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Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation

Nature Structural & Molecular Biology volume 26, pages 1071–1077 (2019)Cite this article

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Abstract

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR–Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.

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Fig. 1: S-GlcNAc mimics O-GlcNAc in the context of an OGA peptide.
Fig. 2: OGT catalyzes S-GlcNAcylation of TAB1S395C in vitro and in cells.
Fig. 3: OGT catalyzes hydrolytically stable cysteine S-GlcNAcylation of OGA in cells, which reduces OGA stability.

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Data availability

Atomic coordinates and structure factors for the CpOGA–(S-GlcNAc peptide) complex structure are deposited in the Protein Data Bank under accession code PDB 6RHE. Source data for Figs. 1c, 2c and 3a–c and Extended Data Figs. 1b, 6 and 7 are available with the paper online. All other data are available upon reasonable request to the corresponding author.

Change history

  • 30 April 2020

    The following figures had source data links to the incorrect file: Fig. 2 was linked to the uncropped blot for Extended Data Fig. 2, Extended Data Fig. 2 was linked to the uncropped blot for Extended Data Fig. 5, Extended Data Fig. 3 was linked to the uncropped blot for Extended Data Fig. 6, Extended Data Fig. 4 was linked to the uncropped blot for Extended Data Fig. 7, Extended Data Fig. 5 was linked to the uncropped blot for Fig. 2, Extended Data Fig. 6 was linked to the uncropped blot for Fig. 3 and the statistical source data for Extended Data Fig. 7, and Extended Data Fig. 7 was linked to the statistical source data for Extended Data Fig. 1. The figures are now linked to the correct source data files.

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Acknowledgements

This work was funded by a Wellcome Trust Investigator Award (grant no. 110061 to D.M.F.v.A) and a Wellcome Trust 4-year PhD studentship (no. 105310/Z/14/Z to A.G.). We thank R. Gourlay and D. Campbell (MRC PPU) for assistance with mass spectrometry. We thank O. G. Raimi for purifying OGT-CK2 linear fusion proteins. We thank ESRF (beamline ID29) for the synchrotron time.

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Authors and Affiliations

  1. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK

    Andrii Gorelik, Sergio Galan Bartual, Vladimir S. Borodkin, Andrew T. Ferenbach & Daan M. F. van Aalten

  2. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK

    Joby Varghese

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Contributions

A.G. and D.M.F.v.A conceived the study. A.G., S.G.B., V.S.B., J.V. and A.T.F. performed experiments and analyzed data. A.G. and D.M.F.v.A. wrote the manuscript with input from all authors.

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Correspondence to Daan M. F. van Aalten.

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The University of Dundee holds a patent for the GlcNAcstatin inhibitor. The authors declare no other competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cysteine-S-GlcNAc can replace Threonine-O-GlcNAc on α-synuclein-derived peptides.

(a) MALDI-TOF mass spectra of synthetic O-/S-GlcNAc-modified peptides derived from α-synuclein with or without CpOGA treatment for 4 and 24 h. Loss of GlcNAc residue is characterized by the reduction in m/z by 203 Da. M–m/z, [M + Na]–m/z with a sodium adduct (23 Da), m/z–mass/charge ratio. (b) Fluorescence polarization assay dose-response curves showing the displacement of a fixed concentration of fluorescent probe (5 nM GlcNAcstatin B-FITC) from CpOGAD298N by increasing concentrations of O- or S-GlcNAcylated peptides derived from α-synuclein. Highest amount of probe bound to CpOGAD298N in the absence of competing O- or S-GlcNAcylated peptides was arbitrarily set as 100%. Data points were fitted to a four-parameter equation for dose-dependent inhibition using Prism (GraphPad). Data are shown as mean ± s.e.m. of n = 3 independent experiments. KD values were calculated as described previously23. Source data for panel b are available with the paper online.

Source data

Extended Data Fig. 2 OGT-mediated Cys-S-GlcNAcylation of the OGT-CK2S347C linear fusion.

