突触后致密部蛋白PSD－93与SynGAP相互作用位点的鉴定

Identification of the interaction sites of the postsynaptic density proteins PSD-93 and SynGAP

目的：鉴定突触后致密部蛋白PSD-93与SynGAP结合位点。方法分别构建PSD－93、SynGAP质粒，免疫印迹检测各个重组质粒是否表达，免疫共沉淀检测PSD－93与SynGAP的结合情况；分别缺失突变PSD－93的95－105aa和170－190aa残基以及SynGAP的670－685aa残基，免疫印迹检测各个突变质粒是否表达，免疫共沉淀检测两者的结合情况。结果 PSD－93（1－199aa）－HA可在细胞中过表达，PSD－93（1－109aa）－HA不表达或者表达蛋白不稳定；SynGAP的3个质粒均可在细胞内过表达；PSD－93（1－199aa）均可与SynGAP的3个片段相结合。 PSD－93（1－199aa）缺失突变95－105aa片段后，可在细胞内过表达，且与SynGAP的3个片段均可结合，而敲除170－190aa片段则不能与SynGAP蛋白结合；同样，突变SynGAP（670－685aa）片段则不能与PSD－93蛋白结合。结论 PSD－93与SynGAP结合的位点分别位于PSD－93的170－190aa序列以及SynGAP的670－685aa序列。

Objective To identify the interaction sites of postsynaptic density proteins PSD-93 and SynGAP. Methods PSD-93 and SynGAP plasmids were constructed and their expression was detected by Western blotting.The binding of PSD-93 and SynGAP was examined by immunoprecipitation.Then, mutated deletion was performed at the 95-105aa and 170-190aa residues of PSD-93 as well as the 670-685aa residues of SynGAP.The expression of each mutant plasmid was detected by Western blotting and their binding by co-immunoprecipitation.Results PSD-93 （ 1-199aa）-HA could bed over-expressed in cells.PSD-93 （1-109aa）-HA could not bed expressed or the ex-pressed protein was unstable.All the three plasmids of SynGAP could be over-expressed in cells.PSD-93 （ 1-199aa）-HA protein could bind to any fragment of SynGAP.After mutant deletion of 95-105aa fragment, PSD-93 （1-199aa） could also be over-expressed in cells, and bind to any fragment of SynGAP.In contrast, it could not bind to SynGAP after mutant deletion of 170-190aa fragment.Similarly, SynGAP could not bind to PSD-93 after SynGAP （670-685aa） fragment was mutated.Conclusions The interaction sites of PSD-93 and SynGAP are located in the 170-190aa sequence of PSD-93 and the 670-685aa sequence of SynGAP.