Zhang N, Meng Y, Mao S, Ni H, Huang C, Shen L, Fu K, Lv L, Yu C, Meekrathok P, Kuang C, Chen F, Zhang Y, Yuan K.
FBXO31-mediated ubiquitination of OGT maintains O-GlcNAcylation homeostasis to restrain endometrial malignancy.
Nature communications2025
16(1)
39894887
Abstract: Protein O-GlcNAcylation is a post-translational modification coupled to cellular metabolic plasticity. Aberrant O-GlcNAcylation has been observed in many cancers including endometrial cancer (EC), a common malignancy in women. However, clinical characterization of dysregulated O-GlcNAcylation homeostasis in EC and interrogating its molecular mechanism remain incomplete. Here we report that O-GlcNAcylation level is positively correlated with EC histologic grade in a Chinese cohort containing 219 tumors, validated in The Cancer Genome Atlas dataset. Increasing O-GlcNAcylation in patient-derived endometrial epithelial organoids promotes proliferation and stem-like cell properties, whereas decreasing O-GlcNAcylation limits the growth of endometrial cancer organoids. CRISPR screen and biochemical characterization reveal that tumor suppressor F-box only protein 31 (FBXO31) regulates O-GlcNAcylation homeostasis in EC by ubiquitinating the O-GlcNAc transferase OGT. Downregulation of O-GlcNAcylation impedes EC tumor formation in mouse models. Collectively, our study highlights O-GlcNAcylation as a useful stratification marker and a therapeutic vulnerability for the advanced, poorly differentiated EC cases.
Xu S, Yin K, Xu X, Fu L, Wu R.
O-GlcNAcylation reduces proteome solubility and regulates the formation of biomolecular condensates in human cells.
Nature communications2025
16(1)
40307207
Abstract: O-GlcNAcylation plays critical roles in the regulation of protein functions and cellular activities, including protein interactions with other macromolecules. While the formation of biomolecular condensates (or biocondensates) regulated by O-GlcNAcylation in a few individual proteins has been reported, systematic investigation of O-GlcNAcylation on the regulation of biocondensate formation remains to be explored. Here we systematically study the roles of O-GlcNAcylation in regulating protein solubility and its impacts on RNA-protein condensates using mass spectrometry-based chemoproteomics. Unexpectedly, we observe a system-wide decrease in the solubility of proteins modified by O-GlcNAcylation, with glycoproteins involved in focal adhesion and actin binding exhibiting the most significant decrease. Furthermore, O-GlcNAcylation sites located in disordered regions and with fewer acidic and aromatic residues nearby are related to a greater drop in protein solubility. Additionally, we discover that a specific group of O-GlcNAcylation events promotes the dissociation of RNA-protein condensates under heat stress, while some enhance the formation of RNA-protein condensates during the recovery phase. Using site mutagenesis, inhibition of O-GlcNAc transferase, and fluorescence microscopy, we validate that O-GlcNAcylation regulates the formation of biocondensates for YTHDF3 and NUFIP2. This work advances our understanding of the functions of protein O-GlcNAcylation and its roles in the formation of biomolecular condensates.
Oh SC, Jeon BC, Jang IH, Song MR, Hwang H, An D, Yue L, Jung Y, Lee Y, Jo S, Park SK, Kim TD.
Genetic manipulation of OGT enhances NK cell-mediated cytotoxicity in tumor immunity.
Journal of advanced research202540914422
Abstract: Natural killer (NK) cells are essential effectors in immune surveillance and cancer immunotherapy, but their function is often compromised by metabolic stress and environmental factors within the tumor microenvironment (TME). O-GlcNAcylation, a post-translational modification, regulates immune responses, yet its impact on NK cell function and therapeutic potential in immune cell-based therapies remains underexplored.
Luna S, Malard F, Pereckas M, Aoki M, Aoki K, Olivier-Van Stichelen S.
Studying the O-GlcNAcome of human placentas using banked tissue samples.
Glycobiology2024
34(4)
38253038
Abstract: O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.
Vang S, Helton ES, Guo Y, Burpee B, Rose E, Easter M, Bollenbecker S, Hirsch MJ, Matthews EL, Jones LI, Howze PH 4th, Rajasekaran V, Denson R, Cochran P, Attah IK, Olson H, Clair G, Melkani G, Krick S, Barnes JW.
O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.
Frontiers in immunology2024
15
38665916
Abstract: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis.
Hou C, Wu C, Wu Z, Cheng Y, Li W, Sun H, Ma J.
Systematic Evaluation of Affinity Enrichment Methods for O-GlcNAc Proteomics.
