Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.
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.
Veillard, F; Sztukowska, M; Mizgalska, D; Ksiazek, M; Houston, J; Potempa, B; Enghild, JJ; Thogersen, IB; Gomis-Rüth, FX; Nguyen, K-A; Potempa, J
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