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.
Luna S, Malard F, Pereckas M, Aoki M, Aoki K, Olivier-Van Stichelen S.
Studying the O-GlcNAcome of human placentas using banked tissue samples.
Glycobiology2024
34(4)
38253038
Abstract: O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.
Vang S, Helton ES, Guo Y, Burpee B, Rose E, Easter M, Bollenbecker S, Hirsch MJ, Matthews EL, Jones LI, Howze PH 4th, Rajasekaran V, Denson R, Cochran P, Attah IK, Olson H, Clair G, Melkani G, Krick S, Barnes JW.
O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.
Frontiers in immunology2024
15
38665916
Abstract: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis.
Hou C, Wu C, Wu Z, Cheng Y, Li W, Sun H, Ma J.
Systematic Evaluation of Affinity Enrichment Methods for O-GlcNAc Proteomics.
Journal of proteome research202439302247
Abstract: O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins plays critical roles in the regulation of diverse biological processes. However, protein O-GlcNAcylation analysis, especially at a large scale, has been a challenge. So far, a number of enrichment materials and methods have been developed for site-specific O-GlcNAc proteomics in different biological settings. Despite the presence of multiple methods, their performance for the O-GlcNAc proteomics is largely unclear. In this work, by using the lysates of PANC-1 cells (a pancreatic cancer cell line), we provided a head-to-head comparison of three affinity enrichment methods and materials (i.e., antibody, lectin AANL6, and an OGA mutant) for site-specific O-GlcNAc proteomics. The enriched peptides were analyzed by HCD product-dependent EThcD (i.e., HCD-pd-EThcD) mass spectrometry. The resulting data files were processed by three different data analysis packages (i.e., Sequest HT, Byonic, and FragPipe). Our data suggest that each method captures a subpopulation of the O-GlcNAc proteins. Besides the enrichment methods, we also observe complementarity between the different data analysis tools. Thus, combining different approaches holds promise for enhanced coverage of O-GlcNAc proteomics.
Hou C, Deng J, Wu C, Zhang J, Byers S, Moremen KW, Pei H, Ma J.
Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins.
Proceedings of the National Academy of Sciences of the United States of America2024
121(47)
39531497
Abstract: As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.
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.
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.
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.
Fan Z, Li J, Liu T, Zhang Z, Qin W, Qian X.
A new tandem enrichment strategy for the simultaneous profiling of O-GlcNAcylation and phosphorylation in RNA-binding proteome.
The Analyst2021
146(4)
33465208
Abstract: RNA-protein interactions play important roles in almost every step of the lifetime of RNAs, such as RNA splicing, transporting, localization, translation and degradation. Post-translational modifications, such as O-GlcNAcylation and phosphorylation, and their "cross-talk" (OPCT) are essential to the activity and function regulation of RNA-binding proteins (RBPs). However, due to the extremely low abundance of O-GlcNAcylation and the lack of RBP-targeted enrichment strategies, large-scale simultaneous profiling of O-GlcNAcylation and phosphorylation on RBPs is still a challenging task. In the present study, we developed a tandem enrichment strategy combining metabolic labeling-based RNA tagging for selective purification of RBPs and HILIC-based enrichment for simultaneous O-GlcNAcylation and phosphorylation profiling. Benefiting from the sequence-independent RNA tagging by ethynyluridine (EU) labeling, 1115 RBPs binding to different types of RNAs were successfully enriched and identified by quantitative mass spectrometry (MS) analysis. Further HILIC enrichment on the tryptic-digested RBPs and MS analysis led to the first large-scale identification of O-GlcNAcylation and phosphorylation in the RNA-binding proteome, with 461 O-GlcNAc peptides corresponding to 300 RBPs and 671 phosphopeptides corresponding to 389 RBPs. Interestingly, ∼25% RBPs modified by two PTMs were found to be related to multiple metabolism pathways. This strategy has the advantage of high compatibility with MS and provides peptide-level evidence for the identification of O-GlcNAcylated RBPs. We expect it will support simultaneous mapping of O-GlcNAcylation and phosphorylation on RBPs and facilitate further elucidation of the crucial roles of OPCT in the function regulation of RBPs.
