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O-GlcNAcylation modulates liquid–liquid phase separation of SynGAP/PSD-95

Abstract

Liquid–liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O-GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O-GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O-GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O-GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O-GlcNAcylation to exert effective regulation. We also showed that O-GlcNAc-dependent LLPS is reversibly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O-GlcNAc cycling may serve as an LLPS-regulating post-translational modification.

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Fig. 1: SynGAP is O-GlcNAcylated.
Fig. 2: EPL synthesis of site-specifically O-GlcNAcylated SynGAP CC-PBM indicates inhibition of LLPS by T1306 O-GlcNAcylation.
Fig. 3: SynGAP Thr1306 O-GlcNAc disrupts interaction with PSD-95.
Fig. 4: O-GlcNAc regulates SynGAP/PSD-95 LLPS in a dominant-negative manner.
Fig. 5: The LLPS between SynGAP CC-PBM and PSD-95 PSG could be reversibly regulated by OGT and OGA.

Data availability

All relevant data presented in this study are provided in the Article, Extended Data figures and Supplementary Information. The data and genetic constructs are also available from the corresponding authors upon request. The crystal structure of the PSD-95 PDZ3-C/SynGAP PBM complex is from https://www.rcsb.org/structure/5JXB (PDB 5JXB). Source data are provided with this paper.

Code availability

MATLAB code can be downloaded from GitHub at https://github.com/XChenlab/LLPS. Alternatively, it is available from the corresponding authors upon request.

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Acknowledgements

We thank Y. Liu for help with the Rosetta simulation. Parts of the MS experiments were performed at the Analytical Instrumentation Center of Peking University (PKUAIC). This work was supported by the National Key R&D Program of China (no. 2018YFA0507600 to X.C., S.D., C.L. and P.Z.) and the National Natural Science Foundation of China (nos. 91753206 and 21521003 to X.C. and no. 21708002 to W.Z.).

Author information

Authors and Affiliations

Authors

Contributions

X.C. conceived the study and supervised the entire project. X.C., P.L. and Y.D. designed the experiments and analysed the data. P.L. and Y.D. performed most of the experiments, unless otherwise specified, with the help of X.Z., Y.W., M. Zeng, L.P., W.Z., P.Z., C.L. and M. Zhang. C.H. performed the protein semisynthesis under the supervision of S.D. The manuscript was written by X.C., P.L. and Y.D., with input from all the authors.

Corresponding authors

Correspondence to Suwei Dong or Xing Chen.

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The authors declare no competing interests.

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Nature Chemistry thanks Xiaoyong Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 O-GlcNAcylation of SynGAP.

a,b, Bar graph showing relative O-GlcNAcylation levels in Fig. 1c (a) and Fig. 1d (b). In a and b, the relative O-GlcNAcylation levels are normalized to that of GFP-SynGAP-α1 with no OGT overexpression and that of GFP-SynGAP CC-PBM with no OGT overexpression, respectively. Error bars represent mean ± SD. Results are from three independent experiments. Unpaired two-tailed Student’s t-test with 95% CI was used to determine difference, p < 0.05 is considered significant. c,d, ETD-MS/MS spectra of two O-GlcNAcylated peptides of recombinant GFP-SynGAP CC-PBM purified from HEK293T co-expressing OGT. The c2 ion unambiguously confirms O-GlcNAcylation at S1159 (c). The c12 and c14 ions unambiguously confirms O-GlcNAcylation at T1306 (d). The matched fragment ions are marked.

Source data

Extended Data Fig. 2 SDS PAGE analysis of recombinant SynGAP CC-PBM, EPL-synthesized SynGAP CC-PBM, SynGAP-S1159OG, and SynGAP-T1306OG.

SynGAP-T1306OG exhibited a slightly higher molecular weight than SynGAP-S1159OG, which can be attributed to the four residual amino acids at the N-terminus of SynGAP-T1306OG. The recombinant and EPL-synthesized SynGAP CC-PBM shown here contain the four amino acids. Of note, the four residual amino acids do not affect the LLPS behaviour of SynGAP CC-PBM/PSD-95 PSG (data not shown). Representative results are shown from three independent experiments.

