Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair

Background: Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe^x) in airway epithelial repair. Objective: To examine whether an sLe^x decoration of EGFR can modulate receptor function during AEC repair. Methods: Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe ^x and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe^x was catalysed by a specific α-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe ^x in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe^x antibody (KM-93) was used. Results: We demonstrated a co-localization of sLe^x with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe^x after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe^x expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe^x and tyrosine-phosphorylated EGFR (pY⁸⁴⁵-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. Conclusion: The present data demonstrate that sLe^x, in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.