Gupta S K, Yurewicz E C, Sacco A G, Kaul R, Jethanandani P, Govind C K
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Mol Hum Reprod. 1998 Nov;4(11):1058-64. doi: 10.1093/molehr/4.11.1058.
Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.
对人透明带糖蛋白进行表征和分类对于理解这些成分在受精过程中的功能至关重要。为实现这一目标，用重组非人类灵长类动物（帽猴，猕猴属）透明带蛋白bmZP1、bmZP2和bmZP3在大肠杆菌中表达后免疫兔子制备抗体。间接免疫荧光显示，针对这三种重组透明带蛋白的抗体与人透明带发生反应。此类抗体被用作特异性探针，在经一维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分离的热溶解人透明带（hSIZP）的蛋白质免疫印迹中进一步表征人透明带糖蛋白。在非还原条件下，人（h）hZP1、hZP2和hZP3分别呈现为60 kDa、100 kDa和53 kDa的条带。在还原条件下，hZP1、hZP2和hZP3的主要反应性分别定位于63 kDa、65 kDa和58 kDa，对53 kDa、96 kDa和138 kDa条带的反应较弱。在二维SDS - PAGE中，hZP1显示由63 - 58 kDa和55 - 45 kDa的两条链组成，每条链由多个异构体组成。与hZP1和hZP3相比，hZP2酸性较弱，由一个65 kDa的主要成分和一个约96 kDa的次要成分组成。与96 kDa成分相比，65 kDa成分显示出更高程度的带电异构体。hZP3在68 - 58 kDa范围内呈现一条宽带。这些研究最终表明，hZP1重链与hZP3重叠，并且在先前的研究中，hZP2可能被误认作hZP1。与先前的报道不同，我们的研究未能区分两种不同的hZP3。这些研究将进一步有助于我们理解人透明带糖蛋白的性质。
