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 和 PSD-95 的液-液相分离 (LLPS) 是两种在神经元突触后密度 (PSD) 中相互作用的丰富蛋白质，与调节兴奋性突触中的 SynGAP PSD 富集有关。然而，潜在的监管机制仍然是神秘的。在这里，我们报告了SynGAP 的O -GlcNAcylation 作为 SynGAP/PSD-95 复合物的 LLPS 的抑制剂。我们为从大鼠脑中分离的内源性 SynGAP 和重组表达的蛋白质鉴定了多个O -GlcNAc 修饰位点。蛋白质半合成用于生成位点特异性O -GlcNAcylated 形式的 SynGAP，体外和基于细胞的 LLPS 测定表明 T1306 OSynGAP 的-GlcNAc 阻断与 PSD-95 的相互作用，从而抑制 LLPS。此外，O -GlcNAcylation 以显性负向方式抑制 SynGAP/PSD-95 LLPS，使亚化学计量的O - GlcNAcylation 发挥有效调节作用。我们还表明O -GlcNAc 依赖性 LLPS 受O - GlcNAc 转移酶 (OGT) 和O -GlcNAc (OGA) 的可逆调节。这些发现表明，OGT 和 OGA 催化的O -GlcNAc 循环可作为 LLPS 调节翻译后修饰。