Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion
Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion
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DOI：
10.1242/dev.094854
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发表时间：
2013-08-01
期刊：
影响因子：
4.6
通讯作者：
Hagihara, Yoshihisa
中科院分区：
文献类型：
--
作者：
Inoue, Naokazu;Hamada, Daizo;Hagihara, Yoshihisa
尽管精子和卵细胞的膜融合是受精的核心事件，但其潜在的分子机制实际上仍然未知。基因破坏研究表明，精子上的 IZUMO1 和卵母细胞上的 CD9 是精卵融合中必需的跨膜蛋白。在这项研究中，我们剖析了 IZUMO1 蛋白，以确定精卵融合功能所需的结构域。我们发现 N 末端的片段（Asp5 至 Leu113）与受精抑制抗体相互作用。它还与卵表面结合并有效抑制体外融合。我们将此片段命名为“IZUMO1 推定功能片段 (IZUMO1(PFF))”。令人惊讶的是，IZUMO1(PPF)仍然保持在Cd9(-/-)卵表面的结合能力。利用圆二色性、沉降平衡和小角X射线散射进行的一系列生物物理测量表明，IZUMO1(PFF)由N端未折叠结构和C端椭圆形螺旋二聚体组成。在不能形成螺旋的 IZUMO1(PFF) 衍生物中没有观察到卵子结合和融合抑制。这些发现表明，IZUMO1 N 末端区域形成螺旋二聚体是其功能所必需的。表达整个IZUMO1分子的Cos-7细胞与卵子结合，IZUMO1在两个细胞之间的界面处积累，但没有观察到融合。这些观察结果表明，IZUMO1 单独不能促进精子-卵子膜融合，但它作为与细胞表面相互作用相关的因子起作用，例如通过与 IZUMO1（PFF-核心）相对应的螺旋区域来束缚膜。
Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment 'IZUMO1 putative functional fragment (IZUMO1(PFF))'. Surprisingly, IZUMO1(PPF) still maintains binding ability on the egg surface of Cd9(-/-) eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1(PFF) is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1(PFF) derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1(PFF-core).