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.
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.
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.
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.
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.
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.
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.
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.
Wu C, Shi S, Hou C, Luo Y, Byers S, Ma J.
Design and Preparation of Novel Nitro-Oxide-Grafted Nanospheres with Enhanced Hydrogen Bonding Interaction for O-GlcNAc Analysis.
ACS applied materials & interfaces2022
14(42)
36240223
Abstract: As an essential modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples.
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.
Song J, Liu C, Wang X, Xu B, Liu X, Li Y, Xia J, Li Y, Zhang C, Li D, Sun H.
O-GlcNAcylation Quantification of Certain Protein by the Proximity Ligation Assay and Clostridium perfringen OGA(D298N)(CpOGA(D298N)).
ACS chemical biology2021
16(6)
34105348
Abstract: O-GlcNAcylation is an O-linked β-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from β-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.
Ramirez DH, Yang B, D'Souza AK, Shen D, Woo CM.
Truncation of the TPR domain of OGT alters substrate and glycosite selection.
Analytical and bioanalytical chemistry2021
413(30)
34725712
Abstract: O-GlcNAc transferase (OGT) is an essential enzyme that installs O-linked N-acetylglucosamine (O-GlcNAc) to thousands of protein substrates. OGT and its isoforms select from these substrates through the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection is unresolved. Here, we report the effects of iterative truncations to the TPR domain of OGT on substrate and glycosite selection with the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Iterative truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization of OGT in cells. The glycoproteome and glycosites modified by four OGT TPR isoforms were examined on the whole proteome and a single target protein, GFP-JunB. We found the greatest changes in O-GlcNAc on proteins associated with mRNA splicing processes and that the first four TPRs of the canonical nucleocytoplasmic OGT had the broadest substrate scope. Subsequent glycosite analysis revealed that alteration to the last four TPRs corresponded to the greatest shift in the resulting O-GlcNAc consensus sequence. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affect the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.
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.
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.
Qin W, Qin K, Fan X, Peng L, Hong W, Zhu Y, Lv P, Du Y, Huang R, Han M, Cheng B, Liu Y, Zhou W, Wang C, Chen X.
Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.
Angewandte Chemie (International ed. in English)2018
57(7)
29237092
Abstract: The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).
Kim DI, Cutler JA, Na CH, Reckel S, Renuse S, Madugundu AK, Tahir R, Goldschmidt HL, Reddy KL, Huganir RL, Wu X, Zachara NE, Hantschel O, Pandey A.
BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.
Journal of proteome research2018
17(2)
29249144
Abstract: Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.
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.
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.
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.