Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Abstract

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 ␣ 5 and ␤ 1 proteins and ␣ 5 ␤ 1 integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of ␣ 5 ␤ 1 to fibronectin. Co-immunoprecipitation of GT1b and ␣ 5 ␤ 1 from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified ␣ 5 ␤ 1 from SCC12 cells and insect recombinant ␣ 5 ␤ 1 , particularly the ␣ 5 subunit, further suggest interaction between ganglioside and ␣ 5 ␤ 1 . The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of ␣ 5 ␤ 1 from SCC12 and insect cells or poorly glycosylated recombinant ␣ 5 ␤ 1 from Escherichia coli cells. The GT1b-␣ 5 ␤ 1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in ␣ 5 ␤ 1 . 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 ␣ 5 ␤ 1 -mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the ␣ 5 subunit of ␣ 5 ␤ 1 integrin. Human keratinocyte motility on a fibronectin (FN)1 matrix is critical for the re-epithelialization of healing wounds, for the spread of cutaneous malignancy, and in cutaneous embryogenesis. Although the molecular events that influence this migration are poorly understood, the interaction between keratinocyte ␣ 5 ␤ 1 integrin and the arginine-glycine-aspartic acid (RGD) site of FN is known to be key (1). Increased expression of ␣ 5 ␤ 1 has been shown in keratinocytes at the migrating edge of wounds in vivo, in lesional skin of patients with psoriasis, and in cultured keratinocytes (2-4). Both receptor clustering on keratinocytes and ligand occupancy of ␣ 5 ␤ 1 are required to activate intracellular signal transduction components (5), including focal adhesion kinase, phosphatidylinositol 3-kinase, protein kinase C, and integrin-linked kinase, leading to cell adhesion to .Gangliosides are glycosphingolipids characterized by the presence of one or more sialic acid moieties in the oligosaccharide chain (9). The role of gangliosides, which are localized to the outer leaflet of the plasma membrane of eukaryotic cells, is largely unknown, but studies with cultured keratinocytes and keratinocyte-derived cells suggest that gangliosides are involved in regulating cellular proliferation, differentiation, and adhesion (10 -13). The discovery that gangliosides inhibit cell attachment and spreading on a FN matrix (14) led investigators to consider gangliosides to be the cell receptors for FN before integrin ␣ 5 ␤ 1 was identified (15,16). Although these early studies showed that the terminal sialic acid residues of gangliosides were critica...

The glycosynapse

“…In contrast, GT1b and GD3 inhibit ␣5␤1-dependent binding of keratinocytes. In this system, N-glycosylation of ␣5␤1 is essential for this inhibitory effect, in which GT1b and GD3 are claimed to interact with the mannose core of N-linked glycan through carbohydratecarbohydrate interaction (91).…”

Deacetylated GM3 Promotes uPAR-Associated Membrane Molecular Complex to Activate p38 MAPK in Metastatic Melanoma

“…Protein concentration was measured using Coomassie Blue R250 (25,34). Total protein (10-30 mg) was applied onto a 10% SDS-PAGE mini gel.…”

Ganglioside GM3 Blocks the Activation of Epidermal Growth Factor Receptor Induced by Integrin at Specific Tyrosine Sites

“…GT1b has been shown to bind specifically to the ␣ 5 subunit of ␣ 5 ␤ 1 integrin, leading to competitive inhibition of the interaction of this integrin with FN and explaining the specificity of the GT1b inhibitory effect (19). GM3, the major ganglioside of epithelial cells (19), predominantly plays a role in regulating mitogenesis through inhibition of EGFR phosphorylation (18,26), a process that requires the presence of EGFR ligand (18,20,24,26). GM3 is also able to regulate the adhesion and migration of several carcinoma and other tumor cell lines grown on FN (27)(28)(29)(30); however, GM3 is unable to bind directly to either the ␣ 5 or ␤ 1 subunits of integrin (19).…”

“…GT1b, the highly sialylated ganglioside of keratinocytes and the keratinocyte-derived squamous carcinoma cell line SCC12, strongly inhibits adhesion, spreading, and migration of cultured epithelial cells specifically when cells are plated on a fibronectin (FN) matrix (17,19,23,25). GT1b has been shown to bind specifically to the ␣ 5 subunit of ␣ 5 ␤ 1 integrin, leading to competitive inhibition of the interaction of this integrin with FN and explaining the specificity of the GT1b inhibitory effect (19). GM3, the major ganglioside of epithelial cells (19), predominantly plays a role in regulating mitogenesis through inhibition of EGFR phosphorylation (18,26), a process that requires the presence of EGFR ligand (18,20,24,26).…”

“…GM3, the major ganglioside of epithelial cells (19), predominantly plays a role in regulating mitogenesis through inhibition of EGFR phosphorylation (18,26), a process that requires the presence of EGFR ligand (18,20,24,26). GM3 is also able to regulate the adhesion and migration of several carcinoma and other tumor cell lines grown on FN (27)(28)(29)(30); however, GM3 is unable to bind directly to either the ␣ 5 or ␤ 1 subunits of integrin (19). We have recently shown that endogenous accumulation of GM3 suppresses SCC12 cell migration and invasion through inhibition of extracellular matrix-promoted matrix metalloproteinase-9 activation and disrupts the association of matrix metalloproteinase-9 with integrin ␣ 5 ␤ 1 (31).…”