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.