We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37°C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/ gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition-profile study showed a zinc-dependent collagenase activity. Using the 45-kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen-binding site, the same results were obtained. These results allow us to define a thiol-dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen-binding domain, a zinc-binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.Keywords : cellular fibronectin ; collagenase; gelatinase; matrix metalloprotease; thimet peptidase.Fibronectin is a well-characterized glycoprotein of blood gene for plasma and cellular fibronectin has been found, both plasma and extracellular matrixes that presents a variety of func-fibronectin are strongly related at the structural level [7]. Thus, tional activities [1]. Recently, some biological activities associ-a similar proteolytic potential could be present in cellular fibroated with a conformational change and/or limited proteolysis nectin, and a study of the expression of these enzymatic properhave been described. For example, gelatinase A [matrix met-ties should be of interest. Nevertheless, from a single gene a alloproteinase (MMP)2], was able to release fibronectin frag-complex pattern of alternative mRNA splicing generates ments that stimulate cell migration and inhibit adipocyte differ-multiple fibronectin polypeptides [8]. The various forms of fientiation [2]. Cathepsin D and thrombin digests of fibronectin bronectin are associated with different biological phenomena, added to cultures of articular-cartilage-tis...

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The proteolytic potential of cellular fibronectin fragments issued from a basement membrane hydrolysate was investigated. Three different gelatinase activities (47, 43 and 37 kDa), located by gelatin zymography, were isolated using successively heparin-agarose, gelatin-agarose and immunopurification with polyclonal antibodies directed against bovine plasma fibronectin. These fragments were also characterized using a monoclonal antibody directed against the extra-domain EDA of cellular fibronectin as a probe. A collagenase activity, reliably indicated by the gelatin zymography pattern, was also found using MCA-Pro-Leu-GIy-Leu-DPA-AIa-Arg-NH2, the intramolecularly quenched fluorogenic substrate of collagenases. From these results, cellular fibronectin was found to be able to exhibit a proteolytic function after limited proteolysis. This MMP-like function could be associated with tissue remodeling in both normal and pathological states, such as metastasis, angiogenesis and tissue repair.