Department of Anatomy and Embryology, Graduate School of Comprehensive Human Science, University of Tsukuba, Tsukuba, Japan.
Department of Animal Genetic Resources, National Gene Bank, Giza, Egypt.
PLoS One. 2019 Apr 1;14(4):e0214687. doi: 10.1371/journal.pone.0214687. eCollection 2019.
Calcium-binding proteins regulate ion metabolism and the necessary signaling pathways for the maturational events of sperm. Our aim is to identify the novel calcium-binding proteins in testis. The gene EFCAB2 (GenBank NM_026626.3, NP_080902.1) was not previously examined, and its properties and exact mechanisms of action are unknown. In this study, we performed phylogenetic and structure prediction analyses of EFCAB2, which displays definitive structural features. Additionally, the distribution, localization, and calcium binding ability of mouse EFCAB2 were investigated. Results revealed extensive conservation of EFCAB2 among different eukaryotic orthologs. The constructed 3D model predicted that mouse EFCAB2 contains seven α-helices and two EF-hand motifs. The first EF-hand motif is located in N-terminal, while the second is located in C-terminal. By aligning the 3D structure of Ca2+-binding loops from EFCAB2 with calmodulin, we predicted six residues that might be involved in Ca2+ binding. The distribution of the Efcab2 mRNA, as determined by northern blotting, was detected only in the testis among mouse tissues. Native and recombinant EFCAB2 protein were detected by western blotting as one band at 20 kDa. In situ hybridization and immunohistochemical analyses showed its localization specifically in spermatogenic cells from primary spermatocytes to elongate spermatids within the seminiferous epithelium, but neither spermatogonia nor somatic cells were expressed. Moreover, EFCAB2 was specifically localized to the principal piece of cauda epididymal sperm flagellum. Furthermore, the analyses of purified recombinant EFCAB2 by Stains-all, ruthenium red staining, and by applying in vitro autoradiography assay showed that the physiological function of this protein is Ca2+ binding. These results suggested that EFCAB2 might be involved in the control of sperm flagellar movement. Altogether, here we describe about EFCAB2 as a novel calcium-binding protein in mouse testis and sperm.
钙结合蛋白调节离子代谢和精子成熟事件所必需的信号通路。我们的目的是鉴定睾丸中的新型钙结合蛋白。基因 EFCAB2（GenBank NM_026626.3，NP_080902.1）以前未被研究过，其性质和确切的作用机制尚不清楚。在这项研究中，我们对 EFCAB2 进行了系统发育和结构预测分析，结果显示其具有明确的结构特征。此外，还研究了小鼠 EFCAB2 的分布、定位和钙结合能力。结果表明，EFCAB2 在不同的真核同源物中广泛保守。构建的 3D 模型预测，小鼠 EFCAB2 含有七个α-螺旋和两个 EF 手模体。第一个 EF 手模体位于 N 端，第二个位于 C 端。通过将 EFCAB2 的钙结合环的 3D 结构与钙调蛋白进行比对，我们预测了六个可能参与钙结合的残基。通过 northern blot 检测到 Efcab2 mRNA 的分布仅在小鼠组织的睾丸中。通过 western blot 检测到天然和重组 EFCAB2 蛋白均在 20 kDa 处出现一条带。原位杂交和免疫组织化学分析显示，它在生精上皮的初级精母细胞到伸长的精子细胞中特异性定位于精子细胞，但精原细胞和体细胞均不表达。此外，EFCAB2 特异性定位于附睾尾部精子鞭毛的主段。此外，通过 Stains-all、钌红染色和应用体外放射自显影分析对纯化的重组 EFCAB2 进行的分析表明，该蛋白的生理功能是钙结合。这些结果表明，EFCAB2 可能参与精子鞭毛运动的控制。总之，我们在这里描述了 EFCAB2 作为一种新型的钙结合蛋白在小鼠睾丸和精子中的作用。
