A trypsin‐like protease from <i>Bacteroides gingivalis</i>: partial purification and characterization

Sorsa, Timo; Uitto, Veli‐Jukka; Suomalainen, Kimmo; Turto, Heikki; Lindy, Seppo

doi:10.1111/j.1600-0765.1987.tb01602.x

Cited by 98 publications

(83 citation statements)

References 34 publications

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“…This is unique among the known activation mechanisms of different members of the matrix metalloproteinase family. An early report by Sorsa and coworkers that the activation of latent PMNL collagenase was induced without change of the molecular mass of the enzyme could not be confirmed [14]. In our opinion this was probably due to the fact that the authors may have used an N-terminally truncated enzyme form during their activation experiments.…”

Section: Discussioncontrasting

confidence: 40%

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Mercurial activation of human polymorphonuclear leucocyte procollagenase

Bläser

Knäuper

Osthues

et al. 1991

European Journal of Biochemistry

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The mechanism of human polymorphonuclear leucocyte (PMNL) procollagenase activation by HgCl, was investigated by kinetic and sequence analysis of the reaction products. HgClz activated PMNL procollagenase by intramolecular autoproteolytic cleavage of the Asn53 -Val54 peptide bond to generate a collagenase species of M , 65000, which was immediately converted into a second intermediate collagenase form by further autoproteolytic cleavage of the Asp64 -Met65 peptide bond within the propeptide domain. This intermediate form (Met65 N-terminus) reached maximum concentrations after 45 min and displayed only about 40% of the maximum available enzymatic activity. Final activation was obtained after autoproteolytic cleavage of either Phe79 -Met80 or Met80 -Leu81 peptide bonds. Furthermore, activation in the presence of TIMP-1 did not suppress the intramolecular autoproteolytic cleavage of the Asn53 -Val54 peptide bond. Complete inhibition of further autoproteolytic decay of the enzyme or generated peptides was observed, which was obviously due to complex formation between the intermediate collagenase form (Val54 N-terminus) and inhibitor, which was visualized using the Western blot technique. Thus PMNL procollagenase activation by HgCl, followed a three-step activation mechanism which is entirely different from the known activation mechanisms of the fibroblast matrix metalloproteinases.

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Section: Discussioncontrasting

confidence: 40%

“…mass of the enzyme; there is evidence for autoproteolytic cleavage, in contrast to a recent report [14]. The reaction products have been further investigated by N-terminal sequence determination, revealing a three-step activation mechanism.…”

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confidence: 69%

Mercurial activation of human polymorphonuclear leucocyte procollagenase

Bläser

Knäuper

Osthues

et al. 1991

European Journal of Biochemistry

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“…Freeze-dried whole cells (200 mg) were suspended in 60 ml 0.14 M-NaC1 containing 10 mM-EDTA, pH 7.3, and incubated with stirring at 37 "C for 30 min (Smalley & Birss, 1987). This serves to dissociate the OM and to inhibit any possible cell-associated protease activity (Sorsa et al, 1987 ;Tsutsui et ul., 1987;Yoshimura et al, 1984) during preparation. After being passed twice through a 25 gauge needle, the cells were pelleted by centrifugation (20000 g for 30 min, 4 "C) leaving the supernatant which contained the crude OM preparation.…”

Section: Methodsmentioning

confidence: 99%

Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation

Smalley¹,

Birss²,

McKee³

et al. 1993

Journal of General Microbiology

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Porphyrornunns gingivalis W50 was grown in a chemostat at pH 7.3 under haemin-limitation and haemin-excess at a constant mean doubling time of 6-9 h. Outer membranes (OM) were extracted from whole cells using EDTA and compared by SDS-PAGE. Haemin-limited cells expressed novel outer membrane proteins (OMPs) of mol. mass 115, 113 and 19 kDa when samples were solubilized at 100 "C. A 46 kDa OMP was observed in haeminexcess cells but not in those from haemin-limited conditions. Tetramethylbenzidine (TMBZ) staining of gels, after OM solubilization at 20 "C, was used to detect haemin-binding proteins (HBPs). HBPs were observed only in OM from haemin-limited cells, The major HBP (mol, mass 32.4 kDa) corresponded to a similar sized Kenacid-bluestained protein which was not observed in haemin-excess-derived OM. Haemin-limited cells and OM displayed a ladder-like series of Kenacid-blue-stained proteins. Lighter TMBZ-stained proteins of mol. mass 51, 53, 56 and 60 kDa, with mobilities corresponding to those of silver-stained LPS components, were observed in haemin-limited OM. No soluble HBPs were detected extracellularly. The greater number of HBPs expressed by cells grown under haemin-limitation may reflect an additional cell surface receptor system for haemin acquisition under low environmental levels of this essential cofactor.

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“…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%

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

1997

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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.

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A trypsin‐like protease from Bacteroides gingivalis: partial purification and characterization

Cited by 98 publications

References 34 publications

Mercurial activation of human polymorphonuclear leucocyte procollagenase

Mercurial activation of human polymorphonuclear leucocyte procollagenase

Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation

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

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