人睾丸蛋白SPAG4L与KASH结构域蛋白Nesprin-3的相互作用

Investigation on the interaction of human testis protein SPAG4L with KASH domain protein Nesprin-3

目的探讨含SUN(Sad,UNC-84)结构域的人睾丸蛋白SPAG4L(sperm-associated antigen 4 like)与具有KASH(Klarsicht,ANC-1 and Syne homology)结构域的核膜血影重复蛋白3(nuclear envelop spectrin repeat proteins 3,Nesprin-3)之间的相互作用。方法用生物信息学方法对SPAG4L蛋白进行分析,通过体外转染实验,观察SPAG4L的亚细胞定位;并用免疫荧光技术、免疫共沉淀和双分子荧光互补实验检测SPAG4L是否与KASH结构域蛋白Nesprin-3存在相互作用。结果生物信息学分析结果表明,SPAG4L蛋白具有跨膜结构;亚细胞定位结果发现,SPAG4L蛋白定位于核膜和胞浆;免疫荧光、免疫共沉淀和双分子荧光互补实验结果表明,SPAG4L蛋白与Nesprin-3蛋白质相互作用,形成LINC(linkers of the nucleoskeleton to the cytoskeleton)复合物。结论 SPAG4L与Nesprin-3能够相互作用,形成LINC复合物,对了解SPAG4L蛋白在精子发生中的作用具有重要的意义。

Objective To investigate the interaction of human testis sperm-associated antigen 4 like （SPAG4L） protein containing Sad, UNC-84（SUN） domain with nuclear envelop spectrin repeat proteins 3 （Nesprin-3） containing Klarsicht, ANC-1 and Syne homology （KASH） domain. Methods SPAG4L protein domains were analyzed with the bioinformatics method. After the SPAG4L plasmid was transfected into Ntera-2 cells, the subcellular localization of SPAG4L was observed. The interaction of SPAG4L with Nesprin-3 was de- termined by immunofluorescence, co-immunoprecipitation（co-IP） and bimolecular fluorescence complementation （BiFC） technology, respectively. Results Bioinformatics analysis results showed that there was a transmembrane structure in SPAG4L protein. Subcellular localization results demonstrated that SPAG4L protein located in the nuclear membrane and cytoplasm. Immunofluorescence, Co-IP, and BiFC results showed that there was an interaction between SPAG4L and Nesprin-3, and that a LINC（ linkers of the nucleoskeleton to the cytoskeleton） complex was formed. Conclusion SPAGdL may interact with Nesprin-3 to form a LINC complex, which is important for understanding the function of SPAG4L protein in spermatogenesis.