| Vitronectin is a multifunctional plasma protein that plays a regulatory role in humoral defense reactions that include coagulation and fibrinolysis. Circulating vitronectin is a mixture of a single-chain form and a two-chain form that is derived from proteolytic cleavage at position 379, near the C-terminus. The sequence that spans residues 340-379 in vitronectin is reported to be the primary binding site for a number of ligands, including the anticoagulant drug, heparin, and the antifibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1). This sequence has also been hypothesized to facilitate vitronectin self-association. Most researchers assume that there are no major functional differences between cleaved and uncleaved vitronectin, but potential changes have not been previously investigated.;The purpose of this study was to elucidate the role of the C-terminal domain in heparin binding, PAI-1 binding, and in multimerization, paying close attention to functional differences that result from proteolytic processing at position 379. A recombinant C-terminal domain was generated in Escherichia coli. The baculovirus system was also used to produce recombinant full-length vitronectin and a truncation mutant, which represents the large fragment of two-chain vitronectin. Kinetic, immunologic and fluorescence assays were used to characterize the fold and function of the recombinant proteins. Also, fluorescently labeled vitronectin and PAI-1 were prepared, and used to evaluate conformational changes that occur as the two molecules interact.;Results of these studies indicate that the entire binding site for heparin is contained between residues 331-380 of vitronectin. PAI-binding alters the conformation of the heparin-binding site, such that the fluorescence of a probe attached to arginines in the site is quenched upon binding. The binding sites for heparin and PAI-1 are distinct, so vitronectin can bind both molecules simultaneously. There are no functional differences between full-length and truncated vitronectin, with respect to heparin binding or PAI-1 binding, indicating that proteolytic processing at position 379 in vitronectin is not a regulatory event for controlling coagulation or the plasminogen activation system. Furthermore both forms of the molecule self-associate, suggesting that the C-terminal 80 amino acids are not needed for adopting the multimeric conformation that stabilizes vitronectin-binding events via multivalent interactions. |
