Fibronectins are high molecular weight multifunctional glycoproteins present in soluble form in plasma, amniotic and other body fluids and in insoluble form in extracellular matrices and basement membrane. The functions of fibronectin that include substrate adhesion, cell spreading, opsonization of foreign particles and wound healing are based on its affinity to cell surfaces and to a number of macromolecules including collagen, heparin, heparan sulfate, actin, DNA, fibrin and to certain bacteria especially S. aureus. Plasma, cell surface and amniotic fluid or placental fibronectins differ primarily in their nature and degree of glycosylation. There is also some evidence that differences exist between these different forms of fibronectins in polypeptide sequences especially in the carboxy terminal heparin-binding (Hep-2) domain that results primarily from differential splicing of fibronectin message. Initial work from this laboratory has resulted in structural analysis of several key domains of plasma fibronectin including a glycosylated 20kDa DNA/heparin-binding domain, a nonglycosylated 14kDa DNA/heparin-binding domain and a 29kDa gelatin-binding domain. Complete sequence has also been obtained on the 29/38kDa, heparin-binding (Hep-2) domains and we have shown sequence differences between two subunits in this region. Progress in the analysis of these domains has permitted us to propose studies on equivalent domains from fetal and transformed fibronectins in terms of their protein and carbohydrate structure and to initiate structure-function studies, including inhibition and cross linking with synthetic peptides corresponding to subregions of each domain. Conversion of key amino acids (identified by above approaches) will be attempted using site-directed mutagenesis to further compare the native and mutagenized domains for structure-function studies. Physical studies including circular dichroism and tryptophan fluorescence will be performed for conformational analysis of these domains and for studying the ligand induced changes in their native conformation. These complementary approaches should provide new insight into the structure-function relationship of different forms of human fibronectins.
