title: Propeptide-mediated inhibition of cognate gingipain proteinases.
abstract: Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i) values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Victoria, Australia.
authors.forename: N Laila
authors.initials: NL
authors.lastname: Huq
authors.forename: Christine A
authors.initials: CA
authors.lastname: Seers
authors.forename: Elena C Y
authors.initials: EC
authors.lastname: Toh
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Dashper
authors.forename: Nada
authors.initials: N
authors.lastname: Slakeski
authors.forename: Lianyi
authors.initials: L
authors.lastname: Zhang
authors.forename: Brent R
authors.initials: BR
authors.lastname: Ward
authors.forename: Vincent
authors.initials: V
authors.lastname: Meuric
authors.forename: Dina
authors.initials: D
authors.lastname: Chen
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Cross
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D016966:Porphyromonas gingivalis / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D053148:Caspase 3; D003546:Cysteine Endopeptidases