Purification and partial characterization of a lysine-specific protease of<i>Porphyromonas gingivalis</i>

Fujimura, Setsuo; Shibata, Yukinaga; Nakamura, Takeshi

doi:10.1111/j.1574-6968.1993.tb06503.x

Cited by 19 publications

(18 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It would appear therefore that the 300 kDa cell-associated complex characterized in this present study has characteristics similar to those of the culture fluid enzyme(s) identified by Bedi (1995) and Fujimura & Nakamura (1987).…”

Section: Discussionmentioning

confidence: 50%

“…In particular, proteases have received a great deal of attention for their ability to degrade a broad range of host proteins including structural proteins and others involved in defence. The proteins that have been shown to be substrates for P. gingivalis proteolytic activity include collagen types I and IV, fibronectin, fibrinogen, laminin, complement and plasma clotting cascade proteins, a,-antitrypsin, a,-macroglobulin, antichymotrypsin, antithrombin 111, antiplasmin, cystatin C, IgG and IgA, (Grenier, 1996;Pike et al, 1996;Carlsson et al, 1984;Fujimura et al, 1993;Grenier et al, 1989;Lantz et al, 1991;Mayrand & Holt, 1988;Smalley et al, 1989a;Sorsa et a/., 1987;Sundqvist et al, 1985). The major proteolytic activities associated with this organism have been defined by substrate specificity and are ' trypsin-like ', that is cleavage on the carboxyl side of arginyl and lysyl residues (Yoshimura et al, 1984) and collagenolytic (Toda et al, 1984) although other minor activities have been reported (Grenier & Mayrand, 1993).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

1997

View full text Add to dashboard Cite

Porphyromonas gingiwalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cellassociated trypsin-like proteolytic activity of P. gingiwalis W50 using mild sonication. Anion-exchange and gel-f iltration FPLC of the sonicate revealed that Arg-and Lys-specific proteinase activity was associated with a 300 kDa complex which could be dissociated into seven bands (48,45,44,39,27,17 and 15 kDa) by SDS-PAGE with the 4 4 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on ArgSepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys-and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octylpD-glucopyranoside and was shown to be an Arg-specif ic, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39,27,17 and 15 kDa proteins of the complex exhibit amino acid sequence homology and are proposed to be haemagglutinindadhesins. The 45 kDa Arg-specif ic proteinase and one of the 44 kDa adhesins as well as the 15,17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtR, with all proteins preceded by an Arg or Lys residue within the polyprotein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex are encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39,15 and 44 kDa adhesins of PrtK all exhibit high homology with the 44,15,17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRl5, PrtRl7, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingiwalis On: Sat, 12 May 2018 21:46:32 P. S. BHOGAL, N. SLAKESKI a n d E. C. REYNOLDS involved in the evasion of host defence and in the assimilation of haem and peptides.

show abstract

Section: Discussionmentioning

confidence: 50%

Section: Introductionmentioning

confidence: 99%

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

1997

View full text Add to dashboard Cite

show abstract

“…The other chelating reagent, EGTA, exerted no inhibitory e¡ect on the RGPs and activated KGP. Activation by these chelators was reported in an extracellular KGP of P. gingivalis [3] and trypsin-like enzyme (Pase-C) of P. gingivalis [4].…”

Section: Discussionmentioning

confidence: 99%

“…RGP and KGP were assayed routinely using benzoyl-L-arginyl p-nitroanilide (Bz-Arg-pNA, Sigma, St. Louis, MO, USA) [14] and toluenesulfonyl-glycyl-L-prolyl-L-lysine p-nitroanilide (tosyl-Gly-Pro-LyspNA, Sigma) [3] as substrates, respectively. The activity against other p-nitroanilide derivatives of amino acids or peptides was examined by the same methods as described above.…”

Section: Assay Of Proteinase Activitiesmentioning

confidence: 99%

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

Fujimura¹

1998

FEMS Microbiology Letters

View full text Add to dashboard Cite

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

show abstract

“…Lysine-specific protease and trypsin-like enzyme were purified from culture supematants by the methods described earlier [19,20].…”

Section: Purification Of Proteases Of Porphyromonas Gingivalis Atcc 3mentioning

confidence: 99%

Some binding properties of the envelope of Porphyromonas gingivalis to hemoglobin

Fujimura

1995

FEMS Immunology and Medical Microbiology

View full text Add to dashboard Cite

Abstmct: Porphyromonas gingiualis was found to bind to hemoproteins (hemoglobin, myoglobin, catalase, cytochrome c) and the binding properties of the envelope of P. gingiualti to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58 pg. No significant binding was observed at 4°C and the binding was inhibited strongly by tosyl-L-lysine chloromethyl ketone, Leupeptin, EDTA and partially by meta-periodate. Heating of the envelope at 70°C for 15 min resulted in complete loss of the binding activity. The binding activity of the envelope was not influenced by the treatment with the endogenous proteases. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for these hemoproteins are common.

show abstract

Purification and partial characterization of a lysine-specific protease ofPorphyromonas gingivalis

Cited by 19 publications

References 22 publications

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

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

Some binding properties of the envelope of Porphyromonas gingivalis to hemoglobin

Contact Info

Product

Resources

About