Jeon BC, Kim YJ, Park AK, Song MR, Na KM, Lee J, An D, Park Y, Hwang H, Kim TD, Lim J, Park SK.
Dynamic O-GlcNAcylation governs long-range chromatin interactions in V(D)J recombination during early B-cell development.
Cellular & molecular immunology2025
22(1)
39627609
Abstract: V(D)J recombination secures the production of functional immunoglobulin (Ig) genes and antibody diversity during the early stages of B-cell development through long-distance interactions mediated by cis-regulatory elements and trans-acting factors. O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins that regulates various protein functions, including DNA-binding affinity and protein-protein interactions. However, the effects of O-GlcNAcylation on proteins involved in V(D)J recombination remain largely unknown. To elucidate this relationship, we downregulated O-GlcNAcylation in a mouse model by administering an O-GlcNAc inhibitor or restricting the consumption of a regular diet. Interestingly, the inhibition of O-GlcNAcylation in mice severely impaired Ig heavy-chain (IgH) gene rearrangement. We identified several factors crucial for V(D)J recombination, including YY1, CTCF, SMC1, and SMC3, as direct targets of O-GlcNAc modification. Importantly, O-GlcNAcylation regulates the physical interaction between SMC1 and SMC3 and the DNA-binding patterns of YY1 at the IgH gene locus. Moreover, O-GlcNAc inhibition downregulated DDX5 protein expression, affecting the functional association of CTCF with its DNA-binding sites at the IgH locus. Our results showed that locus contraction and long-range interactions throughout the IgH locus are disrupted in a manner dependent on the cellular O-GlcNAc level. In this study, we established that V(D)J recombination relies on the O-GlcNAc status of stage-specific proteins during early B-cell development and identified O-GlcNAc-dependent mechanisms as new regulatory components for the development of a diverse antibody repertoire.
Jaiswal R, Liu Y, Petriello M, Zhang X, Yi Z, Fehl C.
A reference dataset of O-GlcNAc proteins in quadriceps skeletal muscle from mice.
Glycobiology2025
35(3)
39927985
Abstract: A key nutrient sensing process in all animal tissues is the dynamic attachment of O-linked N-acetylglucosamine (O-GlcNAc). Determining the targets and roles of O-GlcNAc glycoproteins has the potential to reveal insights into healthy and diseased metabolic states. In cell studies, thousands of proteins are known to be O-GlcNAcylated, but reference datasets for most tissue types in animals are lacking. Here, we apply a chemoenzymatic labeling study to compile a high coverage dataset of quadriceps skeletal muscle O-GlcNAc glycoproteins from mice. Our dataset contains over 550 proteins, and > 80% of the dataset matched known O-GlcNAc proteins. This dataset was further annotated via bioinformatics, revealing the distribution, protein interactions, and gene ontology (GO) functions of these skeletal muscle proteins. We compared these quadriceps glycoproteins with a high-coverage O-GlcNAc enrichment profile from mouse hearts and describe the key overlap and differences between these tissue types. Quadriceps muscles can be used for biopsies, so we envision this dataset to have potential biomedical relevance in detecting aberrant glycoproteins in metabolic diseases and physiological studies. This new knowledge adds to the growing collection of tissues with high-coverage O-GlcNAc profiles, which we anticipate will further the systems biology of O-GlcNAc mechanisms, functions, and roles in disease.
Hao Y, Li Z, Du X, Xie Q, Li D, Lei S, Guo Y.
Characterization and chemoproteomic profiling of protein O-GlcNAcylation in SOD1-G93A mouse model.
Molecular medicine (Cambridge, Mass.)2025
31(1)
40021952
Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to affect the processing of several important proteins implicated in ALS. However, the overall level and cellular localization of O-GlcNAc during ALS progression are incompletely understood, and large-scale profiling of O-GlcNAcylation sites in this context remains unexplored.
Jia L, Zheng H, Feng J, Ding Y, Sun X, Yu Y, Hao X, Wang J, Zhang X, Tian Y, Chen F, Cui J.
Upregulation of protein O-GlcNAcylation levels promotes zebrafish fin regeneration.