Source data

Extended Data Fig. 3 Quantification and FRAP analysis of liquid droplets imaging of SynGAP proteins with PSD-95.

a, Time-lapse images of the PSD-95 PSG or SynGAP CC-PBM protein alone (80 μM PSD-95 PSG with 1% conjugated with TAMRA fluorophore, fluorescence channel; 80 μM SynGAP CC-PBM variants, DIC channel). Under the same experimental conditions as Fig. 2g, no liquid droplet was observed, indicating that PSD-95 PSG or SynGAP CC-PBM alone cannot undergo LLPS. Scale bar, 10 μm. b, Box-and-whiskers plot showing statistical analysis of the liquid droplet areas of Fig. 2g. The horizontal lines mark the maximum, median and minimum values of the data, and boxes mark upper and lower quartiles. For each group, at least 30 fluorescence images from at least three independent experiments were analysed. Differences were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test with 95% CI, and p < 0.05 is considered significant. c-f, Recovery of PSD-95 PSG fluorescence over time after photo-bleaching a small region with the droplet of the complex of PSD-95 PSG (with 1% conjugated with TAMRA) with recombinant SynGAP CC-PBM (d), EPL-synthesized SynGAP CC-PBM (e), or SynGAP-S1159OG(f). The overlayed FRAP curves are shown in c. The results are from at least three independent experiments and represented as mean ± SD.

Source data

Extended Data Fig. 4 LLPS of the mixtures of PSD-95 PSG with different SynGAP CC-PBM proteins at the physiological concentration.

a, Time-lapse fluorescence images showing LLPS of 5 μM PSD-95 PSG and recombinant SynGAP CC-PBM, semisynthetic SynGAP CC-PBM, SynGAP-S1159OG, or SynGAP-T1306OG, with 2% PEG8000 over 20 min. Scale bar, 10 μm. b, Box-and-whiskers plot shows statistical analysis of the liquid droplet areas of a. The horizontal lines mark the maximum, median and minimum values of the data, and boxes mark upper and lower quartiles. For each group, at least 30 fluorescence images from at least three independent experiments were analysed. Differences were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test with 95% CI, p < 0.05 is considered significant. c, SDS-PAGE gel showing the distributions of PSD-95 and SynGAP proteins in the supernatant (S) and pellet (P) in the sedimentation-based assay. 5 μM PSD-95 or SynGAP proteins were mixed for 10 min at r.t. in the presence of the crowding reagent (2% PEG8000) and subjected with the sedimentation-based assays. Bar graph on the right shows quantification of the distributions. d, Bar graph shows quantification results in a. The quantification results are from three independent experiments and represented as mean ± SD. Differences were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test with 95% CI, p < 0.05 is considered significant.

Source data

Extended Data Fig. 5 SEC-SLC analysis of complex formation.

a, Curves showing PSD-95 PSG, SynGAP CC-PBM, and the 1:1 mixture of PSD-95 PSG and SynGAP CC-PBM. b, Curves showing PSD-95 PSG, SynGAP-S1159OG, and the 1:1 mixture of PSD-95 PSG and SynGAP-S1159OG.

Source data

Extended Data Fig. 6 3D structural modelling of the SynGAP PBM-T1306OG/PSD-95 PDZ3-C complex.

a, Crystal structure of the PSD-95 PDZ3-C/SynGAP PBM complex (PDB code: 5JXB). The zoomed-in view shows the interaction between PSD-95 H369 and SynGAP T1306. b, Modelled structure of the PSD-95 PDZ3-C/SynGAP PBM-T1306OG based on the PSD-95 PDZ3-C/SynGAP PBM structure by using Rosseta homology modeling. The zoomed-in view shows that the interaction between PSD-95 H369 and SynGAP T1306 is blocked by the O-GlcNAc moiety. Note that the crystal structure and the modeled structure were obtained with SynGAP PBM fused to PSD-95 PDZ3-C with a flexible linker. The linker holds SynGAP PBM in close proximity to PSD-95 PDZ3-C even when T1306 O-GlcNAc disrupts the interaction.

Extended Data Fig. 7 SDS-PAGE gel showing the distribution of SynGAP CC-PBM, SynGAP CC-PBM-S1159A and SynGAP-T1306A in the supernatant (S) and pellet (P) when mixed with PSD-95 PSG in sedimentation assay.

