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Cleavage of the 45-kDa gelatin-binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40-kDa FN-type-1V collagenase A degrades heat-denatured type-I collagen, laminin and also native collagen type IV. The 27-kDa FN-type-IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N-terminal sequence VYQPQPH-(residues 262 -268 of fibronectin) but, contrary to the FN-type-IV collagenase A, the FN-type;IV collagenase B has lost the C-terminal region of type I repeats, where the major gelatin-binding determinants of fibronectin are located. The FN-type-IV collagenases A and B are sequentially similar to the middle domain (domain 11) of collagenase type IV, secreted by H-ras-transformed human bronchial epithelial cells.Substrate and inhibition specificity of FN-type-IV collagenase A and B are different from those of FNgelatinase and FN-laminase, isolated previously from the central and C-terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix-degrading metalloproteinases.Fibronectin is an adhesion protein that is present in both extracellular matrix and blood plasma. This large 540-kDa glycoprotein is composed by two nearly identical polypeptide chains, each comprised of multiple specific sites for binding various ligands including collagens, fibrin and heparin. Each polypeptide chain of fibronectin is composed of three types of sequential repeats (reviewed in [l -31).The collagen-binding domain is located near the N-terminus within a 45-kDa region that contains four type-I and two type-I1 repeats. Whereas the type-I repeats are found also in other functional domains of fibronectin, the type-I1 units are specific for the gelatin-binding domain [4-61. It was therefore suggested that these type-I1 repeats are responsible for the interaction between fibroncctin and collagen(s), a mechanism which is fundamental to the organization of extracellular matrix.Recent studies, however, have provided evidence that the collagen-binding site is located between the second type-I1 unit and the adjacent type-I unit, within a 12-kDa fragment including the sequence -AAHE- [7], which is also found in the metal-binding site of matrix-degrading metalloproteinases. Moreover, it was recently shown that the major gelatin-binding determinants of fibronectin are located within a 21-kDa region of two type-I repeats that occurs in the C-terminal extremity of this domain 181. The initial idea for the present study was to search for the mechanism of interaction between fibronectin and its ligand, collagen. In particular, we have examined the question of whether this interaction might be analogous to the interaction of a zymogen with its potential substrate. We have previously demonstrated a similar mechanism of interaction between trypsin...