Global O-GlcNAcome Identifies O-GlcNAcylated Proteins Regulating Oligodendrocyte Precursor Cells Differentiation

Oligodendrocyte precursor cells (OPCs) differentiate into myelinating oligodendrocytes to form the myelin sheaths essential for neural function, a process frequently compromised during aging and in diseases like multiple sclerosis. However, the regulatory landscape of myelination remains incompletely understood. Here, we perform a comprehensive proteomic analysis of the O-GlcNAcome in postnatal day 0 mouse OPCs, identifying 165 O-GlcNAcylation sites across 118 proteins. These proteins are mostly nuclear and characterized by single-site modifications. Comparative analysis identifies 74 novel sites and 22 proteins uniquely O-GlcNAcylated in OPCs. Molecular docking suggestes that O-GlcNAcylation might act as a dynamic regulator of OPC differentiation. Notably, global O-GlcNAcylation levels increase during the OPC-to-oligodendrocyte transition. By integrating our findings with multiple sclerosis-related transcriptomic and proteomic datasets, we identify candidate proteins linking O-GlcNAc dysregulation to demyelination. Among these, Vimentin exhibits a pronounced age-dependent discrepancy between decreased protein levels and elevated mRNA expression during differentiation. We validate Threonine 35 and 63 as key O-GlcNAcylation sites on Vimentin. Functional assays confirm that blocking Vimentin O-GlcNAcylation enhances the OPC differentiation. Collectively, our study establishes the first O-GlcNAc landscape for OPCs and suggests O-GlcNAcylation as a pivotal regulator for OPC differentiation.