The lanthanide-enhanced affinity chromatography of clostridial collagenase.
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ABSTRACT:
SUBMITTER: Evans CH
PROVIDER: S-EPMC1144624 | biostudies-other | 1985 Jan
REPOSITORIES: biostudies-other
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Project description: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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Project description: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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Project description:The clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein from Clostridium histolyticum was expressed in Escherichia coli and purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca(2+) in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca(2+) chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca(2+) was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca(2+) chelation, in comparison with the thermal melting point (T(m)). The melting point changed from 54 to 49°C by the Ca(2+) chelation, and it was restored to 54°C by the addition of excess Ca(2+). These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca(2+).
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Project description:The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.
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Project description:Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5?nm toward the collagen N terminus in ~3.8?s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution.
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Project description:Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.
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Project description:Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.
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Project description:The peptide derivative N alpha-(2,4-dinitrophenyl)-L-prolyl-L-leucyl-glycyl-L-prolyl-L-tryptophanyl-D- lysine (Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys) has been found to be a convenient substrate for the assay of clostridial collagenase and Pz-peptidase. The substrate shows a 25-fold enhancement of fluorescence (gamma ex. 283 nm, lambda em. 350 nm) following hydrolysis of the Leu2-Gly3 peptide bond. The value of Km for clostridial collagenase was 17 microM. The substrate for the first time makes possible continuous fluorimetric assays for Pz-peptidase and clostridial collagenase.
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Project description:Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm(3+), whose ionic radius is closest to that of Ca(2+), is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100mum in the presence of 5mm-Ca(2+). Er(3+) and Lu(3+), which both have ionic radii smaller than either Ca(2+) or Sm(3+), inhibit less efficiently, and La(3+), which is slightly larger than Ca(2+) or Sm(3+), inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca(2+)-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca(2+) [ionic radius 0.099nm (0.99 A)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca(2+), requires lanthanide concentrations 50-100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50mum, Sm(3+) lowers the apparent K(m) for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent V(max.) from 0.29 Wünsch-Heidrich unit to 0.018 unit. Thus Sm(3+) enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme-Sm(3+)-substrate complex. Inhibition by Sm(3+) is competitive with regard to Ca(2+). The apparent dissociation constant, K(d), of Ca(2+) is 0.27mm, where the K(i) for Sm(3+) is 12mum. Clostridiopeptidase is more thermolabile in the absence of Ca(2+). With Sm(3+), thermoinactivation of the enzyme at 53 degrees C or 60 degrees C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca(2+) at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.
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