| 1. |
Wilson JJ,
Matsushita O,
Okabe A,
Sakon J,
( 2003 ) A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. PMID : 12682007 : DOI : 10.1093/emboj/cdg172 PMC : PMC154464 Abstract >>
The crystal structure of a collagen-binding domain (CBD) with an N-terminal domain linker from Clostridium histolyticum class I collagenase was determined at 1.00 A resolution in the absence of calcium (1NQJ) and at 1.65 A resolution in the presence of calcium (1NQD). The mature enzyme is composed of four domains: a metalloprotease domain, a spacing domain and two CBDs. A 12-residue-long linker is found at the N-terminus of each CBD. In the absence of calcium, the CBD reveals a beta-sheet sandwich fold with the linker adopting an alpha-helix. The addition of calcium unwinds the linker and anchors it to the distal side of the sandwich as a new beta-strand. The conformational change of the linker upon calcium binding is confirmed by changes in the Stokes and hydrodynamic radii as measured by size exclusion chromatography and by dynamic light scattering with and without calcium. Furthermore, extensive mutagenesis of conserved surface residues and collagen-binding studies allow us to identify the collagen-binding surface of the protein and propose likely collagen-protein binding models.
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2. |
Toyoshima T,
Matsushita O,
Minami J,
Nishi N,
Okabe A,
Itano T,
( 2001 ) Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo. PMID : 11913772 : Abstract >>
The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticumclass I collagenase (ColG) was examined both in vitro and in vivo. CBD bound to insoluble type I, II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney, trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD bound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemet's membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will bind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.
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3. |
Matsushita O,
Koide T,
Kobayashi R,
Nagata K,
Okabe A,
( 2001 ) Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase. PMID : 11121400 : DOI : 10.1074/jbc.M003450200 Abstract >>
Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.
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4. |
Sakurai J,
Katayama S,
Jung CM,
Minami J,
( 1999 ) Identification of metal ligands in the Clostridium histolyticum ColH collagenase. PMID : 10217773 : PMC : PMC93724 Abstract >>
A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.
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5. |
Shi L,
Carson D,
( N/A ) Collagenase Santyl ointment: a selective agent for wound debridement. PMID : 19918145 : DOI : 10.1097/WON.0b013e3181bfdd1a Abstract >>
Enzymatic debridement is a frequently used technique for removal of necrotic tissue from wounds. Proteases with specificity to break down the collagenous materials in necrotic tissues can achieve selective debridement, digesting denatured collagen in eschar while sparing nonnecrotic tissues. This article provides information about the selectivity of a collagenase-based debriding agent, including evidence of its safe and efficacious uses. Recent research has been conducted, investigating the chemical and biological properties of collagenase ointment, including healing in animal models, digestion power on different collagen types, cell migration activity from collagen degradation products, and compatibility with various wound dressings and metal ions. Evidence presented demonstrates that collagenase ointment is an effective, selective, and safe wound debriding agent.
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6. |
Philominathan ST,
Koide T,
Hamada K,
Yasui H,
Seifert S,
Matsushita O,
Sakon J,
( 2009 ) Unidirectional binding of clostridial collagenase to triple helical substrates. PMID : 19208618 : DOI : 10.1074/jbc.M807684200 PMC : PMC2667773 Abstract >>
Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca(2+). We have shown that CBD can be functional to anchor growth factors in local tissue. A (1)H-(15)N HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-l-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-ray scattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.
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7. |
Eckhard U,
Schönauer E,
Ducka P,
Briza P,
Nüss D,
Brandstetter H,
( 2009 ) Biochemical characterization of the catalytic domains of three different Clostridial collagenases. PMID : 18937627 : DOI : 10.1515/BC.2009.004 Abstract >>
Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K(m) values of all three enzymes were within the same order of magnitude, the turnover rate k(cat) of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.