Molecular & cellular proteomics : MCP202540044042
Abstract: As one of the most important post-translational modifications, glycosylation participates in various cellular activities in organisms and is closely associated with many pathogeneses. It has been reported that glycosylation affects liver, spinal cord, and heart tissue regeneration. The zebrafish fin has become a valuable model due to its high regenerative capacity. The molecular mechanism of regeneration has been a hot research topic in the field for a long time. However, studies on the influence of glycosylation during limb regeneration in zebrafish are relatively scarce. We discovered that O-GlcNAc expression, identified by WGA, was elevated during the regeneration of the injured fin in zebrafish using lectin microarray. This phenomenon is due to the upregulation of the expression of OGT enzymes and elevated O-GlcNAcylation levels. To investigate the effects on the fin regeneration when O-GlcNAcylation changes, we used OSMI-1 or Alloxan unilateral microinjection to decrease O-GlcNAcylation and observed that it prevented the fin regeneration. Conversely, the O-GlcNAcylation was impressed by a unilateral microinjection of Thiamet-G or Glucose into the fin, leading to a stimulation of the fin regeneration. To further understand the role of O-GlcNAcylation in fin regeneration, LC-MS/MS was performed to identify O-GlcNAc-glycoproteins. The results demonstrated that the O-GlcNAc glycoproteins, such as THBS4 and HSPG, were involved in the regulation of zebrafish fin regeneration process and were closely associated with certain biological processes, such as stem cell differentiation, ECM-receptor interaction pathway, tissue remodeling, etc. We demonstrated that O-GlcNAc glycoproteins are crucial for zebrafish fin regeneration, during which OGT promotes the process by upregulating the O-GlcNAcylation levels in the zebrafish fin.
Rucli S, Descostes N, Ermakova Y, Chitnavis U, Couturier J, Boskovic A, Boulard M.
Functional genomic profiling of O-GlcNAc reveals its context-specific interplay with RNA polymerase II.
Genome biology2025
26(1)
40128797
Abstract: How reversible glycosylation of DNA-bound proteins acts on transcription remains scarcely understood. O-linked β-N-acetylglucosamine (O-GlcNAc) is the only known form of glycosylation modifying nuclear proteins, including RNA polymerase II (RNA Pol II) and many transcription factors. Yet, the regulatory function of the O-GlcNAc modification in mammalian chromatin remains unclear.
Le Minh G, Merzy J, Esquea EM, Ahmed NN, Young RG, Sharp RJ, Dhameliya TT, Agana B, Lee MH, Bethard JR, Comte-Walters S, Ball LE, Reginato MJ.
GATAD2B O-GlcNAcylation Regulates Breast Cancer Stem-like Potential and Drug Resistance.
Cells2025
14(6)
40136647
Abstract: The growth of breast tumors is driven and controlled by a subpopulation of cancer cells resembling adult stem cells, which are called cancer stem-like cells (CSCs). In breast cancer, the function and maintenance of CSCs are influenced by protein O-GlcNAcylation and the enzyme responsible for this post-translational modification, O-GlcNAc transferase (OGT). However, the mechanism of CSCs regulation by OGT and O-GlcNAc cycling in breast cancer is still unclear. Analysis of the proteome and O-GlcNAcome, revealed GATAD2B, a component of the Nucleosome Remodeling and Deacetylase (NuRD) complex, as a substrate regulated by OGT. Reducing GATAD2B genetically impairs mammosphere formation, decreases expression of self-renewal factors and CSCs population. O-GlcNAcylation of GATAD2B at the C-terminus protects GATAD2B from ubiquitination and proteasomal degradation in breast cancer cells. We identify ITCH as a novel E3 ligase for GATAD2B and show that targeting ITCH genetically increases GATAD2B levels and increases CSCs phenotypes. Lastly, we show that overexpression of wild-type GATAD2B, but not the mutant lacking C-terminal O-GlcNAc sites, promotes mammosphere formation, expression of CSCs factors and drug resistance. Together, we identify a key role of GATAD2B and ITCH in regulating CSCs in breast cancer and GATAD2B O-GlcNAcylation as a mechanism regulating breast cancer stem-like populations and promoting chemoresistance.
