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Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase

Nature Chemical Biology volume 13, pages 1267–1273 (2017)Cite this article

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Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT–substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT–substrate binding and recognition.

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Figure 1: The strategy of GlcNAc electrophilic probes (GEPs) for discerning altered OGT ability of sugar binding compared to protein substrate binding.
Figure 2: MS data show that GEP1 specifically labels the C917 residue of OGT.
Figure 3: LC–MS/MS and X-ray crystallography demonstrate GEP1-derived O-GlcNAcylation on peptide substrates of OGT.
Figure 4: Principle validation of GEP1A fluorescence assay and its application for characterizing additional OGT mutants with altered ability on sugar binding compared to protein substrate binding (or sugar transfer).
Figure 5: Crystal structure of the crosslinked OGT–GEP1–CKII complex.

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Acknowledgements

We thank S. Walker's lab at Harvard Medical School for kindly sharing the expression plasmids of OGT, OGT4.5, OGA, and NUP62. We would like to acknowledge the research funding support from University of Wisconsin–Madison (to J.J.), a Vilas Research Investigator Award (to J.J.), NIH R01 GM121718 (to J.J.), NIH R21 AG055377 (to L.L.), NIH R01 GM117058 (to Y.G.), and NIH R01 HL109810 (to Y.G.). We also thank NIH Shared Instrument Program Grant S10 RR029531 and high-end instrument grant S10 OD018475 for funding the MS instruments.

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  1. Chia-Wei Hu, Matthew Worth, Dacheng Fan, Baobin Li and Hao Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Chia-Wei Hu, Dacheng Fan, Baobin Li, Hao Li, Lei Lu, Xiaofang Zhong, Lingjun Li & Jiaoyang Jiang

  2. Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Matthew Worth, Ying Ge & Lingjun Li

  3. Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Ziqing Lin, Liming Wei & Ying Ge

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Contributions

J.J. oversaw all aspects of the experiments and manuscript preparation. C.-W.H. performed mass spectrometric, crosslinking, and cell culture experiments. M.W. and H.L. synthesized the compounds. D.F. performed mutagenesis, in-gel fluorescence scanning, and enzyme kinetic experiments. B.L. obtained protein crystals and determined structures. L. Lu assisted with protein purification. X.Z., L. Li, Z.L., L.W., and Y.G. provided access to the MS instruments. C.-W.H. and J.J. wrote the manuscript with help from M.W., D.F., B.L., and H.L.

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Correspondence to Jiaoyang Jiang.

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Hu, CW., Worth, M., Fan, D. et al. Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase. Nat Chem Biol 13, 1267–1273 (2017). https://doi.org/10.1038/nchembio.2494

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