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Volume 152, Issue 8

Characterization of proteinase–adhesin complexes of Free

Abstract

Proteinase–adhesin complexes of 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, the Lys-specific proteinase Kgp and adhesin domains RgpA, RgpA, RgpA, Kgp and Kgp. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpA, indicated that this band contained C-terminally truncated Kgp (which has an identical N-terminus to RgpA) as well as the HagA* adhesin. Analysis of the Triton X-114 extracted complexes from the isogenic mutants (RgpA complex) and (Kgp complex) suggested that the Kgp complex consisted of Kgp, Kgp and Kgp/HagA and that the RgpA complex consisted of RgpA, RgpA, HagA*, RgpA and RgpA. Each of the complexes was found to have equilibrium binding constants ( ) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower 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.

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Keyword(s): ABM, adhesin-binding motif , Bz-l-Arg-pNA, benzoyl-l-Arg-p-nitroanilide , CBB, Coomassie brilliant blue , IFA, incomplete Freund's adjuvant , KD, dissociation equilibrium constant , PMF, peptide mass fingerprinting , TFA, trifluoroacetic acid , TLCK, Nα-p-tosyl-lysine chloromethlyketone , wt, wild-type and z-l-Lys-pNA, benzyloxycarbonyl-l-Lys-p-nitroanilide
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2006-08-01
2024-04-19
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Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis
Microbiology 152, 2381 (2006); https://doi.org/10.1099/mic.0.28787-0
/content/journal/micro/10.1099/mic.0.28787-0
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