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

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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.

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.


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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.

Author information

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.

Correspondence to Daan M. F. van Aalten.

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Competing interests

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

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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) doi:10.1038/s41594-019-0325-8

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