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

Abstract

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 Nazidoacetylglucosamine (GlcNAz) and N-azidoacetylgalactosamine (GalNAz) can metabolically label O-GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP-galactose 4epimerases 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 coexpression 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-GlcNAcmodified. 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.

SECRET AGENT O-GlcNAc Modifies GIGANTEA: Methods for Mapping of O-Linked β-N-Acetylglucosamine Modification Sites Using Mass Spectrometry

“…We have previously identified several proteins including RGA, TCP proteins and the coat protein of plum pox virus that are also modified in the E. coli co-expression system as well as plants (Kim et al, 2011;Scott et al, 2006;Steiner et al, 2012) suggesting that GI is also O-GlcNAc modified in plants. Although O-GlcNAc modification of GI was not identified in several recently reported global analyses of proteins modified in plants (Li et al, 2023;Wu et al, 2022;Xu et al, 2017), GI protein abundance is subject to circadian fluctuations, being most abundant in evening (Krahmer et al, 2019) and thus could have been missed in these global analyses.…”

“…sec spy double mutants are embryo lethal suggesting that SEC and SPY have overlapping functions (Hartweck et al, 2002). Recent surveys have identified numerous O-GlcNAc and O-fucose modified plant protein (Bi et al, 2023;Li et al, 2023;Wu et al, 2022;Xu et al, 2017;Zentella et al, 2023).…”

SECRET AGENT O-GlcNAcylates hundreds of proteins involved in diverse cellular processes in Arabidopsis

“…Alternative approaches using click chemistry have been employed to detect O-GlcNAc targets in Arabidopsis seedlings (18). By supplementing GalNAz and GlcNAz, metabolic glycan labeling was induced in Arabidopsis seedlings.…”

“…Our comparative analysis revealed only 68 overlaps in seedling datasets and 75 in overall datasets. Notably, click chemistry enriches targets predominantly associated with cellular amide metabolic processes (18).…”

“…Comparatively, although over 3000 and 1430 O-GlcNAc targets have been identified in humans and mice, respectively (15,16), the database for O-GlcNAc targets in plants remains limited (17)(18)(19)(20). Additionally, given the overlapping or even opposite functions of O-GlcNAcylation and O-fucosylation, identifying more common targets would help in understanding their roles.…”