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.
Srivastava PP, Dasari A, Kumar S, Gupta I, Jain D, Minocha S.
Epigenetic co-regulator HCF-1 promotes lung cancer via O-GlcNAcylation-dependent pathways.
Molecular therapy. Oncology2025
33(4)
41018975
Abstract: The rise in the incidences of lung cancer currently poses a global health challenge, making it crucial to understand the underlying molecular and cellular mechanisms. Host cell factor-1 (HCF-1), a conserved epigenetic transcriptional co-regulator, undergoes proteolytic maturation and glycosylation by O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). Elevated O-GlcNAc and OGT levels have been observed in lung cancer, highlighting their potential significance in disease progression. In human lung cancer tissues, we observed a significant upregulation of HCF-1, which coincides with increased OGT, O-GlcNAc, and Nkx2.1 (a diagnostic marker for non-small cell lung cancer [NSCLSC]) levels. To further explore HCF-1's role mechanistically, we utilized the NSCLC cell lines, where HCF-1 depletion resulted in reduced proliferation, O-GlcNAcylation, Nkx2.1 expression, and O-GlcNAcylated proteins upon wheat germ agglutinin (WGA) pull-down, reinforcing its role in lung cancer progression. Additionally, Nkx2.1-mediated conditional knockout of HCF-1 impaired murine lung development and cell proliferation. Interestingly, OGT inhibition with OSMI-1 also reduced HCF-1, Nkx2.1 levels, and proliferation, suggesting a role for O-GlcNAcylation in HCF-1-mediated signaling cascades. Thus, our findings elucidate the critical role of HCF-1 and O-GlcNAcylation in lung cancer pathogenesis. These insights not only deepen our understanding of lung cancer pathogenesis but also identify potential molecular targets for studies aimed at intervention.
Tan H, Zhang H, Gao J, Wang L, Luan X.
Ferroptosis-induced remodeling of glycosylation the immune microenvironment and improves survival in pancreatic cancer.
World journal of surgical oncology202541350704
Abstract: Pancreatic ductal adenocarcinoma (PDAC) is still an aggressive and rapidly progressive malignant tumor of the digestive system. Emerging treatment strategies have yielded limited benefits, primarily due to the high heterogeneity of tumors and the presence of various mechanisms of drug resistance. Ferroptosis is a newly discovered type of cell death that has emerged as a promising anti-tumor strategy, while our understanding of the interaction between ferroptosis and tumor glycosylation and immune remodeling remains limited.
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, 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.
Xiong Y, Lu Z, Shao Y, Meng P, Wang G, Zhou X, Yao J, Bao H, Lu H.
Rapid and large-scale glycopeptide enrichment strategy based on chemical ligation.
National science review2024
11(11)
39534244
Abstract: Protein glycosylation, the most universal post-translational modification, is thought to play a crucial role in regulating multiple essential cellular processes. However, the low abundance of glycoproteins and the heterogeneity of glycans complicate their comprehensive analysis. Here, we develop a rapid and large-scale glycopeptide enrichment strategy via bioorthogonal ligation and trypsin cleavage. The enrichment process is performed in one tube to minimize sample loss and time costs. This method combines convenience and practicality, identifying over 900 O-GlcNAc sites from a 500 μg sample. Surprisingly, it allows simultaneous identification of N-glycosites, O-GlcNAc sites, O-GalNAc sites and N-glycans via a two-step enzymatic release strategy. Combined with quantitative analysis, it reveals the distinct O-GlcNAcylation patterns in different compartments during oxidative stress. In summary, our study offers a convenient and robust tool for glycoproteome and glycome profiling, facilitating in-depth analysis to elucidate the biological functions of glycosylation.
Wong YK, Wang J, Lim TK, Lin Q, Yap CT, Shen HM.
O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells.
Proteomics2022
22(9)
35083852
Abstract: Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis (FAS) activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and FAS in cancer therapy.
Xu S, Zheng J, Xiao H, Wu R.
Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells.
Analytical chemistry2022
94(7)
35132862
Abstract: Glycoproteins with diverse glycans are essential to human cells, and subtle differences in glycan structures may result in entirely different functions. One typical example is proteins modified with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) (the Tn antigen), in which the two glycans have very similar structures and identical chemical compositions, making them extraordinarily challenging to be distinguished. Here, we developed an effective method benefiting from selective enrichment and the enzymatic specificity to simultaneously identify and distinguish glycoproteins with O-GlcNAc and O-GalNAc. Metabolic labeling was combined with bioorthogonal chemistry for enriching glycoproteins modified with O-GlcNAc and O-GalNAc. Then, the enzymatic reaction with galactose oxidase was utilized to specifically oxidize O-GalNAc, but not O-GlcNAc, generating the different tags between glycopeptides with O-GlcNAc and O-GalNAc that can be easily distinguishable by mass spectrometry (MS). Among O-GlcNAcylated proteins commonly identified in three types of human cells, those related to transcription and RNA binding are highly enriched. Cell-specific features are also revealed. Among glycoproteins exclusively in Jurkat cells, those involved in human T-lymphotropic virus type 1 (HTLV-1) infection are overrepresented, which is consistent with the cell line source and suggests that protein O-GlcNAcylation participated in the response to the virus infection. Furthermore, glycoproteins with the Tn antigen have different subcellular distributions in different cells, which may be attributed to the distinct mechanisms for the formation of protein O-GalNAcylation.