Li G, Meng F, Zhong X, Yu K, Zhang N, Zhang K, Huang H, Li W, Zhang J, Wang W, Ren Y, Liu Y.
DNA probe pulldown screening uncovers O-GlcNAcylation modulation of transcription factor DNA interactions.
Scientific reports2025
15(1)
40596516
Abstract: O-linked β-N-acetylglucosamine (O-GlcNAc), a critical post-translational modification predominantly found in the nucleus, plays a substantial role in regulating gene expression by modulating transcription factors (TFs) activity. However, quantitative analysis investigating the influence of O-GlcNAcylation on protein-DNA interactions at a proteome scale remains undone. Herein, a pulldown screening approach using a consensus TF response element (catTFRE) was employed to unravel the impact of fluctuating levels of O-GlcNAcylation on the DNA binding efficiency of endogenous TFs/co-factors. Utilizing quantitative proteomics, we identified a substantial enhancement in the binding capacity of 241 nuclear proteins (NPs) to DNA sequences due to elevated levels of O-GlcNAcylation, whereas a decrease in DNA binding was observed for 2 NPs concurrently. Intriguingly, the O-GlcNAcylation elevation significantly enhanced the binding of 146 TFs/co-factors to specific DNA sequences. We further established that the O-GlcNAcylation of several Forkhead family TFs, including FOXA1 and FOXC1, notably enhances their binding to specific DNA sequences in living cells. Our research presents an efficacious approach to assessing the impact of O-GlcNAcylation on the interactions between proteins and DNA. This significantly enhances our understanding of the role O-GlcNAcylation plays in the regulation of transcription.
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.
Wang H, Sun J, Sun H, Wang Y, Lin B, Wu L, Qin W, Zhu Q, Yi W.
The OGT-c-Myc-PDK2 axis rewires the TCA cycle and promotes colorectal tumor growth.
Cell death and differentiation202438778217
Abstract: Deregulated glucose metabolism termed the "Warburg effect" is a fundamental feature of cancers, including the colorectal cancer. This is typically characterized with an increased rate of glycolysis, and a concomitant reduced rate of the tricarboxylic acid (TCA) cycle metabolism as compared to the normal cells. How the TCA cycle is manipulated in cancer cells remains unknown. Here, we show that O-linked N-acetylglucosamine (O-GlcNAc) regulates the TCA cycle in colorectal cancer cells. Depletion of OGT, the sole transferase of O-GlcNAc, significantly increases the TCA cycle metabolism in colorectal cancer cells. Mechanistically, OGT-catalyzed O-GlcNAc modification of c-Myc at serine 415 (S415) increases c-Myc stability, which transcriptionally upregulates the expression of pyruvate dehydrogenase kinase 2 (PDK2). PDK2 phosphorylates pyruvate dehydrogenase (PDH) to inhibit the activity of mitochondrial pyruvate dehydrogenase complex, which reduces mitochondrial pyruvate metabolism, suppresses reactive oxygen species production, and promotes xenograft tumor growth. Furthermore, c-Myc S415 glycosylation levels positively correlate with PDK2 expression levels in clinical colorectal tumor tissues. This study highlights the OGT-c-Myc-PDK2 axis as a key mechanism linking oncoprotein activation with deregulated glucose metabolism in colorectal cancer.
Yu SB, Wang H, Sanchez RG, Carlson NM, Nguyen K, Zhang A, Papich ZD, Abushawish AA, Whiddon Z, Matysik W, Zhang J, Whisenant TC, Ghassemian M, Koberstein JN, Stewart ML, Myers SA, Pekkurnaz G.
Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.
Developmental cell202438843836
Abstract: Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
Moniaux N, Geoffre N, Darnaud M, Deshayes A, Dos Santos A, Job S, Lacoste C, Nguyen TS, Friedel-Arboleas M, Guettier C, Purhonen J, Kallijärvi J, Amouyal G, Amouyal P, Bréchot C, Vives R, Buendia MA, Issad T, Faivre J.
