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By their endoproteinase activities, cathepsins B, G, H and L can generate matrix-degrading proteolytic system from fibronectin. All four cathepsins studied cleaved fibronectin in fragments that were either proteolytically active or activated after incubation at pH 7.4 and in the presence of Ca2+. The highest enhancement of the matrix protein-degrading activity was observed after a gelatin-affinity chromatography of each digest. These results suggest that the effect of cathepsins at physiological pH in vivo may be enhanced by the activation of a matrix-degrading proteolytic system from fibronectin.

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-IV 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 II) 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 FN-gelatinase 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.

The N-terminal 70-kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27-kDa N-terminal heparin-binding and 45-kDa collagen-binding domains. The N-terminal residue in the 27-kDa fragment was blocked as in native fibronectin. The 45-kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262-265 of fibronectin) that correspond to the beginning of the collagen-binding domain. In the presence of Ca2+ the purified 27-kDa fragment underwent further processing finally leading to the cleavage of the bond K85-D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca(2+)-dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N-terminal domain of fibronectin.

Fibronectin is one of the major adhesive glycoproteins and bears interaction sites for both cell receptors and the extracellular matrix. Disappearance of fibronectin is the first step of cellular transformation in carcinogenesis. This phenomenon has been ascribed to increased proteolysis of fibronectin or of its cellular receptor. Results obtained during previous studies by the authors have shown that the fibronectin molecule has latent proteolytic activities which become apparent only after the action of other external proteases. Two proteinases, FN-gelatinase and FN-laminase, were identified in cathepsin D fibronectin digest. The acute activity of these two proteases is responsible for degradation of the extracellular matrix. Furthermore, the sequences and functions of both enzymes share a number of features with retroviral proteases.

The purified 190-kDa fibronectin fragment produced by cathepsin D can be spontaneously activated in the presence of CaCl2. This activation generates new proteolytic activities and also results in the formation of several subfragments. One of them exhibits the activity of FN-gelatinase that preferentially splits type I denatured collagen and fibronectin (see preceding paper). In this work we describe the purification and characterization of another fragment (25 kDa), issued from the same autodigest. This fragment may be activated to yield another proteinase, that splits preferentially laminin and denatured collagen type I. This enzyme will be referred as FN-laminase. Purified FN-laminase specifically reacted with antibodies against fibronectin. The specificity of bond cleavage by FN-laminase was studied with various synthetic peptides analogous to collagen repeats. FN-laminase cleaves the Ala-Gly bond in the sequence GPAGPR; the arginine residue in position P3' is important for this cleavage. The enzyme is inhibited by pepstatin A and phenylmethanesulfonyl fluoride, like retroviral aspartic proteinases. It is also inhibited by EDTA. No inhibition was obtained with 1,10-phenanthroline or 4-chloromercuribenzoate, inhibitors of Zn-metalloproteinases or cysteine proteinases, respectively.

Human plasma fibronectin contains a latent proteinase that after activation cleaves gelatin and fibronectin. The autoactivation propensity of the two purified cathepsin D-produced fragments of fibronectin (190 and 120 kDa) was compared. Both polypeptides were spontaneously activated in the presence of Ca2+. This activation was inhibited by EDTA. The active gelatinase was isolated from the autodigest of the 190-kDa fragment. Among various protein substrates, including laminin and native type I and IV collagens, the purified enzyme degraded only gelatin and fibronectin. We have named this proteinase FN-gelatinase. FN-gelatinase is inhibited by phenylmethanesulfonyl fluoride and also by pepstatin A like retroviral aspartic proteinases. The amino-acid composition of the purified enzyme (35 kDa) was compared with the entire fibronectin sequence using the computer programme FIT. The optimal fit indicated that the 35-kDa fragment corresponds to the stretch # 1043-1404. This sequence contains a 93-residue segment (# 1140-1233) analogous to retroviral aspartic proteinases, comprising the sequence DTG of their putative active site.