Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis
Wednesday, 2017/02/22 | 07:56:58
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Shou-Ling Xu, Robert J. Chalkley, Jason C. Maynard, Wenfei Wang, Weimin Ni, Xiaoyue Jiang, Kihye Shin, Ling Cheng, Dasha Savage, Andreas F. R. Hühmer, Alma L. Burlingame, and Zhi-Yong Wang SignificanceStudies in mammalian systems have shown important functions of O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) in a wide range of cellular, physiological, and disease processes. Genetic evidence indicates that O-GlcNAcylation is essential for plant growth and development. However, very few O-GlcNAc–modified proteins have been identified in plants. Here, we report identification of 262 O-GlcNAc–modified proteins in Arabidopsis, revealing both conserved and distinct functions of O-GlcNAc modification in plants. This study uncovers potentially important functions of O-GlcNAcylation in many cellular and developmental pathways and also provides a large number of modification sites for further genetic and molecular dissection of these specific functions. Our study provides the framework of an O-GlcNAc modification network underlying plant growth and development. AbstractGenetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc–modified proteins and sites in the model plant Arabidopsis thaliana. Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc–modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.
See: http://www.pnas.org/content/114/8/E1536.full PNAS February 21 2017; vol.114; no.8: E1536–E1543
Fig. 1. Combined analysis for O-GlcNAcylation and phosphorylation from Arabidopsis inflorescence tissues. (A). Flowchart for the serial enrichment and analysis of in vivo phosphorylated and O-GlcNAc–modified peptides. (B) The UV trace of absorbance at 280 nm of three sequential lectin weak affinity chromatography (LWAC) separations of tryptic digest of proteins extracted from Arabidopsis inflorescence tissues, showing enrichment of O-GlcNAc–modified peptides through their retardation on the column (arrows). In each case, an aliquot of GlcNAc (eluting at 4 mL) was injected to elute any complex glycans. Peptides were collected as a single fraction starting at 1.3 mL, desalted, and rerun for a total of three rounds.
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