Peeters-Joris, Ch.Eeckhout, Y.Vaes, G.Hammani, KhalilKhalilHammani2025-05-142025-05-142025-05-141994https://hdl.handle.net/2078.2/42353Stromelysin (stromelysin-1, matrix metalloproteinase-3, MMP-3, transin, EC 3.4.24.17) is a member of the matrix metalloproteinases (MMP) family that is implicated in several physiological and pathological processes of connective tissue dégradation, including bone résorption and metastasis. This enzyme is secreted as a proenzyme which is able, once activated, to dégradé several proteins of the extracellular matrix, such as proteoglycans, type IV collagen, fibronectin, laminin and elastin, and to potentiate the activity of collagénases and gelatinases. Mouse calvarial bones cultured in the presence of heparin synthesize and release MMP-3 as a proenzyme. We undertook the purification of this enzyme from the medium of these cultures by sequential chromatography through DEAE-Sepharose, heparin-Sepharose, and zinc-chelating-Sepharose. The purified enzyme appeared pure as judged by SDS-PAGE after silver staining. It was recovered in a latent form (56.000 to 59.000) which could be activated by 4-aminophenylmercuric-acetate (APMA) or trypsin with a concomitant decrease of its MW. It had a caseinolytic activity at neutral pH and was able to cleave the cartilage proteoglycan aggrecan. However reverse zymography of purified stromelysin still showed contamination with traces of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This protein was eliminated from the end product by continuous elution electrophoresis using a Prep Cell apparatus. The fractions devoid of TIMP were pooled, concentrated and used as antigenic materiel to immunise a goat. A spécifie polyclonal antiserum for MMP-3 was obtained that inhibits and précipitâtes the enzyme. In parallel to these studies, we undertook the cloning and sequencing of a cDNA for stromelysin by screening a Xgt 11 cDNA library prepared by P. Henriet from mouse calvarial bones. The cDNA of MMP-3 was then subcloned in the vector pET-3a or c in order to express two forms of recombinant MMP-3 : a full length proMMP-3 (pETRM-la) and a truncated form (pETRM-lc) lacking the 82 N-terminal aminoacids i.e. the prepeptide and a part of the propeptide. These two recombinant enzymes were produced, mainly in an insoluble form, in Escherichia coli after induction with IPTG. Both forms of recombinant MMP-3 (respective Mr of 56,000 and 46,000) were characterized by Western immunoblotting and by casein- and gelatin- zymography. Further experiments allowed to recover the active recombinant enzymes after solubilization of the inclusion bodies in 8 M urea and renaturation either by dialysis or by chromatography. After dialysis, the enzyme produced by pETRM-la was partially recovered in a latent form, while pETRM-lc provided a fully active enzyme. The spécifie caseinolytic activity of both recombinant enzymes was similar to that of the natural one. Renaturation and purification by zinc-chelating-Sepharose chromatography provided only low Mr (26,000 and 29,000) fully active fragments of the recombinant enzyme. Finally, in order to overcome the limitations of the catalytic détermination of MMP-3, we developed an ELISA assay using the purified enzymes (recombinant and/or natural) and the spécifie polyclonal antiserum. Valuable molecular and immunological tools were thus developed, which may contribute to further investigations on the properties and rôles of this metalloproteinase in tissue remodelling.text::thesis::master thesisthesis:50095