Chemoproteomic Profiling of O-GlcNAcylation in Arabidopsis Thaliana by Using Metabolic Glycan Labeling

Protein O-GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O-GlcNAcylated proteins have been identified in animals, the plant O-GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O-GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both N-azidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz) can metabolically label O-GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP-galactose 4-epimerases were found to interconvert UDP-GalNAz and UDP-GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide-incorporated O-GlcNAc with alkyne-biotin via click chemistry, the O-GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O-GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O-GlcNAcylated proteins were enriched in various plant-specific processes such as hormone responses. By co-expression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O-GlcNAc transferase, we validated that the MGL-identified proteins were O-GlcNAc-modified. Our work establishes a powerful tool for profiling plant O-GlcNAylation and provides an invaluable resource for investigating the functional role of O-GlcNAc in Arabidopsis.