Sialidase-Mediated Desialylation Regulating EGFR Phosphorylation and Signal Flux

Protein glycosylation and phosphorylation are fundamental post-translational modifications (PTMs) 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. We employed an integrated TMT18-labeled multi-omics pipeline to simultaneously profile the global proteome, phosphoproteome, and N-glycoproteome of A498 cells. Using enzymatic in-situ remodeling with sialidase, we investigated the signaling response to EGF stimulation and the synergistic effects of desialylation with the tyrosine kinase inhibitor (TKI) gefitinib. 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 flux. 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 in ccRCC and beyond.