Xu Lu, Sun Sha
Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, 300071, China.
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
J Membr Biol. 2022 Dec;255(6):723-732. doi: 10.1007/s00232-022-00242-0. Epub 2022 May 20.
Mammalian placenta formation requires continuous fusion of trophoblasts. Human endogenous retrovirus-derived proteins syncytin-1 and syncytin-2 mediate cell-cell fusion of placental cytotrophoblasts to form syncytiotrophoblasts in primates, which is required for normal placenta function and fetal development. Syncytins are post-translationally cleaved by the endoprotease furin into surface (SU) and transmembrane (TM) subunits for activation. Little is currently known about the molecular mechanisms of syncytin-mediated cell-cell fusion, and their functions have not been well studied in vitro. Here, we express tagged syncytin-2 in mammalian HEK293T cells and demonstrate that the tagging greatly influences the cleavage and fusogenic activity of syncytin-2. By detecting the N-terminal tagged SU, we find that it is released into the extracellular space during the fusion process. Furthermore, when N-linked glycosylation and disulfide bond formation are blocked, the cleavage and fusogenic activity of syncytin-2 are inhibited. Finally, we were able to purify functional syncytin-2 from HEK293T cells and incorporate it into proteoliposomes. These findings lay a solid foundation for interogating the molecular mechanisms of syncytin-2-mediated cell-cell fusion in vitro.
哺乳动物胎盘的形成需要滋养层细胞持续融合。人类内源性逆转录病毒衍生蛋白合胞素-1和合胞素-2介导胎盘细胞滋养层细胞间的融合，从而在灵长类动物中形成合体滋养层，这是正常胎盘功能和胎儿发育所必需的。合胞素在翻译后被弗林蛋白酶切割成表面（SU）和跨膜（TM）亚基以激活。目前对于合胞素介导的细胞间融合的分子机制知之甚少，并且它们的功能在体外尚未得到充分研究。在此，我们在哺乳动物HEK293T细胞中表达带标签的合胞素-2，并证明该标签极大地影响合胞素-2的切割和融合活性。通过检测N端带标签的SU，我们发现它在融合过程中释放到细胞外空间。此外，当N-糖基化和二硫键形成被阻断时，合胞素-2的切割和融合活性受到抑制。最后，我们能够从HEK293T细胞中纯化出功能性合胞素-2并将其整合到蛋白脂质体中。这些发现为在体外研究合胞素-2介导的细胞间融合的分子机制奠定了坚实的基础。
