Proteolysis of Aggregated Fibronectin A Model for In Vivo Matrix Degradation

The extracellular matrix (ECM) is a network of several proteins representing both a barrier delimiting the tissues and a substratum for cell adhesion, migration, and differentiation.1 Its degradation by mammalian Zn2+-proteinases referred to as MMPs (for matrix metalloproteinases) is implied in many normal or pathological processes (development, inflammation, metastasis, dissemination, etc.).2 Fibronectin (FN) is a major structural and functional protein of the ECM. It is a dimeric protein mainly composed of three different types of homologous modules that are grouped in compact domains interconnected by flexible strands.3 Each domain presents numerous binding sites (cell binding domain, collagen, heparin, DNA binding domains, etc.) (see FIGURE 1, upper). FN modules are highly resistant to proteolysis, but observable cleaved sites are located on the connecting strands. Furthermore, many FN proteolytic fragments have been shown to present functions that are not observed in the intact protein.4 Precise knowledge of the in vivo mechanisms of FN fragment production is thus of high interest. This study presents an original approach to the kinetics of multiple cleavage site proteolysis of large proteins such as FN (Mr, 500 × 103). We have determined the proteolytic cascade leading to apparition of different FN fragments and the velocity at which cleavages occur, using thermolysin as a model for MMPs. Proteolysis has been studied using FN under two forms: as a soluble form (as found in plasma) and as a reticulated insoluble form that mimics the aggregated form that is incorporated into ECM. Hence, contrary to the usual studies dealing with enzymology under heterogeneous conditions, the substrate is here immobilized and the soluble enzyme diffuses into it.