Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
Department of Quantum Life Science, Graduate School of Science and Engineering, Chiba University, Chiba 263-8522, Japan.
Biomolecules. 2023 Nov 10;13(11):1636. doi: 10.3390/biom13111636.
Mammalian fertilization is a species-selective event that involves a series of interactions between sperm proteins and the oocyte's zona pellucida (ZP) glycoproteins. Bovine ZP consists of three glycoproteins: bZP2, bZP3, and bZP4. In our previous study, we demonstrated that bovine sperm binds to plastic wells coated with recombinant bZP4 and identified that the -terminal domain and the middle region of bZP4 are critical for sperm-binding activity. Here, we investigated the sperm-binding site in the middle region (residues 290 to 340) of bZP4, which includes the hinge region. We showed that bovine sperm binds to bZP4's middle region in a species-selective manner. We mapped the function of bZP4's middle region to its -glycosylation site at Asn-314 using several recombinant mutated proteins. Moreover, we showed that mutations of the -glycosylation sites at Asn-314 close to the hinge region and Asn-146 of the hinge region of bZP4 and bZP3, respectively, reduced the sperm-binding activity of the complex of the bZP3 (from 32 to 178) and bZP4 (from 136 to 464) fragments. Together, these results suggest that ZP's middle regions of bZP3 and bZP4 form one of the sperm-binding sites of bovine ZP.
哺乳动物受精是一个物种选择性事件，涉及一系列精子蛋白与卵母细胞透明带（ZP）糖蛋白之间的相互作用。牛 ZP 由三种糖蛋白组成：bZP2、bZP3 和 bZP4。在我们之前的研究中，我们证明了牛精子与包被有重组 bZP4 的塑料孔结合，并确定 bZP4 的 C 端结构域和中间区域对于精子结合活性是至关重要的。在这里，我们研究了 bZP4 中间区域（残基 290 到 340）的精子结合位点，该区域包括铰链区。我们表明，牛精子以物种选择性的方式与 bZP4 的中间区域结合。我们使用几种重组突变蛋白，将 bZP4 中间区域的功能映射到其在 Asn-314 的 -糖基化位点。此外，我们表明 bZP4 和 bZP3 的铰链区域的 Asn-314 附近的 -糖基化位点以及铰链区域的 Asn-146 的突变分别降低了 bZP3（从 32 到 178）和 bZP4（从 136 到 464）片段复合物的精子结合活性。总之，这些结果表明，bZP3 和 bZP4 的 ZP 中间区域形成了牛 ZP 的一个精子结合位点。
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