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.
Xu S, Sun F, Wu R.
A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation.
Analytical chemistry2020
92(14)
32574038
Abstract: O-GlcNAcylation has gradually been recognized as a critically important protein post-translational modification in mammalian cells. Besides regulation of gene expression, its crosstalk with protein phosphorylation is vital for cell signaling. Despite its importance, comprehensive analysis of O-GlcNAcylation is extraordinarily challenging due to the low abundances of many O-GlcNAcylated proteins and the complexity of biological samples. Here, we developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective as compared to the engineered enzyme and click chemistry reagents. Biological triplicate experiments were performed to validate the effectiveness and the reproducibility of this method, and the results are comparable with the previous chemoenzymatic method using the engineered enzyme and click chemistry. Moreover, because of the promiscuity of the galactosyltransferase, 18 unique O-glucosylated peptides were identified on the EGF domain from nine proteins. Considering that effective and approachable methods are critical to advance glycoscience research, the current method without any sample restrictions can be widely applied for global analysis of protein O-GlcNAcylation in different samples.
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.
Joiner CM, Levine ZG, Aonbangkhen C, Woo CM, Walker S.
Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.
Journal of the American Chemical Society2019
141(33)
31373491
Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.
Qin K, Zhu Y, Qin W, Gao J, Shao X, Wang YL, Zhou W, Wang C, Chen X.
Quantitative Profiling of Protein O-GlcNAcylation Sites by an Isotope-Tagged Cleavable Linker.
ACS chemical biology2018
13(8)
30059200
Abstract: Large-scale quantification of protein O-linked β- N-acetylglucosamine (O-GlcNAc) modification in a site-specific manner remains a key challenge in studying O-GlcNAc biology. Herein, we developed an isotope-tagged cleavable linker (isoTCL) strategy, which enabled isotopic labeling of O-GlcNAc through bioorthogonal conjugation of affinity tags. We demonstrated the application of the isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa cells. Furthermore, we investigated the O-GlcNAcylation sensitivity to the sugar donor by quantifying the levels of modification under different concentrations of the O-GlcNAc labeling probe in a site-specific manner. In addition, we applied isoTCL to compare the O-GlcNAcylation stoichiometry levels of more than 100 modification sites between placenta samples from male and female mice and confirmed site-specifically that female placenta has a higher O-GlcNAcylation than its male counterpart. The isoTCL platform provides a powerful tool for quantitative profiling of O-GlcNAc modification.
Li S, Zhu H, Wang J, Wang X, Li X, Ma C, Wen L, Yu B, Wang Y, Li J, Wang PG.
Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics.
Electrophoresis2016
37(11)
26853435
Abstract: O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.
Drougat L, Olivier-Van Stichelen S, Mortuaire M, Foulquier F, Lacoste AS, Michalski JC, Lefebvre T, Vercoutter-Edouart AS.
Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition.
Biochimica et biophysica acta2012
1820(12)
22967762
Abstract: DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.
Parker BL, Gupta P, Cordwell SJ, Larsen MR, Palmisano G.
Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry.
Journal of proteome research2011
10(4)
21158410
Abstract: A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO(4)-containing peptides were enriched using titanium dioxide chromatography. Modified peptides were analyzed using a combination of higher energy collision dissociation for identification and electron transfer dissociation to localize the site of O-GlcNAc attachment. The enrichment method was developed and optimized using an alpha-crystallin standard protein and then applied to a soluble protein preparation of mouse brain tissue and a nuclear preparation generated from HeLa cells. A total of 42 unique O-GlcNAcylated peptides were identified, including 7 novel O-GlcNAc sites.
Fujiki R, Hashiba W, Sekine H, Yokoyama A, Chikanishi T, Ito S, Imai Y, Kim J, He HH, Igarashi K, Kanno J, Ohtake F, Kitagawa H, Roeder RG, Brown M, Kato S.
GlcNAcylation of histone H2B facilitates its monoubiquitination.
Nature2011
480(7378)
22121020
Abstract: Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.
Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW.
Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis.
Science signaling2010
3(104)
20068230
Abstract: Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
Srikanth B, Vaidya MM, Kalraiya RD.
O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.
The Journal of biological chemistry2010
285(44)
20729549
Abstract: Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.