Systematic Quantification of Protein O-GlcNAcylation Reveals Common and Cell-Type-Specific Responses to N-Glycosylation Inhibition in Human Cells

Both protein O-GlcNAcylation and N-glycosylation are extremely important in human cells and regulate many cellular events. While O-GlcNAcylation is known to act as a stress sensor, its changes in human cells with N-glycosylation perturbations remain to be explored. In this study, we comprehensively and site-specifically studied common and cell-type-specific responses of protein O-GlcNAcylation under N-glycosylation inhibition in three types of human cells (HEK293T, HepG2, and Jurkat cells) by integrating metabolic labeling, bio-orthogonal chemistry, and multiplexed proteomics. In total, more than 1000 O-GlcNAcylated proteins were identified and quantified, and the results demonstrate that under the inhibition of protein N-glycosylation, O-GlcNAcylated proteins related to stress response and translation are commonly changed in different types of cells. Furthermore, O-GlcNAcylation changes are cell-type-specific, and O-GlcNAcylated proteins related to leukocyte proliferation and T-cell activation were upregulated in Jurkat cells, while in HEK293T cells, those associated with ribonucleotide metabolism and ribosome biogenesis were upregulated. Site-specific analysis revealed that O-GlcNAcylation sites in structured regions exhibited larger abundance changes compared with those in intrinsically disordered regions. This study provides valuable insights into the regulation of protein O-GlcNAcylation in human cells under N-glycosylation inhibition, advancing our understanding of protein glycosylation.