O- and S-GlcNAc modifications were detected by Western blot using a pan-specific O-GlcNAc antibody CTD110.6 on OGT-CK2 and OGT-CK2S347C. To remove O-GlcNAc, modified proteins were treated with 3 μM CpOGA for 2 h at 37 °C. Uncropped Western blot images are shown in the Source Data.

Source data

Extended Data Fig. 3 OGT-mediated Cys-S-GlcNAcylation of TAB1S395C is recognized by the CTD110.6 antibody but not by the O-GlcNAc-specific antibody RL2.

OGT reactions were performed with TAB1WT and TAB1S395C for 12 h. The reactions were untreated or treated with 3 μM CpOGA for 2 h at 37 °C. O- and S-GlcNAc modifications were detected by Western blot using pan-specific O-GlcNAc antibodies CTD110.6 and RL2. Uncropped Western blot images are shown in the Source Data.

Source data

Extended Data Fig. 4 S-GlcNAcylation is an enzymatic modification in cells.

IL-1R Hek293 cells were transfected with N-terminally FLAG-tagged TAB1 constructs that were wild type or carried S395C mutations of the O-GlcNAc modification site. The cells were treated with GlcNAcstatin G (GG) for 24 h to elevate O-GlcNAc levels. The lysate was then split in half and one half was treated with CpOGA to remove O-GlcNAc (as monitored by Western blot using an anti-O-GlcNAc antibody RL2). Overexpressed TAB1 levels were assessed with an anti-FLAG antibody. Glycosylation levels of TAB1 were monitored with a site-specific O-GlcNAc TAB1 antibody (gTAB1). Additionally, cells were treated with a precursor (4Ac5S-GlcNAc) of the OGT inhibitor UDP-5S-GlcNAc. Upon 4Ac5S-GlcNAc treatment, disappearance of gTAB1 signal indicates that the modification is enzymatic. Tubulin was used as a loading control. Uncropped Western blot images are shown in the Source Data.

Source data

Extended Data Fig. 5 GalTY289L efficiently labels S-GlcNAc with GalNAz and allows quantification of S-GlcNAc stoichiometry on an in vitro S-GlcNAcylated recombinant TAB1.

GalT labelling of in vitro GlcNAcylated TAB1WT and TAB1S395C with GalNAz was performed followed by reaction with alkyne-. The PEGylation reaction was assessed by Western blot using total TAB1 antibody. Molecular weight shift corresponds to GlcNAc-modified TAB1. A site-specific O-GlcNAc TAB1 antibody (gTAB1) was used as a control to monitor the successful OGT in vitro reaction (24 h) as well as galactosyltransferase labelling efficiency (based on the disappearance of GlcNAc signal). Stoichiometry was quantified using LI-COR Image Studio Software: 38% for O-GlcNAc TAB1 and 32% for S-GlcNAc TAB1. GalT - GalTY289L galactosyltransferase. Uncropped Western blot images are shown in the Source Data.

Source data

Extended Data Fig. 6 Quantification of OGT levels in wild type OGA and OGAS405C mutant mES cells.

OGT levels were monitored by Western blot with an anti-OGT antibody. Tubulin was used as a loading control. Data are shown as mean ± s.e.m. of n = 6 (WT OGA) and n = 9 (OGAS405C) biological replicates. NS–no significant difference (analyzed by two-tailed Student’s t-test). Uncropped Western blot images and quantification source data are available with the paper online.

Source data

Extended Data Fig. 7 Thermal shift assay on wild type and OGAS405C mESC lysates.

OGA levels were monitored by Western blot with an anti-OGA antibody. For quantification, bands from samples at 37 °C (starting temperature) were assigned as 100% OGA. Data are shown as mean ± s.e.m. of n = 6 biological replicates. Uncropped Western blot images and quantification source data are available with the paper online.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Figures 1–5

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Gorelik, A., Bartual, S.G., Borodkin, V.S. et al. Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation. Nat Struct Mol Biol 26, 1071–1077 (2019). https://doi.org/10.1038/s41594-019-0325-8

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