Studies on Protein Structure and Physiological Function of Ovalbumin with Property of Molecular Chaperon

卵清蛋白是具有碳水化合物和磷酸盐残基的主要蛋白，属于丝氨酸蛋白酶抑制剂超家族。然而，卵清蛋白对蛋白酶没有抑制作用，因此，除了作为营养来源的可能贡献外，其功能仍不确定。我们以前报道过卵清蛋白分子从蛋白经蛋黄、羊水、血清等转运到胚胎中，不降解，分布在包括中枢神经系统在内的各个器官中，同时这些分子发生二级构象的变化，达到热稳定状态（这种状态的分子将被称为“hs -卵清蛋白”）。在这里，我们证明了通过目前制备的单克隆抗体可以将hs -卵白蛋白与已知的热稳定分子形式s -卵白蛋白区分开来，并且hs -卵白蛋白来自其在发育中的天然形式（n -卵白蛋白）。我们还建立了体外培养条件，在体外培养条件下，胚胎发生4期及以后的卵清蛋白可以成功培养至少24小时。当用多克隆抗体中和内源性卵清蛋白的残余部分时，分离的胚胎不能正常生长，在脑组织中特别不规则，几乎不会发生正常胚胎在9期发生的神经管闭合。通过在培养早期向培养基中添加过量的卵清蛋白，使胚胎免于畸变。hs -卵白蛋白的抢救率高于n -和s -卵白蛋白。我们的研究结果表明，卵清蛋白对胚胎器官发生是不可或缺的，并且可能在胚胎器官发生中起着至关重要的作用。

Ovalbumin, which is the major albumen protein having carbohydrate and phosphate residues, belongs to the serine protease inhibitor superfamily. However, ovalbumin is not inhibitory against proteases, and, therefore, its function remains uncertain except for a possible contribution as a nutrient source. We reported previously that ovalbumin molecules were transported from albumen to the embryo via egg yolk, amniotic fluid, serum etc, without degradation, and distributed in various organs including the central nervous system, while these molecules undergo changes in secondary conformation and attain a heat-stable state (this state of molecules will be called 'HS-ovalbumin'). Here we showed that HS-ovalbumin is distinguishable from the known heat-stable molecular form named S-ovalbumin by a presently prepared monoclonal antibody and that HS-ovalbumin arises from its native form (N-ovalbumin) in developing eggs. We also established in vitro culture conditions, under which embryos isolated from eggs at and after stage 4 of embryogenesis could be successfully cultured for at least 24 h. When the remnant fractions of endogenous ovalbumin was neutralized by using a polyclonal antibody, the isolated embryos could not grow normally, being particularly irregular in the brain tissue, which hardly undergo the neural tube closure occurring in normal embryos at stage 9. The embryos were rescued from the aberration by adding an excess amount of ovalbumin to the medium at an early period of culture. HS-ovalbumin showed a higher rescue rate than the N-and S-counterparts. Our findings demonstrated that ovalbumin is indispensable to, and probably plays a crucial role in, the embryonic organogenesis.