Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds. Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva. HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen. Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P. gingivalis. Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation. Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy. Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P. gingivalis induced disorders in mice. In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically. Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection.
Gingipains, extracellular cysteine proteinases of Porphyromonas gingivalis, constitute the major virulence factor of this periodontopathogenic bacterium. They are the product of three genes, two coding for an Arg-specific (RgpA and RgpB) and one for a Lys-specific proteinase (Kgp). Proteinase domains of RgpA and RgpB are virtually identical; however, the gene encoding the former enzyme is missing a large segment coding for hemaglutinin / adhesin (HA) domains. The latter domains are present also in Kgp. The tertiary structure of RgpB revealed that the proteinase domain of gingipains has a protein fold referred to as the caspase-hemoglobinase fold. On this basis, they are also evolutionary related to other highly specific proteinases including clostripain, caspases, legumains and separase (clan CD of cysteine peptidases). Gingipains are produced as large preproproteins and are subject to elaborate, not yet fully understood, secretion, glycosylation, activation, and maturation processes. How they traverse the outer membrane is unknown, although it can be hypothesized that they use an autotransporter pathway. Apparently during transport through the periplasm the LPS-like glycan moiety is added at the conserved C-terminal portion of progingipains. At the cell surface pro-gingipains fold into partially active, single-chain zymogens and undergo autocatalytic, intermolecular processing. Two sequential cleavages within the profragment domain enhance zymogen activity and in the case of RgpA and Kgp are followed by excision of the individual HA domains. These domains are further truncated at the C-terminus by concerted action of Kgp and carboxypeptidase and form a non-covalent multidomain, multifunctional complex anchored into the outer membrane by the glycated, C-terminal HA domain. This hypothetical scenario is a reasonable explanation for the occurrence of many forms of gingipains.
Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin-like activity which have been implicated as virulence factors in adult-onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis-derived proteinases are important virulence factors in the development of periodontal disease.
To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Ar-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10' M), or human plasma incubated with RGP-1 (> 10-9 M), induced VPE in a dose-and activitydependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug.The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK).RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMVVK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism. (J. Clin. Invest 1994. 94:361-367.)
Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 M HRGP and KGP treatments for 15 min, and 1 M RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0.3 M HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-␣ production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 g/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation. The Journal of Immunology, 2000, 165: 411-418.A n oral chronic inflammation, i.e., periodontal disease, is one of the major diseases afflicting mankind and caused by a bacterial infection leading to gingival inflammation, destruction of periodontal tissues, loss of alveolar bone, and culminating in tooth loss (1, 2). Porphyromonas gingivalis has been implicated as a principal bacterium not only in adult periodontitis, but also in rapidly progressive periodontitis (1, 2). P. gingivalis possesses a number of putative virulence factors such as LPS, fimbriae, toxic products of metabolism, and proteinases, all of which enable this anaerobe to cause the disease either directly or indirectly by activation of host cells to release inflammatory mediators (3).It is now clear that all of the trypsin-like proteinase activity of P. gingivalis is due to two cysteine proteinases (4). Two types of cysteine proteinases specific for Arg-X (50 and 95 kDa) (5) and Lys-X (105 kDa) bonds have been purified and characterized and are referred to as arginine-specific gingipain (RGP) 3 (3) and lysine-specific gingipain (KGP), respectively (6). The 95-kDa high molecular mass RGP (HRGP or HRgpA) differs from the 50-kDa RGP (RGP2 or RgpB) in that the protein noncovalently complexes with the hemagglutinin/adhesin domain in the same manner as KGP. It has been shown that gingipains play a critical role in the onset of inflammation through enhancement of vascular permeability by activation of the kallikr...
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