Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3.
Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3.
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DOI：
10.1091/mbc.01-09-0440
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发表时间：
2002-02
影响因子：
3.3
通讯作者：
H. Qi;Zev Williams;P. M. Wassarman
中科院分区：
文献类型：
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作者：
H. Qi;Zev Williams;P. M. Wassarman
透明带(ZP)是一种高度组织化的细胞外被物，包裹在所有哺乳动物的卵周围。小鼠卵子ZP由三种被称为mZP1-3的糖蛋白组成，它们是由成长中的卵母细胞专门合成、分泌和组装成ZP的。在这里，我们将mZP2和mZP3的表位标记(Myc和Flag)的cDNA显微注射到正在生长的小鼠卵母细胞的生发泡(核)中。用特异性抗体和激光扫描共聚焦显微镜跟踪通透性和非通透性卵母细胞中新生的重组ZP糖蛋白。当将这些cDNA注入后，合成了表位标记的mZP2(Myc-mZP2)和mZP3(Flag-mZP3)，包装成大的细胞内小泡，并由绝大多数卵母细胞分泌。分泌的糖蛋白仅掺入增厚的ZP的最内层，且该区域的新生糖蛋白的数量随着卵母细胞培养时间的增加而增加。与先前的观察一致，mZP2和mZP3 C末端假定的跨膜区在结合到ZP中的新生糖蛋白中缺失。当mZP3 C端附近的共有的Furin裂解位点发生突变，使其不被Furin切割时，mZP3的分泌和组装就会减少。另一方面，被整合到ZP中的mZP3缺乏突变的Furin裂解位点下游的跨膜结构域，这表明其他一些蛋白酶(S)切除了该结构域。这些结果有力地表明，新生的mZP2和mZP3只整合到ZP的最内层，需要切除糖蛋白的C-末端区域才能组装到卵母细胞ZP。
The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.