Identification of O-GlcNAc proteins and glycosites using bioorthogonal ligation-tryptic cleavage strategy
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Haojie Lu
PROVIDER: PXD048703 | iProX | Thu Jan 18 00:00:00 GMT 2024
REPOSITORIES: iProX
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Project description:Protein O-GlcNAcylation, an extensive post-translational modification (PTM), has been proposed as a nutrient and stress sensor that regulates multiple essential cellular processes. Due to the low stoichiometry of O-GlcNAcylation and the high complexity of biological samples, comprehensive analysis of O-GlcNAcylation is an extraordinary challenge. In this work, we established a novel, simple, site-specific O-GlcNAc identification strategy by integrating bioorthogonal ligation and tryptic-cleavage，and applied it for O-GlcNAc proteomics analysis and applications. In this strategy, the capture step was based on click chemistry reaction between solid material and peptides, and the release step was completed by efficient tryptic cleavage. Benefiting from the simplified workflow and a tryptic-cleavable tag, this strategy would be a versatile tool for the proteomic analysis of O-GlcNAcylation to explore O-GlcNAcylation functions involving in cellular physiology and pathology disease.
2023-12-01 | PXD047478 |
Project description:O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins is an essential modification in physiology and pathology. Although O-GlcNAcylation is functionally critical, its analysis has never been an easy task. Despite the existence of a number of methods developed in the past years, which one(s) might have the best performance is largely unclear. To that end, we proposed to conduct rigorous comparison of several cleavable bioorthogonal biotin-alkyne probes which showed promise for sensitive O-GlcNAc proteomics. In brief, we developed chemoenzymatic labeling/click chemistry-based analytical workflows for O-GlcNAc proteomics, by utilizing four cleavable bioorthogonal probes including photocleavabe-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-biotin-alkyne); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-biotin-alkyne (Dde-biotin-alkyne), and diazobenzene-biotin-alkyne (Diazo-biotin-alkyne). The analytical performance of the probes was evaluated with synthetic O-GlcNAc peptides and then benchmarked by mouse brain lysates for O-GlcNAc proteomics. Besides providing valuable technical insights to O-GlcNAc proteomics methods, our work yielded an unprecedented O-GlcNAc proteome depth of mouse brain. In total, 2,906 O-GlcNAc sites were unambiguously assigned on 878 proteins. Among them, 1,611 sites were newly identified, including 138 O-GlcNAcylated tyrosine residues. Our work will not only help guide selection/development of O-GlcNAc proteomics methods for future studies but also provide an invaluable resource for functional elucidation of protein O-GlcNAcylation in brain biology as well as critical insights into tyrosine O-GlcNAcylation.
2025-10-20 | PXD063995 | Pride
Project description:Bioorthogonal ligation-tryptic cleavage strategy revealed distinct spatial O-GlcNAcylation patterns upon oxidative stress.
2024-01-24 | PXD048827 |
Project description:We applied the chemical reporter-based metabolic labeling method to acquire O-GlcNAc modified proteins chromatin loci. Human breast cancer cell line MCF-7, as well as the genotoxic stress (Adriamycin) adapted cells MCF-7/ADR, were fed with 1 mM GalNAz. Metabolic labeled O-GlcNAz chromatin were crosslinked, sonicated and enriched by bioorthogonal chemistry. Then, the genomic DNA fragments bounded by O-GlcNAc mark were de-crosslinked, and constructed into libraries following by next-generation sequencing (Chemoselective O-GlcNAc chromatin sequencing, COGC-seq). To verify the robustness of this chemical reporter-based metabolic labeling method, we compared the results in MCF-7 and MCF-7/ADR cells with classical lectin succinylated wheat germ agglutinin (sWGA) ChIP-seq strategy. We also analyzed gene expression MCF-7 and MCF-7/ADR cells by RNA-seq.
2020-06-01 | GSE141698 | GEO
Project description:We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation.
2017-07-18 | GSE84502 | GEO
Project description:Bioorthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogs of monosaccharides that contain bioorthogonal functionality, like azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bioorthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g. N-linked vs. O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat disappointing from the standpoint of a mucin-selective MCR, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.
