Biochemical characterization of the catalytic domains of three different clostridial collagenases

Eckhard, Ulrich; Schönauer, Esther; Ducka, Paulina; Briza, Peter; Nüss, Dorota; Brandstetter, Hans

doi:10.1515/bc.2009.004

Cited by 37 publications

(37 citation statements)

References 33 publications

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“…cereus ATCC 14579 ColA ΔPP wt with the recombinant protease domain of ColT from C . tetani (ColT PD ) [12]. Proteolytic inactive ColA ΔPP E501A and ColG from C .…”

Section: Resultsmentioning

confidence: 99%

“…Proteolytic inactive ColA ΔPP E501A and ColG from C . histolyticum exhibiting low peptidolytic activity [12] were included as negative controls. Incubation of FALGPA with ColA ΔPP wt led to a rapid substrate turnover of ~85% after 12 min, which was comparable with the activity of ColT PD (Fig 3A).…”

Section: Resultsmentioning

confidence: 99%

“…histolyticum and ColT from C . tetani revealing a double glycine motif upstream of the HEXXH motif, which is also necessary for the collagenase activity [11, 12]. Upon calcium binding the PKD-like domain of ColG undergoes a conformational domain rearrangement [9, 13].…”

Section: Introductionmentioning

confidence: 99%

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Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

et al. 2016

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Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.

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“…cereus ATCC 14579 ColA ΔPP wt with the recombinant protease domain of ColT from C . tetani (ColT PD ) [12]. Proteolytic inactive ColA ΔPP E501A and ColG from C .…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

et al. 2016

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“…5C). The two forms of VchC contain the collagenase unit:activator (M9N) and peptidase M9 domains, which have been shown in collagenase G from C. histolyticum as the only modules indispensable for collagen degradation (23,64,65). Very limited information is available regarding the tertiary structure of the Vibrio collagenases belonging to the M9A subfamily, and the function and biochemical properties of their domains, as well as the physiological importance of the C-terminal processing, remain to be addressed.…”

Section: Discussionmentioning

confidence: 99%

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

et al. 2015

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bVibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program. Members of the genus Vibrio are important inhabitants of the marine coastal waters, estuaries, and ocean sediments, with some pathogenic species also causing wound infections, primary septicemia, gastroenteritis, and diarrhea in humans (1, 2). Among the more than 200 serogroups of V. cholerae, only O1 and O139 are recognized as agents of cholera, a potentially life-threatening diarrheal disease. Cholera remains a significant public health problem by affecting millions of people annually, predominantly in developing countries that have limited clean water supplies and poor sanitation (3). Infection of the human host occurs upon ingestion of water or food contaminated with the bacterium. Following colonization of the small intestine, V. cholerae utilizes a multicomponent secretory pathway, the type II secretion system (T2SS), to release cholera toxin. Cholera toxin induces acute diarrhea in people infected with cholera, enabling the bacteria to escape from the host into the aquatic reservoir (4). During interepidemic periods, V. cholerae persists freely or in association with various aquatic organisms, including copepods, insect egg masses, shellfish, and vertebrate fish (5-7). Extracellular proteins, including those secreted by the T2SS, have been implicated in facilitating the fitness of V. cholerae in both the human host and aquatic niches (4,6,8). The array of extrace...

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“…The glycine residues are single-bonded, serving as hinge residues and having unique conformational degrees of freedom. Consequently, glycine is important for structural or protease dynamics that may be necessary for catalysis (Eckhard et al 2009 (Imperiali & O'Connor 1999). The HdMP cDNA sequence was submitted to the NCBI GenBank under accession number KJ599465.…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization and gene expression analysis of a metalloprotease from Pacific abaloneHaliotis discus hannai

Kim

Han²,

Moon

et al. 2014

Animal Cells and Systems

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We isolated a metalloprotease (MP) homologue from an abalone muscle cDNA library. A 3284-kb full-length cDNA encoding a predicted polypeptide of 667 amino acids was sequenced. The abalone MP Haliotis discus hannai (HdMP) exhibited a domain structure typical of the peptidase M4 family, a 21-amino acid N-terminal hydrophobic signal sequence followed by a long propeptide sequence of 347 amino acids and the mature protease domain comprising 299 amino acids. The mature region contains features characteristic of a zinc protease, including a zinc-binding motif (HEXXH) and an active site. The protein showed 32-38% amino acid sequence identity with other known MP sequences and with a hypothetical protein from owl limpet. The mRNA transcript is expressed in almost all tissues, with high expression in the mantle and adductor muscle of healthy abalones, and is expressed constitutively during the early developmental stages after fertilization. Lipopolysaccharide or poly I:C stimulation induced the expression of the HdMP transcript in the digestive track and gills of abalones. Collectively, these results suggest that HdMP could play multiple roles in defense, the immune response, growth, and regulation of reproduction.

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Biochemical characterization of the catalytic domains of three different clostridial collagenases

Cited by 37 publications

References 33 publications

Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

Molecular characterization and gene expression analysis of a metalloprotease from Pacific abaloneHaliotis discus hannai

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