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8. |
McCarthy RC,
Spurlin B,
Wright MJ,
Breite AG,
Sturdevant LK,
Dwulet CS,
Dwulet FE,
( 2008 ) Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. PMID : 18374061 : DOI : 10.1016/j.transproceed.2008.01.041 Abstract >>
A collagen degradation activity (CDA) assay was developed to improve the biochemical characterization of purified collagenase used for islet isolation. Purified class I collagenase (CI) or class II collagenase (CII) from Clostridium histolyticum cultures were used in all experiments. The CDA assay was performed by incubating 50 microg/mL of FITC fibrils with CI or CII for 60 minutes at 35 degrees C. The correlation of the molecular species of the enzyme to CDA was determined by fractionating CI:CII mixtures over an anion exchange column. Individual fractions were analyzed for A280, CDA activity, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to correlate chromatographic analysis of these enzyme mixtures to the molecular species of collagenase effective in collagen degradation. CI has approximately 6 to 17 x higher specific activity than CII in this assay. Assays of different individual fractions recovered after anion exchange chromatography showed that the CDA of collagenase was dependent on the molecular species of the enzyme. Only intact CII and CI with molecular weights >or=100 kDa could degrade collagen fibrils. This assay provides a more reliable assessment of the functional activity of collagenase enzymes than peptide substrates currently used today. Fractionation of purified collagenase mixtures by anion exchange chromatography followed by analysis of individual fractions by SDS-PAGE and CDA assays will provide a powerful tool to analyze the molecular species of CI and CII required for islet isolation.
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9. |
Bauer R,
Janowska K,
Taylor K,
Jordan B,
Gann S,
Janowski T,
Latimer EC,
Matsushita O,
Sakon J,
( 2015 ) Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH. PMID : 25760606 : DOI : 10.1107/S1399004714027722 PMC : PMC4356367 Abstract >>
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 ? resolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 ? resolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 ? resolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 ? resolution, R = 16.9%, Rfree = 21.2%; 1.6 ? 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.
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10. |
Eckhard U,
Schönauer E,
Brandstetter H,
( 2013 ) Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T. PMID : 23703618 : DOI : 10.1074/jbc.M112.448548 PMC : PMC3711286 Abstract >>
Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.
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11. |
Eckhard U,
Huesgen PF,
Brandstetter H,
Overall CM,
( 2014 ) Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen. PMID : 24125730 : DOI : 10.1016/j.jprot.2013.10.004 PMC : PMC3985423 Abstract >>
Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified >100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1', proline at P2 and P2', and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage. We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of specific test substrates and selective inhibitors. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?
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12. |
Breite AG,
McCarthy RC,
Dwulet FE,
( 2011 ) Characterization and functional assessment of Clostridium histolyticum class I (C1) collagenases and the synergistic degradation of native collagen in enzyme mixtures containing class II (C2) collagenase. PMID : 22099748 : DOI : 10.1016/j.transproceed.2011.09.059 Abstract >>
Clostridium histolyticum expresses two classes of collagenases, C1 and C2. However, degradation of these enzymes by proteases during the fermentation or purification process may lead to numerous molecular forms that lead to inconsistent release of islets from human pancreata. This report defines the amino acid sequence of the truncated forms of C1 (C1b or C1c) that contain a single collagen-binding domain (CBD) and investigates the synergy between the different forms of C1 collagenase and C2 to degrade native collagen. Highly purified collagenase isoforms were purified from C. histolyticum culture supernatants using established column chromatography techniques and analyzed using high-pressure liquid chromatograph (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The collagen-degrading activity (CDA) assay was used to investigate the synergy between different collagenase molecular forms. MS was used to confirm the sequence of full-length C2 and C1 from the reported gene sequence. These results were correlated with the molecular weights observed on the SDS- PAGE and elution after analytical anion-exchange HPLC. HPLC peaks designated as C1b and C1c were both confirmed to be C1 lacking the terminal CBD. The only difference being the cleavage site leading to a 12 amino acid difference between the two forms. A non-additive synergy in CDA relative to activity of individual collagenases was observed for C2 with each of the three C1 molecular forms. The C1 molecular forms did not display this synergy in the absence of C2. These observations support earlier reports that suggest the two collagenases bind to different portions of the collagen and have different specificities to cut native collagen. Although the implications of this are not yet understood, they are fundamental in advancing the understanding of how collagenases work together along with the neutral protease to breakdown the extracellular matrix for islet isolation.
