Fibronectin binding to gangliosides and rat liver plasma membranes

Abstract

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (G T1b ⋍ G D1b ⋍ G D1a > G M1 ⪢ G M2 ⋍ G D3 ⋍ G M3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367) [14]. Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides G M1, G D1a or G T1b bound more fibronectin than erythrocytres not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent K d for binding to mixed rat liver gangliosides of 7.8 × 10 −9 M was determined. This value compared favorably with the apparent K d for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 × 10 −9 M for fibronectin modified on the tyrosine residue, or 6.4 × 10 −9 M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochim biophys acta 550 (1979) 92) [23], fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysinemodified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43–75% less [ 125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.