Discovery of protein-protein interactions of the lysyl oxidase enzyme: Implications for cardiovascular disease, cancer, and fibrosis

Lysyl oxidase (LOX) is a copper-containing amine oxidase known to catalyze the covalent cross-linking of fibrillar collagens and elastin at peptidyl lysine residues. In addition, its involvement in cancer, wound healing, cell motility, chemotaxis, and differentiation reflect a remarkable functional diversity of LOX. To investigate novel mechanisms of LOX regulation and function, we performed a yeast two-hybrid screen to identify LOX-interacting proteins. From the 58 sequenced positive cDNAs, we identified placental lactogen (PL), fibronectin (FN), and fibulin-1 (FBLN1) as putative LOX-binding proteins. In this study, we present an extensive characterization of the interaction between LOX and FN, as well as evidence suggesting an important biological function in human physiologic and disease processes. GST pull-downs and solid phase binding assays confirmed the LOX-FN interaction. LOX bound to the cellular form of FN (cFN) with a dissociation constant (Kd) of 2.5 nM. This was comparable to our measured Kd of LOX binding to tropoelastin (1.9 nM) and type I collagen (5.2 nM), but LOX demonstrated a much lower binding affinity for the plasma form of FN (pFN). Immunofluorescent microscopy revealed co-localization of FN and LOX in normal human tissues where these proteins may interact in vivo. LOX enzymatic activity assays showed that cFN does not seem to be a substrate of LOX. However, cFN can act as a scaffold for enzymatically active 30-kD LOX. Furthermore, in FN-null mouse embryonic fibroblasts, we observed dramatically decreased proteolytic processing of the 45-kD LOX proenzyme to the 30-kD active form, with a corresponding decrease in LOX enzyme activity. Our results suggest that the FN matrix provides specific microenvironments to regulate LOX catalytic activity.