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13. |
Eckhard U,
Schönauer E,
Nüss D,
Brandstetter H,
( 2011 ) Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis. PMID : 21947205 : DOI : 10.1038/nsmb.2127 PMC : PMC3191118 Abstract >>
Collagen constitutes one-third of body protein in humans, reflecting its extensive role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that have evolved for collagen remodeling. The most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues; but despite intense study, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55-? resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which recognition and unraveling of collagen microfibrils into triple helices, as well as unwinding of the triple helices, are driven by collagenase opening and closing.
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14. |
Eckhard U,
Brandstetter H,
( 2011 ) Polycystic kidney disease-like domains of clostridial collagenases and their role in collagen recruitment. PMID : 21871007 : DOI : 10.1515/BC.2011.099 Abstract >>
Bacterial collagenases exhibit a multimodular domain organization. While the N-terminal collagenase unit harbors the catalytic zinc and suffices to degrade peptidic substrates, collagen substrates come in different types, explaining the requirement for accessory domains such as polycystic kidney disease (PKD)-like domains for efficient catalysis. How the recognition and unfolding of (micro-)fibrillar or triple-helical collagen is accomplished are only poorly understood. Here, we present the crystal structure of the PKD-like domain of collagenase G from Clostridium histolyticum. The �]-barrel structure reveals a two-tier architecture, connected by kinked hinge segments. Together with sheet extension as a generic oligomerization mechanism, this explains the cooperativity among accessory domains as well as their adaptivity to varying substrates.
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15. |
( 1993 ) Collagenase versus placebo in the treatment of Peyronie's disease: a double-blind study. PMID : 8417217 : DOI : 10.1016/s0022-5347(17)35998-0 Abstract >>
We investigated 49 men with Peyronie's disease in a prospectively randomized placebo controlled double-blind study, comparing the effects on plaque size and penile deformity of intralesional purified clostridial collagenase and saline placebo. For the group as a whole, treatment out-performed placebo (p < 0.007). When patients were analyzed with respect to disease severity, those with lesser deformity responded more favorably to treatment. The absolute angular change in patients responding to treatment was small. No significant side effects were noted within a 3-month followup.
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16. |
( 1993 ) The heterodimeric protease clostripain from Clostridium histolyticum is encoded by a single gene. PMID : 8341259 : DOI : 10.1007/bf00276893 Abstract >>
Clostripain (EC 3.4.22.8) is a heterodimeric cysteine endopeptidase with strict specificity for Arg-Xaa peptidyl bonds. It is secreted by Clostridium histolyticum strains. For the first time we present evidence that both polypeptide chains of native clostripain are encoded by a single gene. DNA sequencing of two overlapping genomic DNA fragments revealed a single open reading frame (ORF) of 1581 nucleotides encoding a polypeptide of 526 amino acid residues. The ORF is preceded by canonical transcription signals and both chains of the clostripain heterodimer are completely represented by the deduced coding sequence. Most interestingly, the sequences coding for the light and the heavy chain are joined by a DNA stretch coding for a linker nonapeptide that is preceded by the C-terminal arginyl residue of the light chain and also ends with an arginyl residue. Heterologous expression of the gene in Escherichia coli yielded an enzyme capable of hydrolyzing the clostripain substrates N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-carbobenzoxy-L-arginine p-nitroanilide (Z-Arg-pNA).
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17. |
( 1994 ) Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase. PMID : 7961400 : DOI : 10.1128/jb.176.21.6489-6496.1994 PMC : PMC197002 Abstract >>
The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.