Journal of proteome research202439302247
Abstract: O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins plays critical roles in the regulation of diverse biological processes. However, protein O-GlcNAcylation analysis, especially at a large scale, has been a challenge. So far, a number of enrichment materials and methods have been developed for site-specific O-GlcNAc proteomics in different biological settings. Despite the presence of multiple methods, their performance for the O-GlcNAc proteomics is largely unclear. In this work, by using the lysates of PANC-1 cells (a pancreatic cancer cell line), we provided a head-to-head comparison of three affinity enrichment methods and materials (i.e., antibody, lectin AANL6, and an OGA mutant) for site-specific O-GlcNAc proteomics. The enriched peptides were analyzed by HCD product-dependent EThcD (i.e., HCD-pd-EThcD) mass spectrometry. The resulting data files were processed by three different data analysis packages (i.e., Sequest HT, Byonic, and FragPipe). Our data suggest that each method captures a subpopulation of the O-GlcNAc proteins. Besides the enrichment methods, we also observe complementarity between the different data analysis tools. Thus, combining different approaches holds promise for enhanced coverage of O-GlcNAc proteomics.
Hou C, Deng J, Wu C, Zhang J, Byers S, Moremen KW, Pei H, Ma J.
Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins.
Proceedings of the National Academy of Sciences of the United States of America2024
121(47)
39531497
Abstract: As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.
Luo Y, Wang Y, Tian Y, Zhou H, Wen L.
"Two Birds One Stone" Strategy for the Site-Specific Analysis of Core Fucosylation and O-GlcNAcylation.
Journal of the American Chemical Society2023
145(29)
37340703
Abstract: Core fucosylation and O-GlcNAcylation are the two most famous protein glycosylation modifications that regulate diverse physiological and pathological processes in living organisms. Here, a "two birds one stone" strategy has been described for the site-specific analysis of core fucosylation and O-GlcNAcylation. Taking advantage of two mutant endoglycosidases (EndoF3-D165A and EndoCC-N180H), which efficiently and specifically recognize core fucose and O-GlcNAc, glycopeptides can be labeled using a biantennary N-glycan probe bearing azido and oxazoline groups. Then, a temperature-sensitive poly(N-isopropylacrylamide) polymer functionalized with dibenzocyclooctyne was introduced to facilitate the enrichment of the labeled glycopeptides from the complex mixture. The captured glycopeptides can be further released enzymatically by wild-type endoglycosidases (EndoF3 and EndoCC) in a traceless manner for mass spectrometry (MS) analysis. The described strategy allows simultaneous profiling of core-fucosylated glycoproteome and O-GlcNAcylated glycoproteome from one complex sample by MS technology and searching the database using different variable modifications.
He J, Fan Z, Tian Y, Yang W, Zhou Y, Zhu Q, Zhang W, Qin W, Yi W.
Spatiotemporal Activation of Protein O-GlcNAcylation in Living Cells.
Journal of the American Chemical Society2022
144(10)
35138101
Abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a prevalent protein modification that plays fundamental roles in both cell physiology and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase (OGT). The study of protein O-GlcNAc function is limited by the lack of tools to control OGT activity with spatiotemporal resolution in cells. Here, we report light control of OGT activity in cells by replacing a catalytically essential lysine residue with a genetically encoded photocaged lysine. This enables the expression of a transiently inactivated form of OGT, which can be rapidly reactivated by photo-decaging. We demonstrate the activation of OGT activity by monitoring the time-dependent increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based quantitative proteomics. We further apply this activation strategy to control the morphological contraction of fibroblasts. Furthermore, we achieved spatial activation of OGT activity predominantly in the cytosol. Thus, our approach provides a valuable chemical tool to control cellular O-GlcNAc with much needed spatiotemporal precision, which aids in a better understanding of O-GlcNAc function.
Liu J, Hao Y, Wang C, Jin Y, Yang Y, Gu J, Chen X.
An Optimized Isotopic Photocleavable Tagging Strategy for Site-Specific and Quantitative Profiling of Protein O-GlcNAcylation in Colorectal Cancer Metastasis.
ACS chemical biology2022
17(3)
35254053
Abstract: O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation is a ubiquitous protein post-translational modification of the emerging importance in metazoans. Of the thousands of O-GlcNAcylated proteins identified, many carry multiple modification sites with varied stoichiometry. To better match the scale of O-GlcNAc sites and their dynamic nature, we herein report an optimized strategy, termed isotopic photocleavable tagging for O-GlcNAc profiling (isoPTOP), which enables quantitative and site-specific profiling of O-GlcNAcylation with excellent specificity and sensitivity. In HeLa cells, ∼1500 O-GlcNAcylation sites were identified with the optimized procedures, which led to quantification of ∼1000 O-GlcNAcylation sites with isoPTOP. Furthermore, we apply isoPTOP to probe the O-GlcNAcylation dynamics in a pair of colorectal cancer (CRC) cell lines, SW480 and SW620 cells, which represent primary carcinoma and metastatic cells, representatively. The stoichiometric differences of 625 O-GlcNAcylation sites are quantified. Of these quantified sites, many occur on important regulators involved in tumor progression and metastasis. Our results provide a valuable database for understanding the functional role of O-GlcNAc in CRC. IsoPTOP should be applicable for investigating O-GlcNAcylation dynamics in various pathophysiological processes.