Xie X, Wu Q, Zhang K, Liu Y, Zhang N, Chen Q, Wang L, Li W, Zhang J, Liu Y.
O-GlcNAc modification regulates MTA1 transcriptional activity during breast cancer cell genotoxic adaptation.
Biochimica et biophysica acta. General subjects2021
1865(8)
34019948
Abstract: Chromatin modifier metastasis-associated protein 1 (MTA1), closely associated with tumor angiogenesis in breast cancer, plays an important role in gene expression and cancer cell behavior. Recently, an association between O-GlcNAc transferase (OGT) and MTA1 was identified by mass spectroscopy. However, the potential relationship between MTA1 and O-GlcNAc modification has not yet explored.
Ramirez DH, 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.
Chen Y, Qin H, Yue X, Zhou J, Liu L, Nie Y, Ye M.
Highly Efficient Enrichment of O-GlcNAc Glycopeptides Based on Chemical Oxidation and Reversible Hydrazide Chemistry.
Analytical chemistry2021
93(49)
34846842
Abstract: Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups. The labeled O-GlcNAc glycopeptides could be efficiently enriched based on the equilibrium between the hydrazine and oxime bonds. Good specificity of the glycopeptide enrichment was observed from the mixtures of glycopeptide and non-glycopeptides using the CHO-GlcNAc method. Then, it was applied to analyze O-GlcNAcylation in the nucleus of HeLa cells, and 829 potential O-GlcNAcylation sites on 274 glycoproteins were identified, including the two readers of m6A (YTHDF1 and YTHDF3), which could provide clues for the mechanism of crosstalk between O-GlcNAcylation and other PTMs of proteins and RNA. Thus, this method could be a versatile tool for the proteomic analysis of O-GlcNAcylation.
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.
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.
Phoomak C, Park D, Silsirivanit A, Sawanyawisuth K, Vaeteewoottacharn K, Detarya M, Wongkham C, Lebrilla CB, Wongkham S.
O-GlcNAc-induced nuclear translocation of hnRNP-K is associated with progression and metastasis of cholangiocarcinoma.
Molecular oncology2019
13(2)
30444036
Abstract: O-GlcNAcylation is a key post-translational modification that modifies the functions of proteins. Associations between O-GlcNAcylation, shorter survival of cholangiocarcinoma (CCA) patients, and increased migration/invasion of CCA cell lines have been reported. However, the specific O-GlcNAcylated proteins (OGPs) that participate in promotion of CCA progression are poorly understood. OGPs were isolated from human CCA cell lines, KKU-213 and KKU-214, using a click chemistry-based enzymatic labeling system, identified using LC-MS/MS, and searched against an OGP database. From the proteomic analysis, a total of 21 OGPs related to cancer progression were identified, of which 12 have not been previously reported. Among these, hnRNP-K, a multifaceted RNA- and DNA-binding protein known as a pre-mRNA-binding protein, was one of the most abundantly expressed, suggesting its involvement in CCA progression. O-GlcNAcylation of hnRNP-K was further verified by anti-OGP/anti-hnRNP-K immunoprecipitations and sWGA pull-down assays. The perpetuation of CCA by hnRNP-K was evaluated using siRNA, which revealed modulation of cyclin D1, XIAP, EMT markers, and MMP2 and MMP7 expression. In native CCA cells, hnRNP-K was primarily localized in the nucleus; however, when O-GlcNAcylation was suppressed, hnRNP-K was retained in the cytoplasm. These data signify an association between nuclear accumulation of hnRNP-K and the migratory capabilities of CCA cells. In human CCA tissues, expression of nuclear hnRNP-K was positively correlated with high O-GlcNAcylation levels, metastatic stage, and shorter survival of CCA patients. This study demonstrates the significance of O-GlcNAcylation on the nuclear translocation of hnRNP-K and its impact on the progression of CCA.
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.