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

• Published in: Nature chemistry 2022-07, Vol.14 (7), p.831-840
• Main Authors: Lv, Pinou, Du, Yifei, He, Changdong, Peng, Luxin, Zhou, Xinyue, Wan, Yi, Zeng, Menglong, Zhou, Wen, Zou, Peng, Li, Chenjian, Zhang, Mingjie, Dong, Suwei, Chen, Xing
• Format: Article
• Language: English
• Subjects: 631/378/2591; 639/638/92; Analytical Chemistry; Antibodies; Biochemistry; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Density; Inorganic Chemistry; Laboratories; Life sciences; Liquid phases; Neurons; O-GlcNAcylation; Organic Chemistry; Phase separation; Phosphorylation; Physical Chemistry; Post-translation; Postsynaptic density; Postsynaptic density proteins; Proteins; Regulatory mechanisms (biology); RNA polymerase; Semisynthesis; Synapses; Variance analysis
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/s41557-022-00946-9

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. SynGAP and PSD-95 are two abundant proteins that form a complex and undergo liquid–liquid phase separation (LLPS) in the postsynaptic density of neurons. Now, O -GlcNAcylation of SynGAP has been found to suppress LLPS of the SynGAP/PSD-95 complex, and O -GlcNAc-dependent LLPS was also shown to be dynamically regulated by the enzymes O -GlcNAc transferase and O -GlcNAcase.