The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene

Dargatz, Harald; Diefenthal, Thomas; Witte, Volker; Reipen, Gernot; Wettstein, Diter von

doi:10.1007/bf00276893

Cited by 30 publications

(27 citation statements)

References 29 publications

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“…1). The deduced amino acid sequence of the putative protein encoded by ccp was highly similar to that of alphaclostripain, a cysteine proteinase from Clostridium histolyticum (7). The expression of ccp was previously shown to be positively regulated by the VirR/VirS system (27).…”

Section: Resultsmentioning

confidence: 85%

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

et al. 2008

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Analysis of the complete sequence of the genome of Clostridium perfringens strain 13 resulted in identification of five genes, including pfoA (encoding theta toxin) and vrr (encoding VirR/VirS-regulated RNA), with consensus VirR-binding sequences upstream of the open reading frame (ORF), suggesting that expression of these genes may be regulated directly by the two-component VirR/VirS system. To test this possibility, we examined VirR/VirS system-mediated transcriptional regulation of three genes, virT, ccp (encoding alpha-clostripain), and virU, with the novel VirR-binding sequences. Northern analysis revealed that the steady-state levels (increases or decreases in the amounts of RNA expressed) of virT, ccp, and virU mRNAs were lower in a virR mutant strain than in the wild-type strain, as were the levels of the pfoA and vrr transcripts. The consensus VirR-binding sites were located similarly relative to the transcription start sites in the virT, ccp, and virU promoters. Mutation and overexpression analyses with virT and virU revealed that the virT gene product has a negative effect on expression of pfoA and ccp, whereas the virU gene product positively affects expression of pfoA, virT, ccp, and vrr. Nonsense and frameshift mutations in the virT or virU putative ORF did not affect the regulatory functions, suggesting that virT and virU may encode RNA regulators rather than proteins. These results suggest that a complex regulatory network, perhaps involving several regulatory RNA molecules, governs the expression of the VirR/VirS regulon in C. perfringens.

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Section: Resultsmentioning

confidence: 85%

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

et al. 2008

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“…Fig. 5 shows the aligned structure of the clostripain precursor and the sequence around the catalytically active Cys231, which represents Cys41 of the heavy chain (Dargatz et al, 1993). Clostripain probably does not form specific S' subsite loops as found in common serine proteases (Schellenberger et al, 1993a).…”

Section: Discussionmentioning

confidence: 99%

The specificity of clostripain from Clostridium histolyticum

Ullmann¹,

Jakubke²

1994

European Journal of Biochemistry

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The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using Nu-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-XaaAla-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P; position, low specificity for amino acid amides and Xaa-(Ala),-Gly peptides was found, however, in the Pi position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P: and Pi positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P; -Pi increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of PiPhe. Surprisingly, P:Gly leads to effective P'-S' interactions. However, the opposite result was obtained for PiGly. The high efficiency for Gly-NH, does not fit with the hydrophobicity structure/ activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH, with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P:Pro and PiGly and a negative cooperativity between Pi Pro and PiPhe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.The cysteine protease clostripain (Clostridium histolyticum proteinase B) from the culture filtrate of C. histolyticum is well known for its restricted substrate specificity, the cleavage of Arg-Xaa bonds, including proline in the P; position (subsite nomenclature of Schechter and Berger, 1967) (Ogle andTytell, 1953 ;Labouesse and Gros, 1960; Mitchell, 1964). For this reason this enzyme is important both in sequence analysis and in enzymic peptide synthesis, because proline is normally not well accepted in the P: position by the majority of proteases. Furthermore, a precise characterization of clostripain specificity is important if selective inhibitors are to be designed.Although S subsites have been studied in detail (Cole et al., 1971;Porter et al., 1971;Gilles et al., 1979;

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“…Polyacrylamide gel electrophoresis revealed that the 62-kDa protease, which is specific for the arginyl peptidyl bond, is composed of 15.5-and 48-kDa polypeptides. Interestingly, the structure and substrate specificity of this enzyme are reminiscent of those of the secreted alpha-clostripain from Clostridium histolyticum (Dargatz et al 1993), a homolog (74% amino acid identity) of which is present in C. botulinum (CBO1920). The C. histolyticum alpha-clostripain is a trypsin-like cysteine endopeptidase with strict specificity for the arginyl bond.…”

Section: Neurotoxin Genes and Virulence Factorsmentioning

confidence: 99%

“…The C. histolyticum alpha-clostripain is a trypsin-like cysteine endopeptidase with strict specificity for the arginyl bond. It is synthesized as an inactive prepro-enzyme that undergoes an autocatalytic cleavage to generate 15.4-and 43-kDa polypeptides, which associate to form a heterodimeric active enzyme (Dargatz et al 1993). Furthermore, both the C. histolyticum alphaclostripain and the C. botulinum 62-kDa protease require a reducing agent and calcium for full activity and are susceptible to the same protease inhibitors.…”

Section: Neurotoxin Genes and Virulence Factorsmentioning

confidence: 99%

Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes

Sebaihia¹,

Peck²,

Minton³

et al. 2007

Genome Res.

234

215

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Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp), which carry 3650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbors a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of Clostridium difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.

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The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene

Cited by 30 publications

References 29 publications

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

Characterization of Genes Regulated Directly by the VirR/VirS System in Clostridium perfringens

The specificity of clostripain from Clostridium histolyticum

Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes

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