Chen Y, Tang F, Qin H, Yue X, Nie Y, Huang W, Ye M.
Endo-M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O-GlcNAcylation Analysis.
Angewandte Chemie (International ed. in English)2022
61(23)
35289036
Abstract: To selectively enrich O-linked β-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.
Xie X, Wu Q, Zhang K, Liu Y, Zhang N, Chen Q, Wang L, Li W, Zhang J, Liu Y.
O-GlcNAc modification regulates MTA1 transcriptional activity during breast cancer cell genotoxic adaptation.
Biochimica et biophysica acta. General subjects2021
1865(8)
34019948
Abstract: Chromatin modifier metastasis-associated protein 1 (MTA1), closely associated with tumor angiogenesis in breast cancer, plays an important role in gene expression and cancer cell behavior. Recently, an association between O-GlcNAc transferase (OGT) and MTA1 was identified by mass spectroscopy. However, the potential relationship between MTA1 and O-GlcNAc modification has not yet explored.
Ramirez DH, Aonbangkhen C, Wu HY, Naftaly JA, Tang S, O'Meara TR, Woo CM.
Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.
ACS chemical biology2020
15(4)
32119511
Abstract: O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.
Zhu Y, Willems LI, Salas D, Cecioni S, Wu WB, Foster LJ, Vocadlo DJ.
Tandem Bioorthogonal Labeling Uncovers Endogenous Cotranslationally O-GlcNAc Modified Nascent Proteins.
Journal of the American Chemical Society2020
142(37)
32870666
Abstract: Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide N-acetylglucosamine (GlcNAc). This modification, known as O-GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O-GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O-GlcNAc modification can occur cotranslationally and that this process prevents premature degradation of such nascent polypeptide chains. Here, we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O-propargyl-puromycin (OPP) and target the specific subset of nascent chains that are cotranslationally glycosylated with O-GlcNAc by metabolic saccharide engineering using tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-d-galactopyranose (Ac4GalNAz). Using various combinations of sequential chemoselective ligation strategies, we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous cotranslationally O-GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their cotranslational O-GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O-GlcNAc and other cotranslational protein modifications and should stimulate an improved understanding of the role of O-GlcNAc in cytoplasmic protein quality control and proteostasis.
Liu Y, Chen Q, Zhang N, Zhang K, Dou T, Cao Y, Liu Y, Li K, Hao X, Xie X, Li W, Ren Y, Zhang J.
Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response.
Nature communications2020
11(1)
33214551
Abstract: O-GlcNAc modification plays critical roles in regulating the stress response program and cellular homeostasis. However, systematic and multi-omics studies on the O-GlcNAc regulated mechanism have been limited. Here, comprehensive data are obtained by a chemical reporter-based method to survey O-GlcNAc function in human breast cancer cells stimulated with the genotoxic agent adriamycin. We identify 875 genotoxic stress-induced O-GlcNAc chromatin-associated proteins (OCPs), including 88 O-GlcNAc chromatin-associated transcription factors and cofactors (OCTFs), subsequently map their genomic loci, and construct a comprehensive transcriptional reprogramming network. Notably, genotoxicity-induced O-GlcNAc enhances the genome-wide interactions of OCPs with chromatin. The dynamic binding switch of hundreds of OCPs from enhancers to promoters is identified as a crucial feature in the specific transcriptional activation of genes involved in the adaptation of cancer cells to genotoxic stress. The OCTF nuclear factor erythroid 2-related factor-1 (NRF1) is found to be a key response regulator in O-GlcNAc-modulated cellular homeostasis. These results provide a valuable clue suggesting that OCPs act as stress sensors by regulating the expression of various genes to protect cancer cells from genotoxic stress.
Li J, Li Z, Duan X, Qin K, Dang L, Sun S, Cai L, Hsieh-Wilson LC, Wu L, Yi W.
An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O-GlcNAcylation.
ACS chemical biology2019
14(1)
30620550
Abstract: O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.
Hao Y, Fan X, Shi Y, Zhang C, Sun DE, Qin K, Qin W, Zhou W, Chen X.
Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells.
Nature communications2019
10(1)
31492838
Abstract: Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.