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18. |
Gilles AM,
De Wolf A,
Keil B,
( 1983 ) Amino-acid sequences of the active-site sulfhydryl peptide and other thiol peptides from the cysteine proteinase alpha-clostripain. PMID : 6337850 : DOI : 10.1111/j.1432-1033.1983.tb07174.x Abstract >>
One free -SH group in the heavy chain of alpha-clostripain reacts rapidly with N-tosyllysine chloromethyl ketone which inactivates the enzyme. Iodoacetic acid also reacts with the thiol group required for enzyme activity but more slowly. A tryptic peptide containing the reactive sulfhydryl group labelled with iodo[1-14C]acetic acid was isolated and determined to be Gln-Ser-Val-Asp-Leu-Leu-Ala-Phe-Asp-Ala-Cys-Met. All other cysteine peptides were isolated from the trypsin hydrolysate of the [14C]carboxymethylated enzyme. Moreover N-terminal and C-terminal sequences of both chains of alpha-clostripain were determined. The sequences representing 20% of the primary structure of alpha-clostripain are not homologous with either other cysteine proteinases or with any other protein structure known to date.
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19. |
Gilles AM,
Lecroisey A,
Keil B,
( 1984 ) Primary structure of alpha-clostripain light chain. PMID : 6391922 : DOI : 10.1111/j.1432-1033.1984.tb08579.x Abstract >>
The primary structure of light chain of alpha-clostripain was determined by sequence analysis of peptides derived from tryptic digests purified by reverse-phase high-performance liquid chromatography. The 22 isolated tryptic peptides were aligned by peptides derived from chymotryptic and staphylococcal V8 proteinase digests. The light chain contains 133 amino acids residues and has a relative molecular mass of 15400. The prediction of its secondary structure is given.
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20. |
Bond MD,
Van Wart HE,
( 1984 ) Characterization of the individual collagenases from Clostridium histolyticum. PMID : 6087888 : DOI : 10.1021/bi00308a036 Abstract >>
The six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from Clostridium histolyticum isolated in the preceding paper [Bond, M. D., & Van Wart, H. E. (1984) Biochemistry (first paper of three in this issue)] have been characterized in detail. The molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000. Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. These experiments also reveal that the subspecies of alpha- and gamma-collagenases (alpha1 vs. alpha 2 and gamma 1 vs. gamma 2) have different isoelectric points but the same molecular weights. Microheterogeneity is also observed for the beta- and epsilon-collagenases. The amino acid compositions of all six collagenases have been determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content. Zinc and calcium are the only metals that copurify with the collagenases. The purified enzymes contain approximately 1 mol of zinc/mol of protein and a calcium content that varies from about 2 mol/mol for alpha-collagenase to about 7 mol/mol for beta-collagenase. All of the collagenases are 5-10 times more active against gelatin than collagen. The alpha-, beta-, and gamma-collagenases are significantly less active toward the synthetic peptide substrates examined than the delta-, epsilon, and zeta-collagenases. This property, taken together with data on the stabilities and amino acid compositions of these enzymes, strongly supports their assignment to two distinct classes. This establishes clearly that C. histolyticum does, indeed, produce more than one different type of collagenase.
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21. |
Schönauer E,
Kany AM,
Haupenthal J,
Hüsecken K,
Hoppe IJ,
Voos K,
Yahiaoui S,
Elsässer B,
Ducho C,
Brandstetter H,
Hartmann RW,
( 2017 ) Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases. PMID : 28820255 : DOI : 10.1021/jacs.7b06935 PMC : PMC5607459 Abstract >>
Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.
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22. |
Mookhtiar KA,
Steinbrink DR,
Van Wart HE,
( 1985 ) Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities. PMID : 3002446 : DOI : 10.1021/bi00344a033 Abstract >>
The action of three class I (beta, gamma, and eta) and three class II (delta, epsilon, and zeta) collagenases from Clostridium histolyticum on two series of peptides with collagen-like sequences has been examined. The peptides in the first series all contain 4-nitrophenylalanyl-Gly-Pro-Ala in subsites P1 through P3', but each is successively lengthened in the N-terminal direction by addition of an appropriate residue until subsite P5 is occupied. The second group of peptides all have cinnamoyl-Leu in subsites P2 and P1, respectively, but each is successively lengthened in the C-terminal direction by partial additions of the Gly-Pro-Leu triplet until subsite P6' is occupied. N-Terminal elongation causes the kcat/KM values to rise markedly and to level off after occupancy of subsite P6 for the class I enzymes and subsite P3 for the class II enzymes. C-Terminal elongation produces the best substrates for both classes of enzymes when subsites P3' or P4' are occupied by amino acids with free carboxyl groups. The kcat/KM values for the hydrolysis of both Leu-Gly bonds of cinnamoyl-Leu-Gly-Pro-Leu-Gly-Pro-Leu have been measured for both classes of enzymes. Both rates are large, but both classes preferentially hydrolyze the Leu-Gly bond of the C-terminal triplet. Thus, both classes of enzymes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities.
