Molecular Cloning and Partial DNA Sequencing of the Collagenase Gene of <i>Vibrio alginolyticus</i>

Fukushima, Jun; Takeuchi, Hiroaki; Tanaka, Eiichi; Hamajima, Kenji; Sato, Yoshiaki; Kawamoto, Susumu; Morihara, Kazuyuki; Keil, B.; Okuda, Kenji

doi:10.1111/j.1348-0421.1990.tb01519.x

Cited by 11 publications

(6 citation statements)

References 17 publications

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“…We and another group have independently cloned (32,41) and analyzed (2,10,23) the elastase structural gene (lasB) from the elastase-producing strains P. aeruginosa IFO 3455 and PAO1, respectively. We also cloned and analyzed the elastase gene from several non-elastase-producing Pseudomonas strains (36) and other metalloprotease genes (Pseudomonas alkaline proteinase [1,28] and Vibrio collagenase [9,35]). Recently, the elastase-related lasA gene (30,33,40), the existence of preproelastase and proelastase (2,10,18), a transcriptional activator (lasR gene) (12), and translational control (4) of elastase expression have been described (see reference 11 for a review).…”

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confidence: 99%

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection

et al. 1993

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Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the BaciUlus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.Pseudomonas aeruginosa is an opportunistic pathogen which can cause fatal infection in vulnerable hosts. Several components related to its virulence have been identified and characterized (29). Elastase acts to inactivate a variety of biologically important proteins and processes (24). In addition, there is strong evidence that elastase plays an important role in the pathogenesis of P. aeruginosa infections (14,27). We and another group have independently cloned (32, 41) and analyzed (2, 10, 23) the elastase structural gene (lasB) from the elastase-producing strains P. aeruginosa IFO 3455 and PAO1, respectively. We also cloned and analyzed the elastase gene from several non-elastase-producing Pseudomonas strains (36) and other metalloprotease genes (Pseudomonas alkaline proteinase [1,28] and Vibrio collagenase [9,35]). Recently, the elastase-related lasA gene (30,33,40), the existence of preproelastase and proelastase (2, 10, 18), a transcriptional activator (lasR gene) (12), and translational control (4) of elastase expression have been described (see reference 11 for a review). Moreover, P. aeruginosa elastase has been crystallized and the three-

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confidence: 99%

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection

et al. 1993

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“…In contrast, both the isolation and sequence characterization of a number of different eucaryotic collagenase genes has been described (11). In addition, except for some partial sequence data for a collagenase isolated from Vibrio alginolyticus (9), no information is presently available regarding the nucleotide sequences of the bacterial enzymes.…”

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Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity

1992

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In order to examine the potential role of bacterial collagenases in periodontal tissue destruction, we recently isolated a gene, prtC, from Porphyromonas gingivalis ATCC 53977, which expressed collagenase activity (N. Takahashi, T. Kato, and H. K. Kuramitsu, FEMS Microbiol. Lett. 84:135-138, 1991). The nucleotide sequence of the gene has been determined, and the deduced amino acid sequence corresponds to a basic protein of 37.8 kDa. In addition, Southern blot analysis indicated that the prtC gene is conserved among the three major serotypes of P. gingivalis. The enzyme has been purified to near homogeneity from Escherichia coli clone NTS1following Mono Q anion exchange and sequential gel filtration chromatography. The molecular mass of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ca. 35 kDa, and the active enzyme behaved as a dimer following gel filtration chromatography. The collagenase degraded soluble and reconstituted fibrillar type I collagen, heat-denatured type I collagen, and azocoll but not gelatin or the synthetic collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg. Enzyme activity was enhanced by Ca2' and inhibited by EDTA, sulfhydryl-blocking agents, and the salivary peptide histatin.Preliminary evidence for the existence of a second collagenase expressed by strain 53977 was also obtained.Porphyromonas gingivalis, a gram-negative anaerobic rod-shaped bacterium, has been isolated from the lesions of advanced adult periodontitis (35, 45) and has been implicated as a periodontal pathogen (20,38,47). These organisms exhibit a number of potential virulence traits, including high proteolytic activity (15). The proteases from P. gingivalis may degrade periodontal tissues (36,40,46) as well as inactivate host defense mechanisms (6,31,43). Since type I collagen serves as a major supporting structure for the teeth, it may be particularly relevant that these organisms exhibit collagenase activity (10,46). This activity appears to be localized primarily in the membrane fraction of these organisms (28), but the enzymes have not yet been purified to homogeneity and extensively characterized. However, since the collagenase activity detected in the gingival fluid from diseased sites appears to be primarily of human origin (12,15), there is no direct evidence that bacterial collagenases play a role in periodontal diseases.In order to investigate the potential role of P. gingivalis collagenases in periodontitis, we have recently isolated the prtC gene which expressed collagenase activity from P. gingivalis ATCC 53977 (44). Although extensive biochemical characterization of the collagenase activity from Clostridium histolyticum has been carried out (32), little molecular genetic characterization of the bacterial enzymes has been reported. In contrast, both the isolation and sequence characterization of a number of different eucaryotic collagenase genes has been described (11). In addition, except for some partial sequence data for a collage...

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“…To clarify its molecular size and enzymic characteristics, the primary structure of the collagenase needs to be clarified. Recently we cloned the collagenase gene from Vibrio alginolyticus [16]. In the present study we completely sequenced the collagenase gene and clarified these major issues.…”

Section: Introductionmentioning

confidence: 95%

“…As a first step, we constructed Sau3AI-digested genomic DNA libraries from V. alginolyticus, using pUC18 as a cloning vector [16,19] < 100 < 100 < 100 < 100 25600 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 12800 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 12800 < 100 pLCO-3, reacted repeatedly. These pLCO-1, pLCO-2 and pLCO-3 plasmids contained fragments of V. alginolyticus of approx.…”

Section: Cloning the Collagenase Genementioning

confidence: 99%

Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase

Takeuchi

Shibano

Morihara

et al. 1992

Biochemical Journal

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The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC 18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81 875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases.

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Molecular Cloning and Partial DNA Sequencing of the Collagenase Gene of Vibrio alginolyticus

Abstract: DNA fragments Vibrio alginolyticus chemovar iophagus, at least 7 kb in length, were ligated to Escherichia coli expression vectors.

Cited by 11 publications

References 17 publications

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection

Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity

Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase

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