Song H, Ma J, Bian Z, Chen S, Zhu J, Wang J, Huang N, Yin M, Sun F, Xu M, Pan Q.
Global profiling of O-GlcNAcylated and/or phosphorylated proteins in hepatoblastoma.
Signal transduction and targeted therapy2019
4
31637018
Abstract: O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) and phosphorylation are critical posttranslational modifications that are involved in regulating the functions of proteins involved in tumorigenesis and the development of various solid tumors. However, a detailed characterization of the patterns of these modifications at the peptide or protein level in hepatoblastoma (HB), a highly malignant primary hepatic tumor with an extremely low incidence in children, has not been performed. Here, we examined O-GlcNAc-modified or phospho-modified peptides and proteins in HB through quantitative proteomic analysis of HB tissues and paired normal liver tissues. Our results identified 114 O-GlcNAcylated peptides belonging to 78 proteins and 3494 phosphorylated peptides in 2088 proteins. Interestingly, 41 proteins were modified by both O-GlcNAcylation and phosphorylation. These proteins are involved in multiple molecular and cellular processes, including chromatin remodeling, transcription, translation, transportation, and organelle organization. In addition, we verified the accuracy of the proteomics results and found a competitive inhibitory effect between O-GlcNAcylation and phosphorylation of HSPB1. Further, O-GlcNAcylation modification of HSPB1 promoted proliferation and enhanced the chemotherapeutic resistance of HB cell lines in vitro. Collectively, our research suggests that O-GlcNAc-modified and/or phospho-modified proteins may play a crucial role in the pathogenesis of HB.
Woo CM, Lund PJ, Huang AC, Davis MM, Bertozzi CR, Pitteri SJ.
Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag).
Molecular & cellular proteomics : MCP2018
17(4)
29351928
Abstract: Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.
Huo B, Zhang W, Zhao X, Dong H, Yu Y, Wang J, Qian X, Qin W.
A triarylphosphine-trimethylpiperidine reagent for the one-step derivatization and enrichment of protein post-translational modifications and identification by mass spectrometry.
Chemical communications (Cambridge, England)2018
54(98)
30379171
Abstract: We report a new reagent that is capable of both chemical derivatization and selective enrichment of azide-labeled PTM peptides for sensitive identification by mass spectrometry (MS). Facile sample recovery, enhanced ionization and fragmentation in MS of the enriched PTM peptides are achieved, which leads to the identification of 3293 O-GlcNAc peptides and the location of 1706 sites in HeLa cells and efficiently expands the current mapping scale.
Wang S, Yang F, Petyuk VA, Shukla AK, Monroe ME, Gritsenko MA, Rodland KD, Smith RD, Qian WJ, Gong CX, Liu T.
Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease.
The Journal of pathology2017
243(1)
28657654
Hahne H, Sobotzki N, Nyberg T, Helm D, Borodkin VS, van Aalten DM, Agnew B, Kuster B.
Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry.
Journal of proteome research2013
12(2)
23301498
Abstract: The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.
Hahne H, Gholami AM, Kuster B.
Discovery of O-GlcNAc-modified proteins in published large-scale proteome data.
Molecular & cellular proteomics : MCP2012
11(10)
22661428
Abstract: The attachment of N-acetylglucosamine to serine or threonine residues (O-GlcNAc) is a post-translational modification on nuclear and cytoplasmic proteins with emerging roles in numerous cellular processes, such as signal transduction, transcription, and translation. It is further presumed that O-GlcNAc can exhibit a site-specific, dynamic and possibly functional interplay with phosphorylation. O-GlcNAc proteins are commonly identified by tandem mass spectrometry following some form of biochemical enrichment. In the present study, we assessed if, and to which extent, O-GlcNAc-modified proteins can be discovered from existing large-scale proteome data sets. To this end, we conceived a straightforward O-GlcNAc identification strategy based on our recently developed Oscore software that automatically analyzes tandem mass spectra for the presence and intensity of O-GlcNAc diagnostic fragment ions. Using the Oscore, we discovered hundreds of O-GlcNAc peptides not initially identified in these studies, and most of which have not been described before. Merely re-searching this data extended the number of known O-GlcNAc proteins by almost 100 suggesting that this modification exists even more widely than previously anticipated and the modification is often sufficiently abundant to be detected without enrichment. However, a comparison of O-GlcNAc and phospho-identifications from the very same data indicates that the O-GlcNAc modification is considerably less abundant than phosphorylation. The discovery of numerous doubly modified peptides (i.e. peptides with one or multiple O-GlcNAc or phosphate moieties), suggests that O-GlcNAc and phosphorylation are not necessarily mutually exclusive, but can occur simultaneously at adjacent sites.