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23. |
( 2013 ) Solution structure of clostridial collagenase H and its calcium-dependent global conformation change. PMID : 23561530 : DOI : 10.1016/j.bpj.2013.02.022 PMC : PMC3617444 Abstract >>
Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.
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24. |
( 1999 ) Gene duplication and multiplicity of collagenases in Clostridium histolyticum. PMID : 9922257 : PMC : PMC93460 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.
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25. |
( 2013 ) Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum. PMID : 23144249 : DOI : 10.1128/JB.00010-12 PMC : PMC3553835 Abstract >>
Clostridium histolyticum secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-? resolution (R = 17.4%; R(free) = 23.3%), while the resolution of the previously determined s3b was extended to 1.4 ? (R = 17.9%; R(free) = 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C(�\) = 1.5 ?). All but one residue, whose side chain chelates with Ca(2+), are conserved. The dual Ca(2+) binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca(2+) (holo T(m) = 94.1�XC; apo T(m) = 70.2�XC). holo s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the cis-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca(2+) concentration possibly make both CBDs suitable for targeted growth factor delivery.
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26. |
( 2012 ) Bacterial collagen-binding domain targets undertwisted regions of collagen. PMID : 22898990 : DOI : 10.1002/pro.2145 PMC : PMC3526996 Abstract >>
Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C-terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly �� Ala substitution [(POG)(x) POA(POG)(y)]?, where x + y = 9 and x > 3). ?H-??N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ??N-labeled CBD demonstrated that the minicollagen binds to a 10 ? wide 25 ? long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ??N-labeled CBD. CBD binds to either the Gly �� Ala substitution site or to the C-terminus of each minicollagen. Small-angle X-ray scattering measurements revealed that CBD prefers to bind the Gly �� Ala site to the C-terminus. The HSQC NMR spectra of ??N-labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.
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27. |
( N/A ) The potential of collagenase as a new therapy for separation of human retained placenta: hydrolytic potency on human, equine and bovine placentae. PMID : 9699958 : Abstract >>
The purpose of this study was to determine to what degree bacterial collagenase may digest human placentae compared to equine and bovine placentae. Placenta samples from human, equine and bovine were incubated with bacterial collagenase solution at various concentrations. The degree of hydrolysis and collagen breakdown was measured by the release of total proteins and hydroxyproline into the incubation media. Also, whole placentae were injected via umbilical cord arteries with collagenase solution (200 U/ml, 200 ml total volume in human and 1000 ml in equine) and hydrolysis determined chemically and subjectively. Human and equine placental collagens were the most sensitive to collagenase digestion. Overall mean collagenase activity determined by the release of hydroxyproline from human placenta was 1.6 times and in equine placenta three times greater than in bovine placenta, while the breakdown of non-collagenous proteins remained negligible. When injected into whole placenta, the collagenase digested placentae evenly within 6-12 h. At 24 h, placentae were liquefied, although, umbilical blood vessels resisted collagenase digestion. Bacterial collagenase was highly effective in breaking down human placenta collagen. Intraplacental injections of collagenase via umbilical cord arteries may help to detach retained placenta in women as it does in mares and cows.
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28. |
( 1998 ) Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain. PMID : 9618531 : DOI : 10.1073/pnas.95.12.7018 PMC : PMC22723 Abstract >>
The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.
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( 1998 ) A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. PMID : 9452493 : DOI : 10.1074/jbc.273.6.3643 Abstract >>
The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.
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