penultimate amino acid residue from the N-terminus (P1 position), and the N-terminal residue (P2-position) has effects to some extent on activity towards P. gingivalis DPP7 and DPP11 . Pro at the P1'-position, the C-terminal side residue of the hydrolyzed peptide bond, is never accepted by any DPP, thus peptides with NH 2 -Xaa1-Xaa2-Pro3-are resistant to all DPPs. P1'-position and its C-terminal adjacent P2'-position residues, except for Pro, are least involved in substrate recognition.

Structural analysis of human DPP4 revealed a limited interaction with P1'-position Ser and P2'-position Lys of YP/SKPDNPGE, first ten residues of neuropeptide (Aertgeerts et al., 2004) . Similarly, P1'-and P2'-position residues of Leu-Asp/-Val-Trp exhibit few interactions with P. endodontalis DPP11 . These findings indicate that both P1' and P2' residues are not tightly associated with DPP4 and DPP11, and presumably other DPPs as well. Polypeptides with N-terminal modification are not acceptable for DPPs. In P. gingivalis and human DPP4, this is due to the necessity of the ionic interaction between the N-terminal α-amino group of the substrate and carboxy groups of Glu195 and Glu196 (Rea et al., 2017) . 

DPP3 belongs to the M49.001 peptidase family and is widely distributed among eukaryotic (Ellis & Nuenke, 1967) and prokaryotic organisms, and is known to readily hydrolyze NH 2 -Arg1-Arg2-|-Xaa3-. P. gingivalis DPP3 exhibits a 19.0% amino acid identity to the human entity and shows the highest activity for a synthetic substrate, Arg-Arg-4-methycoumaryl-7-amide (MCA), followed by Leu-Arg-, Ala-Arg-, and Lys-Ala-MCA, the same as the eukaryotic entity . P. gingivalis DPP3 is composed of 906 and 886 amino acid residues in the strains ATCC 33277 and W83, respectively, which are substantially larger than 675 residues of Bacteroides thetiotaomicron DPP3 and 737 residues of human DPP3

due to the C-terminally tagged sequence. The additional sequence is predicted at high confidence to have α-α superhelix fold, belonging to Armadillo-type fold family similar to the AlkD family of bacterial glycosylases (Hromić-Jahjefendić et al., 2017). The genes encoding DPP3 with AlkD domain are specifically found within the genus

There are two lines of evidence indicating that P. gingivalis DPP3

is not responsible for dipeptide production from nutritional proteins.

First, it does not contain a signal sequence, thus seems to be localized in cytoplasm, and second, P. gingivalis cells show little hydrolysis for Arg-Arg-MCA (Ohara- . Accordingly, we speculate that DPP3 functions for degradation of cytosolic proteins and that degradation of nutritional peptides at Arg-Xaa bonds is already completed by Arg-gingipains.

DPP4 is widely distributed in a variety of species from eukaryotic (S9.003) (Misumi et al., 1992) to bacterial (S9.009) (Ahrén et al., 2000) . Human DPP4/CD26 hydrolyzes the peptide bond at the carboxyl side of Pro2 most preferentially and Ala2 at a reduced rate. DPP4 is a key factor that modulates postprandial hyperglycemia by degrading incretin peptides, i.e., glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which induce secretion of insulin from pancreatic β cells. DPP4 inhibitors, a class of oral antidiabetic agents, such as sitagliptin, vildagliptin, and alogliptin, suppress degradation of incretins and improve glucose tolerance and insulin secretion (Ahrén et al., 2000; Gallwitz, 2019; Weber, 2004) . Human DPP4 is a type II membrane protein, in which the N-terminal hydrophobic sequence represents an uncleavable signal peptide, and functions as a membrane-anchoring domain (Misumi et al., 1992) , and the entity expressed in lymphocytes, and endothelial and epithelial cells as a functional receptor for human Middle East respiratory syndrome coronavirus (MERS-CoV) (Raj et al., 2013) .

