Catechol-based matrix metalloproteinase inhibitors with additional antioxidative activity.

New catechol-containing chemical entities have been investigated as matrix metalloproteinase inhibitors as well as antioxidant molecules. The combination of the two properties could represent a useful feature due to the potential application in all the pathological processes characterized by increased proteolytic activity and radical oxygen species (ROS) production, such as inflammation and photoaging. A series of catechol-based molecules were synthesized and tested for both proteolytic and oxidative inhibitory activity, and the detailed binding mode was assessed by crystal structure determination of the complex between a catechol derivative and the matrix metalloproteinase-8. Surprisingly, X-ray structure reveals that the catechol oxygens do not coordinates the zinc atom.

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure.

We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.

The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases.

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl2- and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.