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Versatile O-GlcNAc Transferase Assay for High-Throughput Identification of Enzyme Variants, Substrates, and Inhibitors

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Versatile O-GlcNAc Transferase Assay for High-Throughput Identification of Enzyme Variants, Substrates, and Inhibitors
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† Department of Science Education-Chemistry Major, Daegu University, Gyeongbuk 712-714, South Korea
§ §Laboratory of Cell Biochemistry and Biology and #Laboratory of Bioorganic Chemistry, NIDDK, National Institute of Health, Bethesda, Maryland 20892 United States
∥ Department of Pharmaceutical Science and Technology, Catholic University of Daegu, GyeongBuk 712-702, South Korea
‡ Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University, Changwon 641-773, South Korea
Chemical Biology Research Center, Bio-Therapeutics Research Institutes, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongwon 363-883, South Korea
*E-mail: [email protected]. Telephone Number: 301-496-0943/Fax Number301-496-9431.
*E-mail: [email protected]
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2014, 25, 6, 1025–1030
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https://doi.org/10.1021/bc5001774
Published May 27, 2014

Copyright © 2014 American Chemical Society. This publication is licensed under these Terms of Use.

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Abstract

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The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors. In this study we describe a novel, single-well OGT enzyme assay that utilizes 6 × His-tagged substrates, a chemoselective chemical reaction, and unpurified OGT. The high-throughput Ni-NTA Plate OGT Assay will facilitate discovery of potent OGT-specific inhibitors on versatile substrates and the characterization of new enzyme variants.

Copyright © 2014 American Chemical Society

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Cited By

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This article is cited by 20 publications.