Porphyromonas gingivalis DPP4 exhibits the substrate specificity similar to that of human DPP4 (Banbula et al., 2000) and a very overall structure to human entity (Rea et al., 2017) , despite a limited amino acid identity (23.8%) to human DPP4 (Kiyama et al., 1998) .

Periodopathic bacterial DPP4 as well as human DPP4 degrades incretin and modulates blood glucose levels in mice . This issue will be described in detail in Section 9. P. gingivalis DPP4 also degrades substance P, fibrin inhibitory peptide, IL-1β, IL-2, and β-casomorphin in vitro (Banbula et al., 2000) . Mice injected with the P. gingivalis W83 strain were found to develop abscesses to a greater extent and died more frequently than those challenged with a dpp4-deficient strain (Kumagai et al., 2000) . However, this phenomenon may not represent virulence potential, but the nutri- gingivalis DPP4 entering into the bloodstream is inefficiently inhibited by prescribed DPP4 inhibitors for type 2 diabetes mellitus patients (Rea et al., 2017) .

DPP5 was initially found in Aspergillus fumigatus (Beauvais et al., 1997) , then later in P. gingivalis as the first bacterial species . Based on the discovery of P. gingivalis DPP5, its wide distribution among bacteria and archaea, as well as in eukaryotes including higher animals and plants has been revealed.

The amino acid sequence of P. gingivalis DPP5 is 28.5% identical to that of A. fumigatus DPP5. P. gingivalis DPP5 preferentially removes dipeptides with Ala and hydrophobic residues at the P1 position from synthetic dipeptidyl MCA substrates. Lys-Ala-MCA has been identified as the most potent substrate, though no native substrate has yet been identified.

DPP6 removes the N-terminal dipeptide L-Ala-D-Glu by hydrolysis of the γ-D-glutamyl-(L)-diamino acid bond (Guinand et al., 1979) . 

DPP7, a member of S46.001, was initially discovered in P. gingivalis and does not exist in eukaryotic organisms. In this respect, it should be noted that human DPP7 is not related to bacterial DPP7, but rather is an isozyme of DPP2 (Bezerra et al., 2012) . DPP7 releases the N-terminal dipeptide NH 2 -Xaa1-Zaa2 (Zaa, hydrophobic residues) (Banbula et al., 2001; , and hydrolyzes insulin B chain, type I collagen, and azocasein in vitro (Banbula et al., 2001) Since the P1-position specificity of DPP7 overlaps with that of DPP5, findings of a specific synthetic substrate for DPP7 were eagerly anticipated and Phe-Met-MCA finally identified . Although both DPP5 and DPP7 show a common hydrophobic P1

preference, DPP5 has no apparent amino acid preference at the P2 position (Ohara- , in contrast to the hydrophobic P2 preference of DPP7 . Thus, the difference in P2-position preference between DPP5 and DPP7 amplifies the repertoire of peptide substrates of P. gingivalis. For example, P. gingivalis DPP7 hydrolyzes Leu-Arg-, Leu-Gln-, and Leu-Glu-MCA to some extent, while those are scarcely hydrolyzed by DPP5 . Hence, hydrolyzation of Leu-Gly-β-naphthylamine, which has been observed in five P. gingivalis strains, is likely mediated by DPP7 (Suido et al., 1986 ).

A metabolite analysis of P. gingivalis indicated that glutamic acid/ glutamine (Glx)-and aspartic acid/asparagine (Asx)-containing peptides were the most intensively consumed (Takahashi et al., 2000) , and those experimental results were supported by computational analysis of the genome-scale metabolic network using flux balance calculation (Mazumdar et al., 2009 ). However, how Asp-and

Glu-containing dipeptides are produced in the bacterium was not elucidated, until being revealed by the finding of novel Asp-and Gluspecific DPP 11 (Ohara- Nemoto et al., 2011) .

