N-glycoproteomic profiling reveals aberrant ITGB1 glycosylation in hyperoxia-induced bronchopulmonary dysplasia

Background Bronchopulmonary dysplasia (BPD) is a prevalent chronic lung disease in preterm infants, characterized by arrested alveolarization. Extracellular matrix (ECM) remodeling and receptor signaling are critical for alveolarization and are strictly regulated by protein N-glycosylation. However, the specific role of the vital post-translational modification in BPD pathogenesis remains unexplored. The present research aims to delineate a N-glycoproteomic landscape and elucidate the glycosylation-associated pathogenic mechanisms in a murine BPD model. Methods To establish BPD models, newborn mice were exposed to continuous hyperoxia (85% O 2 ) for 14 days. Lung developmental impairment was quantified using Radial Alveolar Count (RAC) and Mean Linear Intercept (MLI). A comprehensive dual-omics profiling strategy was conducted, utilizing an Orbitrap Exploris 480 platform for global proteomics and a timsTOF Pro mass spectrometer for N-glycoproteomics. Key molecular targets were further validated by lectin histochemistry, Western blot, and AlphaFold-based spatial structure modeling. Results Hyperoxia exposure successfully induced BPD-like lung injury, substantiated by a marked decline in RAC coupled with a concurrent elevation in MLI (P < 0.001). Conjoint omics analysis identified 612 N-glycoproteins and 1105 modification sites. After normalizing to global protein abundance, we revealed distinct glycosylation alterations, characterized by the identification of 16 upregulated and 100 downregulated modification sites. Furthermore, lectin histochemistry confirmed an overall reduction and redistribution of core-mannose N-glycans on the cell surface in BPD lungs. Bioinformatics analysis demonstrated that differentially glycosylated proteins were predominantly enriched in ECM-receptor interactions, integrin-mediated cell adhesion, and PI3K-Akt signaling. Subsequent Protein-Protein Interaction network analysis revealed Integrin beta-1 (ITGB1) as a crucial hub node; notably, we identified a marked downregulation of its site-specific N-glycosylation. Spatial modeling pinpointed a crucial, disease-associated aberrant glycosylation site at Asn212, located merely 17.3Å from the MIDAS binding pocket. This site-specific hypo-glycosylation is postulated to unmask the ITGB1 interface, potentially perturbing physiological ligand binding. Conclusion This study provides the first exhaustive N-glycoproteomic map of hyperoxia-induced BPD. Our results highlight dysregulated N-glycosylation, particularly the site-specific hypo-glycosylation of ITGB1 at Asn212. Based on computational predictions, it is hypothesized to severely disrupts the ECM-receptor networks and integrin affinity, which correlates with potentially driving alveolarization arrest. These insights provide a novel post-translational perspective on BPD pathogenesis and offer new avenues for future investigation into potential therapeutic targets.