Kim Jingon, Hwang Jae Yeon
Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea.
Department of Molecular Biology, Pusan National University, Busan 46241, Korea.
Anim Biosci. 2025 Apr;38(4):655-664. doi: 10.5713/ab.24.0631. Epub 2024 Dec 13.
Mammalian sperm acquire fertilizing ability in the female reproductive tract and develop hyperactivated motility, which is indispensable for male fertility. Hyperactivated motility is initiated by Ca2+ influx via the sperm-specific ion channel, CatSper. CATSPER1, a CatSper pore subunit, possesses a long N-terminal intracellular domain and its degradation correlates with unsuccessful sperm migration in the female tract. However, the cellular function and molecular significance of the CATSPER1 N-terminal domain are not well understood. Here, we identify the interactome of the CATSPER1 N-terminal domain and propose a function for the intracellular domain in mammalian sperm.
To identify CATSPER1 N-terminus interactome, we produced recombinant CATSPER1-N-terminus in bacterial system. The purified protein was incubated with testicular lysates and eluted together with testicular interacting proteins. The elutes were subjected to proteomic analysis and CATSPER1-N-terminus interactome was profiled. Identified proteins were further analyzed by functional annotation.
We purified the partial CATSPER1 N-terminal domain and identified 57 testicular proteins as domain interactomes using mass spectrometry analysis. Functional annotation analysis revealed that 106 gene ontologies were significantly enriched, 16 of which were related to redox processes. We found that antioxidant enzymes, such as PARK7 and PRDX2, 4, and 6, were included in the enriched redox-related gene ontologies.
These results suggest that the CATSPER1 N-terminus could function in defending against oxidative stress to support the successful migration of mammalian sperm to fertilizing sites in the female reproductive tract.
哺乳动物精子在雌性生殖道中获得受精能力并发展出超活化运动能力，这对雄性生育能力至关重要。超活化运动能力由钙离子通过精子特异性离子通道CatSper内流引发。CATSPER1是CatSper孔道亚基，具有一个长的N端胞内结构域，其降解与精子在雌性生殖道中迁移失败相关。然而，CATSPER1 N端结构域的细胞功能和分子意义尚不清楚。在此，我们鉴定了CATSPER1 N端结构域的相互作用组，并提出了该胞内结构域在哺乳动物精子中的功能。
为鉴定CATSPER1 N端相互作用组，我们在细菌系统中产生重组CATSPER1-N端。将纯化的蛋白与睾丸裂解物孵育，并与睾丸相互作用蛋白一起洗脱。对洗脱物进行蛋白质组学分析，描绘出CATSPER1-N端相互作用组。对鉴定出的蛋白质进一步进行功能注释分析。
我们纯化了部分CATSPER1 N端结构域，并通过质谱分析鉴定出57种睾丸蛋白作为结构域相互作用组。功能注释分析显示106个基因本体显著富集，其中16个与氧化还原过程相关。我们发现抗氧化酶，如PARK7和PRDX2、4和6，包含在富集的氧化还原相关基因本体中。
这些结果表明，CATSPER1 N端可能在抵御氧化应激中发挥作用，以支持哺乳动物精子成功迁移至雌性生殖道中的受精部位。
