title: Characterization of proteinase-adhesin complexes of Porphyromonas gingivalis.
abstract: Proteinase-adhesin complexes of Porphyromonas gingivalis wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpA(cat), the Lys-specific proteinase Kgp(cat) and adhesin domains RgpA A1, RgpA A2, RgpA A3, Kgp A1 and Kgp A2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpA A4, indicated that this band contained C-terminally truncated Kgp A1 (which has an identical N-terminus to RgpA A4) as well as the HagA A1* adhesin. Analysis of the Triton X-114 extracted complexes from the P. gingivalis isogenic mutants kgp (RgpA complex) and rgpA (Kgp complex) suggested that the Kgp complex consisted of Kgp(cat), Kgp A1 and Kgp A2/HagA A2 and that the RgpA complex consisted of RgpA(cat), RgpA A1, HagA A1*, RgpA A2 and RgpA A3. Each of the complexes was found to have equilibrium binding constants (K(D)) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower K(D) values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia.
authors.forename: Rishi D
authors.initials: RD
authors.lastname: Pathirana
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia.
authors.forename: Neil M
authors.initials: NM
authors.lastname: O'Brien-Simpson
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Veith
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Riley
authors.affiliation: Cooperative Research Centre for Oral Health Science, School of Dental Science, The University of Melbourne, 720 Swanston Street, Victoria, 3010, Australia.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000080867:Gingipain Cysteine Endopeptidases; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols; D016966:Porphyromonas gingivalis / Q000201:enzymology*
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D003546:Cysteine Endopeptidases