Sasanami Tomohiro, Ohtsuki Mamoru, Ishiguro Tsukasa, Matsushima Kayoko, Hiyama Gen, Kansaku Norio, Doi Yukio, Mori Makoto
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan.
Cells Tissues Organs. 2006;183(1):41-52. doi: 10.1159/000094905.
The extracellular matrix surrounding avian oocytes, referred to as the perivitelline membrane (PL), exhibits a three-dimensional network of fibrils between granulosa cells and the oocyte. We previously reported that one of its components, ZPC, is synthesized in granulosa cells that are specifically incorporated into the PL; this incorporation might be mediated by a specific interaction with ZPB1, another PL constituent, which is synthesized in the liver. In order to extend our previous findings, we established an expression system for quail ZPB1 using a mammalian cell line, and several ZPB1 mutants lacking the zona pellucida (ZP) domain or the glutamine-rich repeat region were produced. Western blot analysis of the immunoprecipitated materials with anti-ZPC antiserum indicated that ZPB1 was coimmunoprecipitated with the antiserum in the presence of ZPC. Ligand blotting also revealed the specific binding of ZPC and ZPB1 and indicated that the binding of these two components might be mediated via an ionic interaction. An analysis using recombinant ZPB1 demonstrated that the ZPB1 lacking the ZP domain did not bind to ZPC, whereas the mutant missing the glutamine-rich repeat region retained its capacity for binding. Furthermore, although the ZPB1 lacking the N-terminal half of the ZP domain was able to bind to ZPC, the deletion of the C-terminal half completely abolished ZPB1 binding to ZPC. These results suggested that the C-terminal half of the ZP domain of ZPB1 contains a binding site for ZPC, and that it appears to be involved in insoluble PL fibril formation in the quail ovary.
鸟类卵母细胞周围的细胞外基质，即卵周膜（PL），在颗粒细胞和卵母细胞之间呈现出三维纤维网络。我们之前报道过，其成分之一ZPC是在颗粒细胞中合成的，并被特异性地整合到PL中；这种整合可能是由与ZPB1（另一种在肝脏中合成的PL成分）的特异性相互作用介导的。为了扩展我们之前的研究结果，我们使用哺乳动物细胞系建立了鹌鹑ZPB1的表达系统，并产生了几种缺乏透明带（ZP）结构域或富含谷氨酰胺重复区域的ZPB1突变体。用抗ZPC抗血清对免疫沉淀物质进行的蛋白质印迹分析表明，在ZPC存在的情况下，ZPB1与抗血清共免疫沉淀。配体印迹也揭示了ZPC和ZPB1的特异性结合，并表明这两种成分的结合可能是通过离子相互作用介导的。使用重组ZPB1进行的分析表明，缺乏ZP结构域的ZPB1不与ZPC结合，而缺失富含谷氨酰胺重复区域的突变体保留了其结合能力。此外，虽然缺乏ZP结构域N端一半的ZPB1能够与ZPC结合，但删除C端一半则完全消除了ZPB1与ZPC的结合。这些结果表明，ZPB1的ZP结构域的C端一半包含一个与ZPC结合的位点，并且它似乎参与了鹌鹑卵巢中不溶性PL纤维的形成。
