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

卵清蛋白属于丝氨酸蛋白酶抑制剂超家族，是含有碳水化合物和磷酸盐残基的主要蛋白。然而，卵清蛋白对蛋白水解酶没有抑制作用，因此，除了可能作为营养来源的贡献外，其功能仍不确定。我们以前报道过，卵清蛋白分子通过蛋黄、羊水、血清等不降解地从蛋白转运到胚胎，并分布在包括中枢神经系统在内的各种器官中，而这些分子经历了次级构象的变化并达到了热稳定状态(这种状态被称为HS-卵清蛋白)。在这里，我们发现HS-卵白蛋白与已知的热稳定的分子形式S卵白蛋白是可以区分的，并且HS-卵白蛋白来源于其天然形态(N-卵清蛋白)在发育的卵子中。我们还建立了体外培养条件，在此条件下，从胚胎发生第4阶段和之后的卵子中分离出的胚胎可以成功培养至少24小时，当用多克隆抗体中和内源性卵白蛋白的残留部分时，分离的胚胎不能正常生长，尤其是脑组织中不规则，几乎不经历正常胚胎在第9阶段的神经管闭合。在培养早期，通过在培养液中添加过量的卵白蛋白，使胚胎免于畸形。HS-卵清蛋白的抢救率高于N-和S-卵白蛋白。我们的发现表明，卵清蛋白在胚胎器官发生中是不可或缺的，并且可能起着关键作用。

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.