80 μM PSD-95 and SynGAP CC-PBM variant were mixed for 10 min at r.t. and then subjected to the sedimentation-based assay. Representative results are shown from three independent experiments.

Source data

Extended Data Fig. 8 FRAP analysis of LLPS of GFP-SynGAP/RFP-PSD-95 in living cells.

a, Representative time-lapse fluorescence images showing the recovery of GFP-SynGAP fluorescence in a punctum over a few minutes. The fluorescence of GFP was selectively bleached at 0 s and the RFP fluorescence remained unchanged. Scale bar: 5 μm. Representative results are shown from three independent experiments. b, Quantification of the recovery of GFP-SynGAP fluorescence over time in the punctum shown in a.

Source data

Extended Data Fig. 9 Dominant-negative effect of SynGAP T1306 O-GlcNAcylation on LLPS of PSD-95 PSG/SynGAP CC-PBM.

a, Schematic showing the procedures for forming the overall 25% O-GlcNAcylated SynGAP CC-PBM trimers with no, one, two, and three O-GlcNAc at the ratio of 42.2%:42.2%:14.0%:1.6%. b, Time-lapse fluorescence images showing LLPS of PSD-95 PSG with SynGAP CC-PBM at indicated concentrations and O-GlcNAcylated ratios over 10 min. Scale bar, 10 μm. c, SDS-PAGE gel showing the distributions of SynGAP CC-PBM and PSD-95 PSG in the supernatant (S) and pellet (P). 80 μM PSD-95 PSG were mixed with 80 μM partially O-GlcNAylated SynGAP CC-PBM with varied O-GlcNAcylation stoichiometry ranging from 10% to 100%. d, SDS-PAGE gel showing the distribution of SynGAP CC-PBM and PSD-95 PSG in the supernatant (S) and pellet (P). PSD-95 PSG at varied concentrations was mixed with SynGAP CC-PBM or 25% O-GlcNAcylated SynGAP CC-PBM at indicated concentrations. Unpaired two-tailed Student’s t-test with 95% CI was used to determine difference, p < 0.05 is considered significant. In c and d, bar graphs showing quantification of the distributions. The quantification results were represented as mean ± SD. In b-d, representative results are shown from three independent experiments.

Source data

Extended Data Fig. 10 OGA and OGT treatment of SynGAP-T1306OG and SynGAP.

a, Representative in-gel fluorescence scanning showing O-GlcNAcylation of SynGAP CC-PBM, SynGAP-T1306OG, and SynGAP-T1306OG treated with 10 μM OGA at r.t. for 4 h. The proteins were incubated with Y289L GalT1 and UDP-GalNAz, reacted with alkyne-Cy5. b, SDS-PAGE gel showing the O-GlcNAcylation levels of SynGAP CC-PBM, SynGAP-T1306OG, and SynGAP-T1306OG treated with 10 μM OGA for 4 h. The proteins were incubated with Y298L GalT1 and UDP-GalNAz, and reacted with alkyne-PEG2k. Note that SynGAP CC-PBM was non-O-GlcNAcylated. The calculated stoichiometry was shown below the gel. Incomplete GalT1-based enzymatic reaction and click reaction could contribute to the apparent stoichiometry. Nevertheless, OGA treatment removed the majority of O-GlcNAc from SynGAP-T1306OG. c, In-gel fluorescence scanning showing SynGAP CC-PBM incubated with OGT at varied concentrations and 5 mM UDP-GlcNAc (left panel) or with 10 μM OGT and UDP-GlcNAc at varied concentrations (right panel) overnight. d, SDS-PAGE gel showing the O-GlcNAcylation levels of SynGAP incubated with 10 μM OGT and 5 mM UDP-GlcNAc overnight. After OGT treatment, the proteins were incubated with Y298L GalT1 and UDP-GalNAz, and reacted with alkyne-PEG2k. The calculated stoichiometry was shown below the gels. In a and c, coomassie brilliant blue (CBB)-stained gels were shown as the loading control. In a-d, representative results are shown from three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–9 and procedures for solid-phase peptide synthesis.

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Lv, P., Du, Y., He, C. et al. O-GlcNAcylation modulates liquid–liquid phase separation of SynGAP/PSD-95. Nat. Chem. 14, 831–840 (2022). https://doi.org/10.1038/s41557-022-00946-9

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