Zhu Guo-Zhang, Miller Brent J, Boucheix Claude, Rubinstein Eric, Liu Christopher C, Hynes Richard O, Myles Diana G, Primakoff Paul
Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, CA 95616, USA.
Development. 2002 Apr;129(8):1995-2002. doi: 10.1242/dev.129.8.1995.
Gamete fusion is the fundamental first step initiating development of a new organism. Female mice with a gene knockout for the tetraspanin CD9 (CD9 KO mice) produce mature eggs that cannot fuse with sperm. However, nothing is known about how egg surface CD9 functions in the membrane fusion process. We found that constructs including CD9's large extracellular loop significantly inhibited gamete fusion when incubated with eggs but not when incubated with sperm, suggesting that CD9 acts by interaction with other proteins in the egg membrane. We also found that injecting developing CD9 KO oocytes with CD9 mRNA restored fusion competence to the resulting CD9 KO eggs. Injecting mRNA for either mouse CD9 or human CD9, whose large extracellular loops differ in 18 residues, rescued fusion ability of the injected CD9 KO eggs. However, when the injected mouse CD9 mRNA contained a point mutation (F174 to A) the gamete fusion level was reduced fourfold, and a change of three residues (173-175, SFQ to AAA) abolished CD9's activity in gamete fusion. These results suggest that SFQ in the CD9 large extracellular loop may be an active site which associates with and regulates the egg fusion machinery.
配子融合是启动新生物体发育的基本第一步。四跨膜蛋白CD9基因敲除的雌性小鼠（CD9基因敲除小鼠）产生的成熟卵子无法与精子融合。然而，关于卵子表面CD9在膜融合过程中的功能却一无所知。我们发现，包含CD9大细胞外环的构建体与卵子一起孵育时会显著抑制配子融合，但与精子一起孵育时则不会，这表明CD9通过与卵膜中的其他蛋白质相互作用发挥作用。我们还发现，向发育中的CD9基因敲除卵母细胞注射CD9 mRNA可使产生的CD9基因敲除卵子恢复融合能力。注射小鼠CD9或人CD9的mRNA（其大细胞外环在18个残基上不同）可挽救注射的CD9基因敲除卵子的融合能力。然而，当注射的小鼠CD9 mRNA包含一个点突变（F174突变为A）时，配子融合水平降低了四倍，三个残基的变化（173 - 175，SFQ变为AAA）则消除了CD9在配子融合中的活性。这些结果表明，CD9大细胞外环中的SFQ可能是一个与卵子融合机制相关并对其进行调节的活性位点。