  1. Junfeng Ma, Ci Wu, Gerald W. Hart. Analytical and Biochemical Perspectives of Protein O-GlcNAcylation. Chemical Reviews 2021, 121 (3) , 1513-1581. https://doi.org/10.1021/acs.chemrev.0c00884
  2. Christin Bednarek, Ilona Wehl, Nicole Jung, Ute Schepers, Stefan Bräse. The Staudinger Ligation. Chemical Reviews 2020, 120 (10) , 4301-4354. https://doi.org/10.1021/acs.chemrev.9b00665
  3. Juan Hu, Yueying Li, Ying Li, Bo Tang, and Chun-yang Zhang . Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity. Analytical Chemistry 2017, 89 (23) , 12992-12999. https://doi.org/10.1021/acs.analchem.7b04065
  4. Lulu Chen, Mengxue Hu, Luojun Chen, Yihan Peng, Cai Zhang, Xin Wang, Xiangpan Li, Yi Yao, Qibin Song, Jing Li, Huadong Pei. Targeting O-GlcNAcylation in cancer therapeutic resistance: The sugar Saga continues. Cancer Letters 2024, 588 , 216742. https://doi.org/10.1016/j.canlet.2024.216742
  5. Léa El Hajjar, Clarisse Bridot, Marine Nguyen, François-Xavier Cantrelle, Isabelle Landrieu, Caroline Smet-Nocca. The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study. 2024, 237-269. https://doi.org/10.1007/978-1-0716-3629-9_14
  6. Yang Liu, Ya-Jie Hu, Wen-Xuan Fan, Xin Quan, Bin Xu, Shi-Ze Li. O-GlcNAcylation: The Underestimated Emerging Regulators of Skeletal Muscle Physiology. Cells 2022, 11 (11) , 1789. https://doi.org/10.3390/cells11111789
  7. Lara K. Abramowitz, John A. Hanover. Chronically Elevated O-GlcNAcylation Limits Nitric Oxide Production and Deregulates Specific Pro-Inflammatory Cytokines. Frontiers in Immunology 2022, 13 https://doi.org/10.3389/fimmu.2022.802336
  8. Elizabeth O. Akinbiyi, Lara K. Abramowitz, Brianna L. Bauer, Maria S. K. Stoll, Charles L. Hoppel, Chao-Pin Hsiao, John A. Hanover, Jason A. Mears. Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass. Scientific Reports 2021, 11 (1) https://doi.org/10.1038/s41598-021-01512-y
  9. Chenghua Wei, Rui Sun, Yuning Jiang, Xiaoyu Guo, Ye Ying, Ying Wen, Haifeng Yang, Yiping Wu. Protease-protection strategy combined with the SERS tags for detection of O-GlcNAc transferase activity. Sensors and Actuators B: Chemical 2021, 345 , 130410. https://doi.org/10.1016/j.snb.2021.130410
  10. Xinjian Yin, Jiaxin Li, Senhua Chen, Yuping Wu, Zhigang She, Lan Liu, Yue Wang, Zhizeng Gao. An Economical High‐Throughput “FP‐Tag” Assay for Screening Glycosyltransferase Inhibitors**. ChemBioChem 2021, 22 (8) , 1391-1395. https://doi.org/10.1002/cbic.202000746
  11. Cyril Balsollier, Roland J. Pieters, Marko Anderluh. Overview of the Assays to Probe O-Linked β-N-Acetylglucosamine Transferase Binding and Activity. Molecules 2021, 26 (4) , 1037. https://doi.org/10.3390/molecules26041037
  12. Kamau Fahie, Bhargavi Naryanan, Fiddia Zahra, Steve Fernandes, Natasha E. Zachara. Transformative Technologies to Advance Our Understanding of the Functions of O-GlcNAc. 2021, 288-302. https://doi.org/10.1016/B978-0-12-819475-1.00091-2
  13. Matthew G. Alteen, Christina Gros, Richard W. Meek, David A. Cardoso, Jil A. Busmann, Gontran Sangouard, Matthew C. Deen, Hong‐Yee Tan, David L. Shen, Cecilia C. Russell, Gideon J. Davies, Phillip J. Robinson, Adam McCluskey, David J. Vocadlo. A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase. Angewandte Chemie 2020, 132 (24) , 9688-9696. https://doi.org/10.1002/ange.202000621
  14. Matthew G. Alteen, Christina Gros, Richard W. Meek, David A. Cardoso, Jil A. Busmann, Gontran Sangouard, Matthew C. Deen, Hong‐Yee Tan, David L. Shen, Cecilia C. Russell, Gideon J. Davies, Phillip J. Robinson, Adam McCluskey, David J. Vocadlo. A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O ‐GlcNAc Transferase. Angewandte Chemie International Edition 2020, 59 (24) , 9601-9609. https://doi.org/10.1002/anie.202000621
  15. Amanda L. Garner. cat-ELCCA: catalyzing drug discovery through click chemistry. Chemical Communications 2018, 54 (50) , 6531-6539. https://doi.org/10.1039/C8CC02332H
  16. Eun Ju Kim. In Vitro Biochemical Assays for O‐GlcNAc‐Processing Enzymes. ChemBioChem 2017, 18 (15) , 1462-1472. https://doi.org/10.1002/cbic.201700138
  17. Nathan J. Cox, Thomas R. Meister, Michael Boyce. Chemical Biology of O -GlcNAc Glycosylation. 2017, 94-149. https://doi.org/10.1039/9781782623823-00094
  18. Andrew Ruba, Weidong Yang. O-GlcNAc-ylation in the Nuclear Pore Complex. Cellular and Molecular Bioengineering 2016, 9 (2) , 227-233. https://doi.org/10.1007/s12195-016-0440-0
  19. Thomas Zielinski, Melvin Reichman, Preston S. Donover, Robert G. Lowery. Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal. ASSAY and Drug Development Technologies 2016, 14 (4) , 240-251. https://doi.org/10.1089/adt.2016.711
  20. Jie Shi, Suhela Sharif, Rob Ruijtenbeek, Roland J. Pieters, . Activity Based High-Throughput Screening for Novel O-GlcNAc Transferase Substrates Using a Dynamic Peptide Microarray. PLOS ONE 2016, 11 (3) , e0151085. https://doi.org/10.1371/journal.pone.0151085
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2014, 25, 6, 1025–1030
Click to copy citationCitation copied!
https://doi.org/10.1021/bc5001774
Published May 27, 2014

Copyright © 2014 American Chemical Society. This publication is licensed under these Terms of Use.

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