Current Protein and Peptide Science, Vol. 4, No. 6, 2003
Contents
Gingipains
Guest Editor: Kenji Yamamoto
Molecular Genetics of Porphyromonas
gingivalis: Gingipains and other Virulence Factors Pp.389-395
K. Nakayama
Gingipains,
the Major Cysteine Proteinases and Virulence Factors of Porphyromonas
gingivalis: Structure, Function and Assembly of Multidomain Protein Complexes Pp.397-407
Jan Potempa, Aneta
Sroka, Takahisa Imamura and James Travis
Porphyromonas
gingivalis Gingipains: The Molecular Teeth of a Microbial Vampire Pp.409-426
N.M.
O’Brien-Simpson, P.D. Veith, S.G. Dashper and E.C. Reynolds
Glycosylation
of the Arg-gingipains of Porphyromonas gingivalis and Comparison with
Glycoconjugate Structure and Synthesis in other
Alexandra Gallagher,
Joseph Aduse-Opoku, Minnie Rangarajan,
Jennifer M. Slaney and Michael A. Curtis
The
Biphasic Virulence Activities of Gingipains: Activation and Inactivation of
Host Proteins Pp.443-450
Takahisa Imamura,
James Travis and Jan Potempa
Suppression
of Virulence of Porphyromonas gingivalis by Potent Inhibitors Specific for
Gingipains Pp.451-458
Tomoko Kadowaki and
Kenji Yamamoto
Abstracts
Back to top] Molecular Genetics of Porphyromonas gingivalis: Gingipains and
other Virulence Factors
K. Nakayama
Porphyromonas gingivalis is a black-pigmented anaerobic gram-negative bacterium that is a major pathogen of chronic adult periodontitis, an inflammatory disease of tooth-supporting tissues. P. gingivalis possesses a number of potential virulence factors. Among them, cell-surface-associated and secreted proteinases such as Arg-gingipain and Lys-gingipain have received much attention because they can degrade various host proteins and cause inflammation. Molecular genetic analysis is extremely powerful to evaluate the significance of each virulence factor in a pathogenic microorganism. This review will describe the introduction of molecular genetics to analysis of pathogenesis of P. gingivalis and the findings that have been obtained using knockout mutants of various potential virulence factors, especially proteinases.
Back to top] Gingipains, the
Major Cysteine Proteinases and Virulence Factors of Porphyromonas gingivalis:
Structure, Function and Assembly of Multidomain Protein Complexes
Jan Potempa, Aneta
Sroka, Takahisa Imamura and James Travis
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.
Back to top]
Porphyromonas
gingivalis Gingipains: The Molecular Teeth of a Microbial Vampire
N.M.
O’Brien-Simpson, P.D. Veith, S.G. Dashper and E.C. Reynolds
The gingipains are cell surface Arg- and Lys-specific proteinases of the bacterium Porphyromons gingivalis, which has been associated with periodontitis, a disease that results in the destruction of the teeth’s supporting tissues. The proteinases are encoded by three genes designated rgpA, rgpB and kgp. Arg-specific proteolytic activity is encoded by rgpA/B and the Lys-specific activity by kgp. RgpA and Kgp are polyproteins comprising proteinases with C-terminal adhesin domains that are proteolytically processed. After processing, the domains remain non-covalently associated as complexes on the cell surface. RgpB is also a cell surface proteinase but does not associate with adhesin domains. Using gene knockout P. gingivalis mutants, the proteolytic processing of the gingipain domains has been shown to involve the gingipains themselves as well as C-terminal processing by a carboxypeptidase. A motif in the C-terminal domain of each protein/polyprotein has been identified that is suggested to be involved in attachment to LPS on the cell surface. RgpB lacks a C-terminal adhesin binding motif found in the catalytic domains of RgpA and Kgp. This adhesin binding motif is proposed to be responsible for the non-covalent association of the RgpA and Kgp catalytic domains into the cell surface complexes with the processed adhesin domains. The RgpA-Kgp proteinase-adhesin complexes, through the adhesin domains A1 and A3, have been implicated in colonization of P. gingivalis by binding to other bacteria in subgingival plaque and also binding to crevicular epithelial cells. The RgpA-Kgp complexes also bind to fibrinogen, laminin, collagen type V, fibronectin and hemoglobin. Amino acid sequences likely to be involved in binding to these host proteins have been identified in adhesin domains A1 and A3. It is proposed that these adhesins target the proteolytic activity to host cell surface matrix proteins and receptors. The continual cycle of binding and degradation of the surface proteins/receptors on epithelial, fibroblast and endothelial cells by the RgpA-Kgp complexes in the gingival tissue leading to cell death would contribute to inflammation, tissue destruction and vascular disruption (bleeding). P. gingivalis has an obligate growth requirement for iron and protoporphyrin IX, which it preferentially utilizes in the form of hemoglobin. Kgp proteolytic activity is essential for rapid hydrolysis of hemoglobin and it is suggested therefore that a major role of the RgpA-Kgp complexes is in vascular disruption and the binding and rapid degradation of hemoglobin for heme assimilation by P. gingivalis. The RgpA-Kgp complexes also have a major role in the evasion and dysregulation of the host’s immune response. It is proposed that host pro-inflammatory cytokines and cellular receptors close to the infection site may be rapidly and efficiently degraded by the gingipains while the proteinases at lower concentrations distally could result in the promotion of an inflammatory response through activation of proteinase-activated receptors and cytokine release. The culmination of this dysregulation would be tissue destruction and bone resorption. In animal models of disease the RgpA-Kgp complex when used as a vaccine to produce a high titre antibody response protects against challenge with P. gingivalis. Using recombinant domains of RgpA and Kgp as vaccines, it has been demonstrated that the A1 and A3 domains confer protection.