2022-02-17 | PXD027333 | Pride
Project description:Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O- glcnAcylation) via overexpression of the O-glcnAc–regulating enzymes O- glcnAc transferase (OGT) or O- glcnAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O- glcnAcylation either by pharmacological or genetic manipulation also alters metabolic function. Sustained O-glcnAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-glcnAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-Seq in SH-SY5Y cells indicated transcriptome reprogramming and down regulation of the NRF2-mediated antioxidant response. Sustained O-glcnAcylation in mice brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-glcnAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-glcnAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.
2017-07-20 | GSE101642 | GEO
Project description:Tissue fibrosis is induced by chronic inflammation accompanied by repeated injuries. Chronic inflammation alters fibroblasts and stellate cells, maintaining the tissues in myofibroblasts and activating stellate cells. This activation promotes the augmentation of these cells and the abundant production of extracellular matrices, such as collagen. Fibrotic tissue dysfunction is eventually induced by expelling parenchymal cells with these abundant collagen depositions. To recover from tissue fibrosis, the activation of myofibroblasts and stellate cells is suppressed by selectively targeting these cells. Here we show that O-N-acetylglucosamine (GlcNAc)-modified proteins leaked from dead cells and GlcNAc-bearing polymers mimicking the GlcNAc moiety of these proteins suppress the activation of these cells; administration of the GlcNAc-bearing polymer into carbon tetrachloride-treated mice can improve liver fibrosis. We found that a multivalent GlcNAc moiety structure elicited antifibrotic activities in myofibroblasts by interacting with cell surface vimentin and desmin. Therefore, since these cells highly express cytoskeletal proteins, vimentin and desmin, on the cell surface and have GlcNAc-binding activity, we suggest that O-GlcNAc-modified proteins leaked from dead cells can function as suppressors of fibrosis and hyperplasia in spontaneous recovery. Moreover, we anticipate that GlcNAc-bearing polymers mimicked by O-GlcNAc-modified proteins will be useful as new therapeutic tools for fibrosis and hyperplasia.
2022-03-30 | GSE199656 | GEO
Project description:Variants in the human β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) gene give rise to an intellectual disability (ID) syndrome termed OGT congenital disorder of glycosylation (OGT-CDG). The mechanisms by which loss of OGT and/or protein O-GlcNAcylation lead to this syndrome are not understood, but symptoms associated with the syndrome suggest a developmental origin. Here, we establish and characterise two lines of mouse embryonic stem cells carrying different patient mutations and show that these mutations lead to disrupted O-GlcNAc homeostasis. Using quantitative proteomics on these cells in the pluripotent state, we identify candidate proteins/pathways that could underpin this syndrome. In addition to the increased levels of OGT and decreased levels of OGA reflecting disrupted O-GlcNAc homeostasis, we find that expression of the ID gene Zscan4 is upregulated. This is associated with increased levels of the OGT:Ten Eleven (Tet) - protein complex that regulates DNA methylation and Zscan4 expression. These data uncover a potential mechanism contributing to the developmental aspects of OGT-CDG.
2025-10-03 | PXD064425 | Pride
Project description:Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analog of the HS constituent GlcNAc and studied the compoundM-bM-^@M-^Ys metabolic fate and its effect on angiogenesis. The 4-deoxy analog was activated intracellularly into UDP-4-deoxy-GlcNAc and HS expression was inhibited up to ~96% (IC50 = 16 M-BM-5M). HS chain size was reduced, without detectable incorporation of the 4-deoxy analog, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Micro-injection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis which hampers pro-angiogenic signaling and neo-vessel formation. 9 samples were analyzed: 3 biological replicates from untreated SKOV3 cells, 3 biological replicates from SKOV3 cells treated with peracetylated GlcNAc, 3 biological replicates from SKOV3 cells treated with peracetylated 4-deoxy-GlcNAc
2013-08-23 | E-GEOD-48284 | biostudies-arrayexpress