STRUCTURE/FUNCTION STUDIES ON FIBRONECTIN/LIGAND BINDING

本提案的目标是结构和生物化学

The objectives of this proposal are to structurally and biochemically define the sites on fibronectin (Fn) that dictate binding to fibrin, heparin, and immunoglobulins (Ig) and employ this information to explore the [patho]physiological role for these ligand-binding interactions in wound repair (fibrin and heparin-binding) and fibrin clot formation, and immune complex (IC) deposition in tissues (fibrin and Ig-biding). We have determined the tertiary structure of the 4th and 5th type 1 structural repeats (4F1.5F1; 93 amino acids) in the N- terminal of Fn, by 1H NMR, and shown that this module pair harbors the fibrin, heparin, and Ig binding sites. A separate fibrin-binding site is in the C-terminal 10th-12th (10F1-12F1) type 1 repeats. We have produced recombinant Fn type 1 modules and functional proteolytic fragments of native Fn for use in the following Specific Aims: Aim 1). To define more precisely the fibrin, heparin, and Ig-binding activity/sites on Fn by solid phase assays (ELISA), by quantitating binding affinity constants and kinetics of binding by real time biospecific interaction analysis, and by implicating interactive residues critical to ligand binding by site directed mutagenesis. This information will be used to explore the [patho]physiological significance of these interaction in vitro and in vivo in the following specific aims: Aim 2). Fn is associated with immune complexes (Ics) in the circulation of patients with a variety of immune complex-related diseases (ICD). Since Fn and fibrin accumulate in the ECM in glomerulonephritis, we hypothesize that the interaction of Fn with Ig and, plasma Fn, as component of Ics, with fibrin, may contribute to the deposition of Ics from the circulation into tissues. To test this hypothesis, cell-generated Fn-containing extracellular matrices (ECMs) will be used as an in vitro model for IC deposition in tissues. Ig- binding (IgBP) and fibrin-binding (FBP) type 1 modules will be tested for their ability to inhibit Ics from biding to ECMs and in preventing IC deposition in normal mice injected with preformed IC's. By immunohistochemical analysis, the co-distribution of Fn, Ics, and fibrin will further support our hypothesis. Aim3). We hypothesize that the interaction of Fn with fibrin is essential for tissue remodeling and specifically in Fn/fibrin matrix assembly and cellular migration on Fn/fibrin wound provisional matrices. To test these hypotheses, FBPs from the N-and C-terminal of Fn will be used in in vitro clot binding and wound repair assays and in vivo, in porcine wound repair. The study of the structure/function relationship of Fn/fibrin and Fn/Ig- binding interactions should elucidate mechanisms of the function of Fn in wound repair and the pathogenesis of ICD.