A previous study noted that P. gingivalis DPP11 encoded by PGN_0607 in strain ATCC 33277 may be an isozyme of DPP7, because of its 38.4% amino acid identity to DPP7 encoded by PGN_1479 (Banbula et al., 2001) . However, it was later shown that the product of PGN_0607 is a novel DPP specific for acidic amino acid residues Asp and Glu, exclusively distinct from the hydrophobic specificity of DPP7, thus DPP11 was classified as an S46.002

peptidase. S46 family peptidases are solely distributed in bacteria, and DPP11 as well as DPP7 prefer hydrophobic residues at the P2 position .

The acidic P1-position residue is recognized by Arg673 in P.

gingivalis DPP11. Biochemical studies have revealed that there are three subtypes of DPP11; (a) Porphyromonas-type DPP11, which is Asp-preferential, (b) Bacteroides-type DPP11, which shows a high preference for Asp and scarcely accepts Glu, and (c) Shewanella-type DPP11, in which S1 Ser673 instead of Arg673, primarily exhibits higher preference for Glu .

In DPP7, Arg673 of DPP11 is replaced by Gly666, which allows the recognition of bulky hydrophobic P1 residue (Ohara-Nemoto et al., 2011) . Furthermore, Gly666Arg substitution in recombinant DPP7 resulted in a partial acquisition of hydrolyzing activity against acidic residues . Also, elucidation of the three-di- 

The existence of multiple DPPs should be beneficial for complete degradation of polypeptides into dipeptides. However, there appear to be two types of peptide bonds resistant to bacterial DPPs identified to date. First, N-terminally-acylated polypeptides are resistant to all DPPs as well as prolyl tripeptidyl-peptidase A (PTP-A), even though plasma proteins serving as potential nutrients for P. gingivalis are frequently N-terminally modified. Second, polypeptides with Pro at the third position from the N-terminus are resistant to DPPs.

The first issue is surmounted by acylpeptidyl-oligopeptidase (AOP), which preferentially degrades polypeptides with N-terminal modification into di-and tri-peptides (Nemoto et al., 2016) . The second one is also surmounted by PTP-A, which liberates NH 2 -Xaa1-Xaa2-Pro3 from polypeptides (Banbula et al., 1999; Ito et al., 2006) .

In other words, AOP and PTP-A have possibly evolved to complete dipeptide production by DPPs in P. gingivalis.

AOP, which belongs to the S09 family, preferentially degrades N-terminally acylated polypeptides to liberate acylated-di-and tri-peptides (Nemoto et al., 2016) . The most potent synthetic substrate for P. gingivalis AOP is benzyloxycarbonyl-Lys-Met-MCA.

The structure of acylated compounds is not of concern, whereas concealment of the N-terminal α-amino group is essential for efficient degradation. Although acylaminoacyl-peptidase (AAP), which specifically removes N-terminal acylated amino acids from a polypeptide, has been previously reported in the human and archaea Aeropyrum pernix (Kiss et al., 2007) , the amino acid sequence identity (15.8%) between P. gingivalis AOP and Aeropyrum pernix AAP is limited, while AOP scarcely removes acylated N-terminal amino acids but dipeptides. Modeling of P. gingivalis AOP revealed the hydrophobic S1 site of AOP in accord with its hydrophobic P1 preference as well as an N-anchor region determining the substrate specificity with a more open active site than those of DPPs accepting a broad range of N-terminal different steric groups (Nemoto et al., 2016) .

Comparisons of three AOPs from P. gingivalis, Bacteroides dorei,

and Lysinibacillu sphaericus revealed that the activity of P. gingivalis AOP was most significantly enhanced by acylation of substrates, further supporting the removal of acylated N-terminus as the primary role of that AOP .

