Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen.

Abstract

Fibronectin mediates the adhesion of cells to col1agen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectinmediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GDia were the most effective inhibitors followed by GD1b >> GM1 >> GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GDia modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin. Most cells require an appropriate substratum for growth in culture. A variety of cells use fibronectin, a large extrinsic matrix protein to adhere either to plastic (1) or to collagen substrates (2). An identical or very similar protein, cold insoluble globulin, is present in serum, and the serum form of fibronectin promotes the attachment of freshly trypsinized cells. Fibronectin prepared from either cells or serum is equally active in promoting cell attachment (3). With collagen substrates, fibronectin binds to the collagen substratum first (2, 4). Then the cells, in the presence of Ca2+ or Mg2+ in an energy-dependent process (5), adhere to the fibronectin-collagen complex. Fibronectin binds to all mammalian collagens that have been tested so far and it also binds to fibrin (6). In the al(I) chain of collagen, it binds to a specific amino acid sequence lacking carbohydrate (7)(8)(9)(10). These interactions are not cation dependent and can be broken by 1 M KBr (11) or 6 M urea (12). Fibrinogen (13) and collagen affinity columns (11, 12) have proved useful in the purification of fibronectin from serum and from conditioned culture medium.Little is known about the interaction of fibronectin with the cell surface or the nature of the membrane component(s) to which fibronectin binds. One approach to their identification is to test various cell surface components for their ability to interact with the fibronectin-collagen complex and thereby prevent cell attachment. Gangliosides are well-defined membrane components, and a specific ganglioside, GM,, functions as the membrane receptor for cholera toxin (14). This role for GM, was elucidated from the original observation that gangliosides block the biological effects of the toxin (15). We now report that certain gangliosides block cell adhesion promoted by the fibronectin in serum.MATERIALS ...

Change in the topographical distribution of GM3 during cell spreading and growth : Immunostaining with monoclonal antibody against GM3.

“…The reactivity of the GM3 in SAM with this antibody was diminished by trypsin treatment of SAM, which suggests that GM3 is not attached directly to the substratum, but indirectly to it through a protein component(s). The fact that the antibody against GM3 did not inhibit cell attachment and spreading on fibronectin confirmed that GM3 is the poorest effector for those phenomena (11,13).…”

“…Using the conventional antibody against GM3, Hakomori et al found that the reactivity of GM3 to that antibody was higher in * To whom correspondence should be addressed These observations suggest that GM3 may have an important function(s) in cellto-cell or cell-to-substrate interaction. GM3 has, however, been reported to be the poorest effector for cell attachment (11) and spreading (13) to fibronectin and in the organization of extra cellular fibronectin into the fibrillar form (17).…”

Disialoganglioside GD2 distributes preferentially into substrate-associated microprocesses on human melanoma cells during their attachment to fibronectin.

“…A number of reports have indicated that the exogenous addition of polysialogangliosides to fibronectin-attached cells caused them to round up and detach from the substrate (18,29,49). These studies led to the conclusion that gangliosides may act as specific fibronectin receptors.…”

“…Direct evidence is provided that GD2 is involved in M21 cell attachment to fibronectin, since treatment of these cells with anti-GD2 monoclonal antibodies causes cell rounding and detachment from a fibronectin substrate. Moreover, scanning electron microscopy demonstrates that this loss of attachment of fibronectin is characterized by a perturbation of the cell attachment-promoting microprocesses that in the presence of these antibodies lose contact with the fibronectin substrate.umber of studies have indicated that the carbohydrate moiety of gangliosides plays a direct role in cellsubstratum interactions, thus implicating them as putative cell surface receptors for fibronectin and collagen (8,18,29,49). In a previous paper, we used monoclonal antibodies (Mabs) ~ that specifically recognize carbohydrate determinants on the gangliosides GD2 and GD32 (8) to localize them on the surface of human melanoma cells and their focal adhesion plaques.…”

“…umber of studies have indicated that the carbohydrate moiety of gangliosides plays a direct role in cellsubstratum interactions, thus implicating them as putative cell surface receptors for fibronectin and collagen (8,18,29,49). In a previous paper, we used monoclonal antibodies (Mabs) ~ that specifically recognize carbohydrate determinants on the gangliosides GD2 and GD32 (8) to localize them on the surface of human melanoma cells and their focal adhesion plaques.…”