The Essential Role of N-Glycosylation in Integrin αV and uPAR Interaction in Glioblastoma

BACKGROUND Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults, characterized by poor patient survival rates. The glycoproteins Integrin αV (IαV), and the Urokinase-type plasminogen activator receptor (uPAR) are key contributors to tumor malignancy in GBM, and although their interaction is well-described, the role of glycans in this process has been scarcely evaluated. Better understanding this interaction could enhance our knowledge of the disease and lead to potential new therapeutics. METHODS We investigated the interaction between IαV and uPAR in human GBM, A172 and LN229, and low-grade glioma, SW1088, cell lines. Expression of these proteins was confirmed via confocal microscopy and co-immunoprecipitation. The role of N-glycosylation was evaluated using the inhibitor Swainsonine (SW) and glycosidase PNGase F. Glycoproteomic analysis by mass spectrometry identified glycosylation sites and differential structures on IαV. The impact of sialic acids and specific glycan structures was assessed using Neuraminidase (NeuA) and lectin binding assays. RESULTS The expression of IαV and uPAR, as well as their interaction, was confirmed in GBM cells but not in low-grade glioma cells, even when uPAR was overexpressed. SW and PNGase treatments markedly reduced IαV/uPAR interaction, highlighting the importance of N-glycosylation. Mass spectrometry analysis showed six glycosylation sites on IαV in GBM cells, with complex and hybrid N-glycans, while only oligomannose N-glycans were detected in low-grade glioma cells. NeuA treatment also reduced IαV/uPAR interaction, underscoring the role of sialic acids. Lectin assays suggested β1–6 branched glycans at specific sites are crucial for this interaction. Inhibition of N-glycosylation and sialic acid removal both decreased AKT phosphorylation, indicating a significant role of these glycans in integrin/uPAR signaling. CONCLUSIONS Our results demonstrate for the first time the interaction between IαV and uPAR in GBM cells, highlighting the critical role of N-glycosylation, particularly β1–6 branched glycans and sialic acids.