PTP-A was first discovered in P. gingivalis and belongs to S9.017, and has been shown to hydrolyze the Pro3-Xaa4 bond (Banbula et al., 1999) . Reflecting the similarity of substrate specificities, the amino acid sequence of P. gingivalis PTP-A is 23.5% identical to that of P. gingivalis DPP4. An unblocked N-terminus is required for its activity and no cleavage occurs with substrates with Pro at the P1' position. Synthetic substrates, Ala-Ala-Pro-and

Gly-Ala-Pro-p-nitroanilide, are used for the assay of PTP-A, and it has been proposed that PTP-A is involved in the degradation of type I collagen in connection with periodontitis inflammation (Ito et al., 2006) . However, because the Gly-Xaa-Pro repeat is located at the center of the collagen sequences, it is ambiguous whether this exopeptidase truly degrades collagens in host tissues. Hence, the primary role of PTP-A is likely to provide oligopeptides without Pro at the third position from the N-terminus for DPPs. Genome analysis (Naito et al., 2008) as well as biochemical studies up to date failed to find tripeptidyl-peptidases except for PTP-A, which strongly suggests the absence of other tripeptidylpeptidases in P. gingivalis.

The enzymatic activities of DPPs, AOP, and PTP-A are scarcely detected in bacterial culture supernatant, indicating that they are cell-associated. In addition, we previously showed the periplasmic localization of DPP5 by subcellular fractionation . Although the subcellular localization of other DPPs, AOP, and PTP-A has not been biochemically verified, they possess typical signal sequences for export from the inner membrane but do not possess a conserved C-terminal domain (CTD) of 70-80 amino acids, a prerequisite for secretion via the Type-9

secretion system (T9SS) (Sato et al., 2010; Seers et al., 2006; Slakeski et al., 2011) . In accord with signal sequence prediction of Met1-Ala21 in P. endodontalis DPP11, the N-terminal Asp22 was identified in an endogenous form (Ohara-Nemoto et al., 2011) .

Accordingly, DPPs are transported through the inner membrane and localized in the periplasmic space. P. gingivalis AOP also possesses a potent signal sequence. Although the probability is less significant, Met1-Ala38 in P. gingivalis PTP-A may correspond to the signal peptide. Proteome analysis of P. gingivalis W50 also suggested the localization in the lumen of the vesicle (periplasm) of DPP4 (PG_0503), DPP5 (PG_1004), DPP7 (PG_0491), and DPP11

(PG_1283) (Veith et al., 2014) . Moreover DPP4 seems to be is enriched in outer membrane vescicle under heme excess conditions (Veith et al., 2018) . In contrast, DPP3 and DPP6 seem to be located in the cytoplasm, and thus, not involved in the degradation of extracellular proteins in the periplasm. Their roles should be elucidated in future studies.

Taken together, the degradation pathway of extracellular proteins in P. gingivalis, which is initiated by gingipains and finally reach dipeptides mediated by DPPs with the help of AOP and PTP-A, is schematically illustrated in Figure 1 . We currently suppose that oligopeptidyl substrates and dipeptidyl MCA are incorporated across the outer membrane via RagAB.

Genome information for P. gingivalis indicates the existence of a single copy of each of three amino acid/oligopeptide transporters,

i.e, serine/threonine transporter (SstT), proton-dependent oligopeptide transporter (POT), and oligopeptide transporter (OPT) (Naito et al., 2008; Nelson et al., 2003) . Among them, only P. gingivalis SstT, which incorporates amino acids with most efficiently Ser and Thr, has been reported . (ydgH, ydgR, yhiP, yidL) (Ernst et al., 2009; Weitz et al., 2007) , and Helicobacter pylori, limited to utilization of carbohydrates as carbon sources, has five POT (dppA, B, C, D, F) and

four OPT (oppA, B, C, D) paralogs (Salama et al., 2000; Weinberg & Maier, 2007) . The presence of multiple sets of transporter genes suggests that these bacteria readily respond to changes in nutritional conditions, while P. gingivalis resides in the consistently poor nutritional environment of subgingival dental plaque.

