Collagen‐binding domain of human plasma fibronectin contains a latent type‐IV collagenase

Abstract

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...

Expression of collagenase/gelatinase activity from basement‐membrane fibronectin

“…Without gelatin, active bands of higher molecular When a sample processed without purified fibronectin fragment motryptic fragment; lane 10, gelatin incubated for 72 h as a control un-was assayed using both methods, proteolytic activity was not der the same conditions as in lanes 8 and 9. observed. Both results might be associated with the collagenase/ gelatinase activity of basement-membrane fibronectin, as reported previously for basement-membrane and plasma fibronectin [5,6,10]. When the purified material was analyzed by SDS/ found with purified fibronectin fragments from basement-mem-PAGE under reducing conditions, proteins of 66 kDa and 55 kDa brane hydrolysate: two major bands of activities were observed, were found, and after 72 h proteins of 45, 30 and 27 kDa were which corresponded to proteins of 47 kDa and 37 kDa.…”

“…4 C, D). The inhibifor the gelatine-binding fragment of plasma fibronectin [6,24], tion profile was superimposable for both kinds of fibronectin is probably the true processing pattern. Thus, the molecular fragments: only 1-10 phenantroline and EDTA reversibly inhibmasses of the gelatinases located on the zymogram are overestiited the enzymatic activity.…”

“…activities (47, 43 and 37 kDa) after limited proteolysis. The isoPlasma fibronectin has been described as a potent proteolytic lation of fibronectin fragments expressing such activities was system activated by limited proteolysis [5,6]. Since only one performed using successive of three affinity methods, and the characterization of the purified material was carried out by gela- and were associated with a 43-kDa band in some samples.…”

Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin

“…3c). The proteolytic potential of plasma fibronectin has been investigated in some previous reports [6,7]. However, these properties could be more important at the cellular level [8,13].…”

Matrix Assembly Induction and Cell Migration and Invasion Inhibition by a 13-Amino Acid Fibronectin Peptide

“…In particular, the FN collagen binding domain (COL-domain), in addition to binding with collagens and gelatin (3,(37)(38)(39)(40), has been shown to possess a number of other biological activities including the expression of collagenase activity (35,(41)(42), the promotion of odontoblast differentiation (43), and the substratum-dependent stimulation of fibroblast migration (44).…”