Comparative properties of envelope-associated arginine-gingipains and lysine-gingipain of Porphyromonas gingivalis

Abstract

Two arginine specific proteinases (Arg-gingipain [RGP-A, RGP-B]) and a lysine specific proteinase (Lys-gingipain [KGP]) were purified from materials of the envelope of Porphyromonas gingivalis solubilized by a detergent by the sequential procedures of ion-exchange chromatography, affinity chromatography, and isoelectric focusing. Each purified enzyme showed a single stained band on SDS-PAGE. The three enzymes were commonly activated by reducing reagents such as mercaptoethanol, dithiothreitol and cysteine. RGP-B was activated markedly by glycyl-glycine and KGP was activated significantly by EDTA. Both RGP-A and RGP-B were inhibited by p-hydroxymercuribenzoate, tosyl-L-lysine chloromethyl ketone (TLCK), N-ethylmaleimide, EDTA, leupeptin, L-trans-epoxy-succinylleucylamido-(4-guanidino)butane and antipain. KGP was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and TLCK. The molecular masses of RGP-A and RGP-B were the same (43 kDa), and that of KGP was 48 kDa. The hydrolytic activities of RGPs and KGP to chromogenic synthetic substrates were limited to the compounds with arginine and lysine in the P-1 positions, respectively. When IgG was treated with the three enzymes separately, it was demonstrated that two new fragments of 34 kDa and 15 kDa were generated in each reaction product. Hemoglobin was almost exhaustively digested by RGP-B, but substantial degradation could not be induced by RGP-A or KGP treatment. z

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

“…3). This discrepancy might be due to the fact that the pH of the paper point buffer (pH 7.5) is more optimal for gingipain activity than the pH we measured in our P. gingivalis cultures, pH 8.5 (data not shown) (12). Another explanation may be that the amount of gingipains in gingival crevicular fluid is higher, possibly due to the presence of host proteins which might trigger the production of these virulence factors.…”

Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin

“…This is the first report documenting this phenomenon. The protease inhibitors TLCK and NEM, which are inhibitory towards Arg-and Lys-gingipains [9], prevented haemoglobin oxidation. It must be concluded that either or both classes of gingipain play a role in accelerating methaemoglobin formation and, hence, µ-oxo bishaem generation.…”

“…Incubations of bacterial cells (75 µg of protein:ml −" ) with oxyhaemoglobin were carried out in NaCl\Tris with 0.1 mM tosyllysylchloromethylketone (TLCK) and 2 mM N-ethylmaleimide (NEM) to inhibit cellular gingipain activity [9]. The changes in both the Soret band intensity and λ max value, and the decrease in A &(' , were monitored with time at 37 mC as described above.…”

Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

“…In this study, we describe a simple method of extraction and purification of the major cell-asscoiated proteinase-adhesin complexes from P. gingivalis using Triton X-114 and a single Arg-affinity chromatography procedure. Although other authors have used detergents such as CHAPS or Triton X-100 in the extraction step for the proteinase-adhesin complexes from P. gingivalis, these detergents were used to solubilize proteins after sonication (Fujimura et al, 1998;Kontani et al, 1996). Takii et al (2005) used sucrosemonolaurate to extract outer-membrane proteins from P. gingivalis, but as with all other reports these authors used multiple chromatography procedures to purify the proteinase-adhesin complexes.…”