Dipeptides incorporated into bacterial cells are cleaved by a dipeptidase to two amino acids, which enter the metabolic pathway of the cell (Figure 1 ). To date, little is known regarding dipeptidases in P.

gingivalis and other periodontopathic bacteria. We recently identified Arg-preferential dipeptidase A [locus tag, PIOMA14_1_1238, MEROPS code, BAU17746] in Pre. intermedia, which belongs to C69.001 (Sarwar et al., 2020) . Pre. intermedia dipeptidase A most preferentially degrades Arg-Leu and Arg-Phe, and does other dipeptides, such as Leu-Leu and Glu-Glu, moderately. It never degrades tripeptides and does not show any amino peptidase ativity (Sarwar et al., 2020) . Dipeptidase A/PepDA (MER0233043) from

Lactobacillus helveticus, the representative enzyme of the C69.001 members, was reported to possess a hydrophobic P1 preference (Dudley et al., 1996; Vesanto et al., 1996) . However, a later reexamination revealed that it also possesses an Arg preference. Pre. intermedia encodes one additional C69-family member (BAU18827), though no hydrolyzing activity has been observed (Sarwar et al., 2020) .

P. gingivalis carries a single C69 family member, PGN_1103, of which the deduced sequence is 13.1% and 47.3% identical to that of BAU17746 and BAU18827 of Pre. intermedia, respectively.

Therefore, PGN_1103 is likely to be an ortholog of BAU18827 and again results of our preliminary search of PGN_1103 revealed no dipeptidase activity present. (Paschoalin et al., 2013) and when present, efficiency for producing dipeptides should be facilitated. P. gingivalis truly possesses two potential Dcp genes, PGN_1776 (MER0034589) and PGN_0788 (MER003588), though they have no signal sequences. Accordingly, these potential Dcps may be cytosolic proteins and are unlikely to be involved in dipeptide production in the periplasm.

Currently, the two-way relationship between periodontal disease and type 2 diabetes mellitus is accounted for by chronic inflammation, in which an elevated level of tumor necrosis factor-α derived from the liver by lipopolysaccharide of periodontopathic Gramnegative bacteria, results in insulin resistance (Soorya et al., 2014; Takano et al., 2010; Teeuw et al., 2010) . We recently proposed that an additional and direct link exists between the two diseases, in which periodontopathic bacterial DPP4 degrades incretins (Ohara- Nemoto et al., 2017) . Since incretins are multifunctional (Seino & Yabe, 2013) and other bioactive peptides including gastrointestinal hormones, neuropeptide, and chemokines could be potential targets, periodontopathic bacterial DPPs may modulate human homeostasis via inactivation of these molecules.

Human DPP4 cleaves the peptide bond between the second Ala and third Glu of GLP-1 and GIP, causing rapid inactivation (Mentlein et al., 1993) . Hence, DPP4 inhibitors are widely used to treat type 2 diabetes mellitus patients. Because of the common enzymatic features, the involvement of periodontopathic bacterial DPP4 in modulation of blood glucose level is reasonably inferred.

Porphyromonas gingivalis cells were found to efficiently de- 

Infection by C. canimorsus caused by a severe dog or cat bite occasionally induces bleeding and coagulation abnormalities. Hack et al. (2017) reported that. C. canimorsus DPP7, which degrades and inactivates coagulation factor X at the N-termini of its light and heavy chains, is responsible for these conditions. Their study strongly suggests that P. gingivalis and Capnocytophaga gingivalis DPP7 may retard the prothrombin time and activated partial thromboplastin time. This capability of DPP7 should be profitable for periodontopathic bacteria, because retardation of coagulation may provide hemoglobin and plasma proteins as nutrition sources for periodontopathic asaccharolytic bacteria. This issue should be further investigated in the future.

Porphyromonas gingivalis, an asaccharolytic Gram-negative rod, utilizes extracellular proteins as its sole carbon and energy sources. 

None to declare.

The peer review history for this article is available at https://publo ns.com/publo n/10.1111/omi.12317.

Takayuki K. Nemoto https://orcid.org/0000-0002-5007-6260

Yuko Ohara Nemoto https://orcid.org/0000-0002-2822-7296

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How to cite this article: Nemoto TK

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