Attenuation of EGFR-Mediated Cellular Signaling by Sialidase-Mediated Desialylation.

Protein glycosylation and phosphorylation are fundamental protein modifications that coordinate cellular signaling. While receptor tyrosine kinases (RTKs) like the epidermal growth factor receptor (EGFR) are heavily glycosylated, the systems-level crosstalk between extracellular sialylation and intracellular phosphorylation dynamics remains poorly understood. Using enzymatic cell surface in-situ desialylation with sialidase, we investigated the signaling response to EGF stimulation and the synergistic effects of desialylation with the tyrosine kinase inhibitor (TKI) gefitinib using an integrated multi-omics pipeline to simultaneously characterize the global proteome, phosphoproteome, and N-glycoproteome. Our analysis revealed that cell surface desialylation significantly attenuates EGF-induced signaling, specifically suppressing over 200 phosphosites within the MAPK cascade and actin cytoskeleton organization modules. Comparative profiling demonstrated that sialidase treatment exerts a distinct regulatory program that is non-redundant with canonical TKI inhibition. Stoichiometric analysis confirmed that the depletion of sialylated N-glycoforms at specific EGFR residues (N413, N444) directly correlates with reduced phosphorylation at key activation sites (Y1197). Finally, an integrated glyco-phospho network analysis identified CD44, MET, and integrin signaling hubs as central nodes regulated by the sialylation. This study establishes cell surface sialylation as a critical rheostat for EGFR-mediated signaling. By bridging the gap between the extracellular glycoprotein and intracellular kinase networks, we identify glycan remodeling as a potent strategy to sensitize RTK-driven malignancies to therapy. Our findings provide a robust data foundation for developing glycoconjugate-targeted interventions and beyond.