Elucidating the Role of Heparin in Fibronectin Module III-13 Self-Association

Abstract

The extracellular matrix (ECM) creates a dynamic 3D environment for cells, providing both mechanical and biochemical structure. Proper development of the ECM is dependent on the initial formation of a fibronectin (FN) matrix, and other matrix components depend on this FN matrix for ECM incorporation. FN matrix assembly is facilitated by unfolding of the compact FN dimer and which exposes hidden binding sites along FN. FN-FN interactions form fibrils, and over time these fibrils are converted into an insoluble form. We identified one type III module of FN, III-13, as important for formation of insoluble fibrils. III-13 not only demonstrates structural features that permit self-association, but it also lies in the center of FN’s heparin-binding (HepII) domain, and heparin was recently found to promote formation of the FN matrix. We hypothesized that heparin binding could be related to self-association. To test ability of III-13 to self- associate, we measured the thermal unfolding of III-13 by fluorimetry and used dynamic light scattering to detect different size populations that III-13 can form, both with and without the addition of heparin. We discovered that when heated III-13 experiences a conformational change before fully denaturing, and that this conformational change facilitates the formation of III-13 multimers. Heparin binding to III-13 increased the temperature needed to reach the conformational change, indicating that heparin stabilizes III-13, but with increasing temperature III-13 formed multimers and aggregates. Because one heparin chain can bind multiple III-13 modules, a model is proposed in which heparin brings III-13 monomers together, increasing the chance that they will form connections as they partially unfold. Although FN matrix formation does not occur at high temperatures in vivo, we discuss other factors that can induce III-13 unfolding and how heparin assists in the order and organization of this process.