Comprehensive and Site-Specific Characterization of Protein N-Glycosylation in AD Samples Reveals Its Potential Roles in Protein Aggregation and Synaptic Dysfunction

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Emerging evidence strongly suggests that protein glycosylation is strongly related to this disease. However, the extent and functional consequences of site-specific N-glycosylation alterations in AD remain to be further explored. Here, we employed a dendrimer boronic acid (DBA)-based enrichment strategy combined with multiplexed proteomics to systematically analyze protein N-glycosylation in post-mortem human brain tissues. We identified 3,105 N-glycosylation sites on 1,299 glycoproteins from nine AD cases and nine healthy controls, and performed a systematic and site-specific investigation of glycosylation alterations in AD. Glycoproteins involved in cholesterol efflux were upregulated, whereas those associated with chemical synaptic transmission and ion transport were significantly downregulated in AD compared to control brain samples. We observed widespread dysregulation of N-glycosylation across multiple protein domains, particularly in the ConA-like lectins/glucanases and Zn-dependent exopeptidases domains. Notably, we identified 161 N-glycosylation sites located within aggregation-prone regions (APRs), and reduced glycosylation at APRs on plaque-associated glycoproteins may be associated with protein aggregation and plaque formation. Additionally, downregulated N-glycosylation sites were enriched in synaptic membrane proteins, such as Ca²⁺ ion channels, GABA-gated chloride channels, and glutamate receptors, implicating glycosylation loss in synaptic dysfunction. Our findings suggest that the loss of N-glycosylation may contribute to the pathogenesis of AD through impairing synaptic transmission and promoting protein aggregation. This study provides novel insights into glycosylation-dependent mechanisms of neurodegeneration, highlighting N-glycosylation as a potential therapeutic target for AD treatment.