O-GlcNAcylation in mitochondrial protein in rat astrocytes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ
ORGANISM(S): Rattus Norvegicus (rat)
TISSUE(S): Brain, Fibroblast
DISEASE(S): Disease Free
SUBMITTER: 
Kazuhide Hayakawa
LAB HEAD: Kazuhide Hayakawa
PROVIDER: PXD045316 | Pride | 2023-10-20
REPOSITORIES: Pride
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2012-07-05 | E-GEOD-36322 | biostudies-arrayexpress
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Project description:O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous posttranslational 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 labeling strategy (MGL) 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 1,591 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 a serine/threonine-enriched peptide, causing ambiguous site assignment. The identified O-GlcNAcylated proteins are involved in multiple biological processes such as transcription, translation and plant hormone signaling. 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.
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Project description:We have found that histone H2B is GlcNAcylated at residue S112 by O-GlcNAc transferase and that H2B S112 GlcNAcylation fluctuates in response to extracellular glucose level. We have also found that H2B S112 GlcAcylation promotes H2B K120 ubiquitination. To investigate whether these histone modification correlate to transcriptional activation, we performed comprehensive gene expression analysis using Affymetrix GeneChip in HeLa cell cultured with different conditions, i.e. without glucose, with glucose and with FBS. HeLa cells were cultured in DMEM with the following three conditions, 1) DMEM without glucose for 24 hours, 2) DMEM without glucose for 24 hours and followed by treatment with 4.5 g/L glucose for another 24 hours, 3) normal culture condition (DMEM with FBS). Total RNA was purified from these cells and each RNA was linearly amplified and hybridized to Affymetrix GeneChip.
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