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Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.

ABSTRACT: BACKGROUND:Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments. METHODS:Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis. RESULTS:PDRgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2nM to 0.85nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PDRgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex. CONCLUSION:Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains. GENERAL SIGNIFICANCE:Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity.

SUBMITTER: Veillard F

PROVIDER: S-EPMC3773880 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.

Veillard Florian F Sztukowska Maryta M Mizgalska Danuta D Ksiazek Mirosław M Houston John J Potempa Barbara B Enghild Jan J JJ Thogersen Ida B IB Gomis-Rüth F Xavier FX Nguyen Ky-Anh KA Potempa Jan J

Biochimica et biophysica acta 20130410 8

<h4>Background</h4>Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular ...[more]

PMID: 23583629

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Project description:Background and objectiveSeveral studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low-density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.Material and methodsPlasma lipoproteins were isolated from whole blood treated with wild-type and gingipain-mutant (lacking either the Rgp- or Kgp gingipains) P. gingivalis by density/gradient-ultracentrifugation and were studied using 2-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis-induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid-reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.ResultsPorphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB-100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high-density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.ConclusionPorphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.

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Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.

Publications

Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.

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