Back to top] Glycosylation of
the Arg-gingipains of Porphyromonas gingivalis and Comparison with
Glycoconjugate Structure and Synthesis in other Bacteria
Alexandra Gallagher,
Joseph Aduse-Opoku, Minnie Rangarajan,
Jennifer M. Slaney and Michael A. Curtis
Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.
Back to top]
The Biphasic
Virulence Activities of Gingipains: Activation and Inactivation of Host
Proteins
Takahisa Imamura,
James Travis and Jan Potempa
Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. Rgps (HRgpA and RgpB) and Kgp are specific for -Arg-Xaa- and -Lys-Xaa- peptide bonds, respectively. 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 cata- lytic subunit of HRgpA. The multiple virulence activities of gingipains are reviewed in view of the biphasic mechanisms: activation and inactivation of host proteins. Rgps enhanced vascular permeability through prekallikrein activation or direct bradykinin release in combination with Kgp. This Rgp action is potentially associated with gingival edema and crevicular fluid production. Rgps activate the blood coagulation system, leading to progression of inflammation and consequent alveolar bone loss in the periodontitis site. Rgps also activate protease-activated receptors and induce platelet aggregation, which, together with the coagulation-inducing activity, may explain an emerging link between periodontitis and cardiovascular disease. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains in human plasma, being involved in the bleeding tendency at the diseased gingiva. Gingipains stimulate expression of matrix metalloproteinases (MMPs) in fibroblasts and activate secreted latent MMPs that can destroy periodontal tissues. Gingipains degrade cytokines, components of the complement system and several receptors, including macrophage CD14, T cell CD4 and CD8, thus perturbing the host-defense systems and thereby facilitating sustained colonization of P. gingivalis. Gingipains are potent virulence factors of P. gingivalis, and in many regards their pathogenic activities constitute new mechanisms of bacterial virulence.
Back
to top] Suppression of Virulence of Porphyromonas
gingivalis by Potent Inhibitors Specific for Gingipains
Tomoko Kadowaki and
Kenji Yamamoto
Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is implicated as a major etiologic agent of adult periodontal disease. This bacterium is asaccharolytic and possesses strong potency for proteolysis. It produces a novel class of cysteine proteinases, termed gingipains, in the cell-associated and secretory forms. Gingipains consist of arginine-X-specific cysteine proteinases (Arg-gingipains, Rgps) and lysine-X-specific cysteine proteinase (Lys-gingipain, Kgp). Previous studies using various P. gingivalis mutants deficient in Rgp- and/or Kgp-encoding genes have revealed that both enzymes are important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. Mammalian internal proteinase inhibitors such as cystatins, a1-antichymotrypsin, and tissue inhibitor of metalloproteinases (TIMPs) have little or no effects on the proteolytic activities of these enzymes, suggesting the evasion of the bacterium from host defense mechanisms. Recent epidemiological reports have shown a significant relation between periodontal diseases and systemic diseases such as cardiovascular diseases and diabetes. Thus, the development of potent inhibitors specific for gingipains provides new therapeutic approaches to treat periodontal diseases and the related systemic diseases. More recently, we have developed novel synthetic inhibitors specific for Rgp and Kgp, based on the specificity and efficacy of cleavage of histatins by each enzyme. We have also isolated a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. Here we summarized the usefulness of these new inhibitors in providing a broader application in studies of this important class of enzymes.