Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α ₅ Modulates α₅β₁ Function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant α₅ and β₁ proteins and α ₅β₁ integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of α ₅β₁ to fibronectin. Co-immunoprecipitation of GT1b and α₅β₁ from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified α₅β₁5 from SCC12 cells and insect recombinant α₅β₁, particularly the α₅ subunit, further suggest interaction between ganglioside and α₅β₁. The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of α₅β₁ from SCC12 and insect cells or poorly glycosylated recombinant α₅β ₁ from Escherichia coli cells. The GT1b-α₅β ₁ interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in α₅β₁. The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate α ₅β₁-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the α₅ subunit of α₅β₁ integrin.