Chemoproteomic profiling of O‐GlcNAcylated proteins and identification of O‐GlcNAc transferases in rice

Abstract

Summary O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) is a ubiquitous post‐translation modification occurring in both animals and plants. Thousands of proteins along with their O‐GlcNAcylation sites have been identified in various animal systems, yet the O‐GlcNAcylated proteomes in plants remain poorly understood. Here, we report a large‐scale profiling of protein O‐GlcNAcylation in a site‐specific manner in rice. We first established the metabolic glycan labelling (MGL) strategy with N‐azidoacetylgalactosamine (GalNAz) in rice seedlings, which enabled incorporation of azides as a bioorthogonal handle into O‐GlcNAc. By conjugation of the azide‐incorporated O‐GlcNAc with alkyne‐biotin containing a cleavable linker via click chemistry, O‐GlcNAcylated proteins were selectively enriched for mass spectrometry (MS) analysis. A total of 1591 unambiguous O‐GlcNAcylation sites distributed on 709 O‐GlcNAcylated proteins were identified. Additionally, 102 O‐GlcNAcylated proteins were identified with their O‐GlcNAcylation sites located within serine/threonine‐enriched peptides, causing ambiguous site assignment. The identified O‐GlcNAcylated proteins are involved in multiple biological processes, such as transcription, translation and plant hormone signalling. Furthermore, we discovered two O‐GlcNAc transferases (OsOGTs) in rice. By expressing OsOGTs in Escherichia coli and Nicotiana benthamiana leaves, we confirmed their OGT enzymatic activities and used them to validate the identified rice O‐GlcNAcylated proteins. Our dataset provides a valuable resource for studying O‐GlcNAc biology in rice, and the MGL method should facilitate the identification of O‐GlcNAcylated proteins in various plants.

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).…”

Maresin-2 Fine-tunes ULK1 O-GlcNAcylation to Improve Post Myocardial Infarction Remodeling

“…Prior experiments have validated that O-GlcNAc transferase (OGT) is a highly conservative enzyme whose expression was not altered in a dynamic and reversible manner 34 . We cultured HEK293T cells with different concentration maresin-2; the GFAT1 expression level were enhanced in gradualness while the expression level of OGT, OGA, GLUT1, GLUT4 were not in statistically altered (Fig6.A&B).…”

E. coli-expressed SECRET AGENT O-GlcNAc modifies threonine 829 of GIGANTEA

“…sec spy double mutants are embryo-lethal, suggesting that SEC and SPY have overlapping functions ( Hartweck et al., 2002 ). Recent surveys have identified a number of O -GlcNAc- and O -fucose-modified plant proteins ( Xu et al., 2017 ; Wu et al., 2022 ; Bi et al., 2023 ; Li et al., 2023 ; Zentella et al., 2023 ). Consistent with SEC and SPY having overlapping functions, O -GlcNAc and O -fucose modifications can occur at the same position ( Xu et al., 2017 ; Bi et al., 2023 ).…”