Tumor suppressive role of the antimicrobial lectin REG3A targeting the O-GlcNAc glycosylation pathway.
Hepatology (Baltimore, Md.)202438975812
Abstract: Antimicrobial proteins of the REG3 family provide a first line of protection against infections and transformed cells. Their expression is inducible by inflammation, which makes their role in cancer biology less clear, since an immune- inflammatory context may preexist or coexist with cancer, as occurs in hepatocellular carcinoma (HCC). The aim of this study is to clarify the role of REG3A in liver carcinogenesis and to determine whether carbohydrate-binding functions are involved.
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.
Jo S, Esch N, Nguyen A, Wong A, Mohan R, Kim C, Blandino-Rosano M, Bernal-Mizrachi E, Alejandro EU.
Loss of O-GlcNAcylation modulates mTORC1 and autophagy in β cells, driving diabetes 2 progression.
JCI insight2024
9(23)
39388284
Abstract: Type 2 diabetes (T2D) arises when pancreatic β cells fail to produce sufficient insulin to control blood glucose appropriately. Aberrant nutrient sensing by O-GlcNAcylation and mTORC1 is linked to T2D and the failure of insulin-producing β cells. However, the nature of their crosstalk in β cells remains unexplored. Recently, O-GlcNAcylation, a posttranslation modification controlled by enzymes O-GlcNAc transferase/O-GlcNAcase (OGT/OGA), emerged as a pivotal regulator for β cell health; deficiency in either enzyme causes β cell failure. The present study investigates the previously unidentified connection between nutrient sensor OGT and mTORC1 crosstalk to regulate β cell mass and function in vivo. We show reduced OGT and mTORC1 activity in islets of a preclinical β cell dysfunction model and islets from humans with obesity. Using loss or gain of function of OGT, we identified that O-GlcNAcylation positively regulated mTORC1 signaling in β cells. O-GlcNAcylation negatively modulated autophagy, as the removal of OGT increased autophagy, while the deletion of OGA decreased it. Increasing mTORC1 signaling, via deletion of TSC2, alleviated the diabetic phenotypes by increasing β cell mass but not β cell function in OGT-deficient mice. Downstream phospho-protein signaling analyses revealed diverging effects on MKK4 and calmodulin signaling between islets with OGT, TSC2, or combined deletion. These data provide evidence of OGT's significance as an upstream regulator of mTORC1 and autophagy, crucial for the regulation of β cell function and glucose homeostasis.
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.
Hao Y, Li X, Qin K, Shi Y, He Y, Zhang C, Cheng B, Zhang X, Hu G, Liang S, Tang Q, Chen X.
Chemoproteomic and Transcriptomic Analysis Reveals that O-GlcNAc Regulates Mouse Embryonic Stem Cell Fate through the Pluripotency Network.
Angewandte Chemie (International ed. in English)2023
62(17)
36852467
Abstract: Self-renewal and differentiation of embryonic stem cells (ESCs) are influenced by protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification, but the underlying mechanism remains incompletely understood. Herein, we report the identification of 979 O-GlcNAcylated proteins and 1340 modification sites in mouse ESCs (mESCs) by using a chemoproteomics method. In addition to OCT4 and SOX2, the third core pluripotency transcription factor (PTF) NANOG was found to be modified and functionally regulated by O-GlcNAc. Upon differentiation along the neuronal lineage, the O-GlcNAc stoichiometry at 123 sites of 83 proteins-several of which were PTFs-was found to decline. Transcriptomic profiling reveals 2456 differentially expressed genes responsive to OGT inhibition during differentiation, of which 901 are target genes of core PTFs. By acting on the core PTF network, suppression of O-GlcNAcylation upregulates neuron-related genes, thus contributing to mESC fate determination.
Li Y, Yang Z, Chen J, Chen Y, Jiang C, Zhong T, Su Y, Liang Y, Sun H.
OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.
Molecules (Basel, Switzerland)2023
28(5)
36903375
Abstract: O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.
Hung YW, Ouyang C, Ping X, Qi Y, Wang YC, Kung HJ, Ann DK.
Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis.
Journal of biomedical science2023
30(1)
37217939
Abstract: Nutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2α O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear.
Shan X, Jiang R, Gou D, Xiang J, Zhou P, Xia J, Wang K, Huang A, Tang N, Huang L.
Identification of a diketopiperazine-based O-GlcNAc transferase inhibitor sensitizing hepatocellular carcinoma to CDK9 inhibition.
The FEBS journal2023
290(18)
37247228
Abstract: O-GlcNAcylation (O-linked β-N-acetylglucosaminylation) is an important post-translational and metabolic process in cells that is implicated in a wide range of physiological processes. O-GlcNAc transferase (OGT) is ubiquitously present in cells and is the only enzyme that catalyses the transfer of O-GlcNAc to nucleocytoplasmic proteins. Aberrant glycosylation by OGT has been linked to a variety of diseases including cancer, neurodegenerative disorders and diabetes. Previously, we and others demonstrated that O-GlcNAcylation is notably elevated in hepatocellular carcinoma (HCC). The overexpression of O-GlcNAcylation promotes cancer progression and metastasis. Here, we report the identification of HLY838, a novel diketopiperazine-based OGT inhibitor with the ability to induce a global decrease in cellular O-GlcNAc. HLY838 enhances the in vitro and in vivo anti-HCC activity of CDK9 inhibitor by downregulating c-Myc and downstream E2F1 expression. Mechanistically, c-Myc is regulated by the CDK9 at the transcript level, and stabilized by OGT at the protein level. This work therefore demonstrates that HLY838 potentiates the antitumor responses of CDK9 inhibitor, providing an experimental rationale for developing OGT inhibitor as a sensitizing agent in cancer therapeutics.
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.
Lin CH, Liao CC, Wang SY, Peng CY, Yeh YC, Chen MY, Chou TY.
Comparative O-GlcNAc Proteomic Analysis Reveals a Role of O-GlcNAcylated SAM68 in Lung Cancer Aggressiveness.
Cancers2022
14(1)
35008409
Abstract: O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.
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.
Jiang T, Yang J, Yang H, Chen W, Ji K, Xu Y, Yu L.
SLC35B4 Stabilizes c-MYC Protein by O-GlcNAcylation in HCC.
Frontiers in pharmacology2022
13
35308201
Abstract: UDP-GlcNAc is a sugar substrate necessary for the O-GlcNAcylation of proteins. SLC35B4 is one of the nucleotide sugar transporters that transport UDP-GlcNAc and UDP-xylose into the endoplasmic reticulum and Golgi apparatus for glycosylation. The roles of SLC35B4 in hepatocellular carcinoma (HCC) tumorigenesis remain unknown. We find that the expression levels of SLC35B4 are higher in HCC tissues than adjacent non-tumor tissues. SLC35B4 is important for the proliferation and tumorigenesis of HCC cells. Mechanistically, SLC35B4 is important for the O-GlcNAc modification of c-Myc and thus the stabilization of c-Myc, which is required for HCC tumorigenesis. Therefore, SLC35B4 is a promising therapeutic target for treating HCC.
Li F, Yang G, Tachikawa H, Shao K, Yang Y, Gao XD, Nakanishi H.
Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT.
The Journal of general and applied microbiology2021
67(1)
33229814
Abstract: O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.
Luanpitpong S, Poohadsuan J, Klaihmon P, Kang X, Tangkiettrakul K, Issaragrisil S.
Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.
Stem cells (Dayton, Ohio)2021
39(6)
33544938
Abstract: Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation prolongedly decreases O-GlcNAcylation and induces the acquisition of CD34+ CD41a+ MK-like progenitors and its progeny CD34- CD41a+ /CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co-immunoprecipitation analyses, we further identify c-Myc as a direct downstream target of O-GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O-GlcNAc/c-Myc axis. Our data indicate that O-GlcNAcylation posttranslationally regulates c-Myc stability by interfering with its ubiquitin-mediated proteasomal degradation. Depletion of c-Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin-α4 and integrin-β7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O-GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.