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Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

ABSTRACT: Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome. C. histolyticum colG is more similar to C. perfringens colA than to colH in terms of domain structure. Both colG and colA have a homologous gene, mscL, at their 3' ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C. histolyticum and C. perfringens and that further divergence of the parent gene produced colG and colA.

SUBMITTER: Matsushita O

PROVIDER: S-EPMC93460 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

Matsushita O O Jung C M CM Katayama S S Minami J J Takahashi Y Y Okabe A A

Journal of bacteriology 19990201 3

Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments ...[more]

PMID: 9922257

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Project description:Clostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4?Å resolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9?Å resolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8?Å resolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4?Å resolution, R = 16.9%, Rfree = 21.2%; 1.6?Å resolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved ?-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent ?-strand. This ?-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

Project description:To determine the safety and efficacy of collagenase clostridium histolyticum (CCH) injection for the treatment of palmar Dupuytren disease nodules.In this 8-week, double-blind trial, palpable palmar nodules on one hand of adults with Dupuytren disease were selected for treatment. Patients were randomly assigned using an interactive web response system to receive a dose of 0.25 mg, 0.40 mg, or 0.60 mg (1:1:1 ratio) and then allocated to active treatment (CCH) or placebo (4:1 ratio). All patients and investigators were blinded to treatment. One injection was made in the selected nodule on Day 1. Caliper measurements of nodule length and width were performed at screening and at Weeks 4 and 8. Investigator-reported nodular consistency and hardness were evaluated at baseline and Weeks 1, 4, and 8. Investigator-rated patient improvement (1 [very much improved] to 7 [very much worse]) and patient satisfaction were assessed at study end.In the efficacy population (n = 74), percentage changes in area were significantly greater with CCH 0.40 mg (-80.1%, P = 0.0002) and CCH 0.60 mg (-78.2%, P = 0.0003), but not CCH 0.25 mg (-58.3%, P = 0.079), versus placebo (-42.2%) at post-treatment Week 8. Mean change in nodular consistency and hardness were significantly improved with CCH versus placebo at Weeks 4 and 8 (P ? 0.0139 for all). At Week 8, investigator global assessment of improvement was significantly greater with CCH 0.40 mg and 0.60 mg (P ? 0.0014) but not statistically significant with CCH 0.25 mg versus placebo (P = 0.13). Most patients were "very satisfied" or "quite satisfied" with CCH 0.40 mg and 0.60 mg. Contusion/bruising (50.0% to 59.1%) was the most common adverse event with CCH treatment.In patients with Dupuytren disease, a single CCH injection significantly improved palmar nodule size and hardness. The safety of CCH was similar to that observed previously in patients with Dupuytren contracture.ClinicalTrials.gov identifier: NCT02193828 . Date of trial registration: July 2, 2014 to December 5, 2014.

Dataset Information

Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

Publications

Gene duplication and multiplicity of collagenases in Clostridium histolyticum.

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