Glycosyltransferase function during fertilization

受精过程中糖基转移酶的功能

• 批准号: 6864810
• 负责人: BARRY D SHUR
• 金额: $ 34.2万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6864810
• 关键词: G protein; N acetylglucosaminidase; acrosome; binding proteins; biological signal transduction; enzyme activity; fertilization; galactosyltransferases; laboratory mouse; membrane proteins; phosphorylation; protein kinase; receptor; receptor binding; sperm; zona pellucida glycoproteins

DESCRIPTION (provided by applicant): My laboratory is interested in the molecular basis of cellular interactions during mammalian fertilization. During the past 20 years, we have used a variety of biochemical and molecular approaches to characterize an unusual sperm surface receptor (Beta-1,4-galactosyltransferase I, GaIT) for glycoside ligands in the egg zona pellucida. With the exception of some early work, HD 23479 has supported all of our studies of GaIT function during fertilization. GaIT is traditionally viewed as a biosynthetic enzyme in the Golgi complex, although a small amount also appears on the surface of somatic cells. Sperm are unusual in that all of their GaIT is expressed on the sperm surface, where it selectively binds to the egg coat glycoprotein, ZP3. In response to ZP3-induced aggregation, GaIT activates, directly or indirectly, a pertussis toxin-sensitive G-protein cascade, contributing to the acrosome reaction. Our recent studies have examined the consequences of altering GaIT expression, both positively and negatively, on heterologous cells and on mouse sperm. In this regard, Xenopus oocytes expressing murine GaIT bind ZP3, and aggregation of GaIT by ZP3 or anti-GaIT antibodies induces G-protein activation and subsequent activation of the Xenopus oocyte. Similarly, sperm that overexpress GaIT bind more ZP3 and undergo accelerated G-protein activation and acrosome reactions. In contrast, sperm that are null for GaIT do not bind ZP3 and do not undergo ZP3-dependent acrosome reactions. These results show clearly that Gall functions as a ZP3 receptor and is capable of activating a pertussis toxin-sensitive G-protein cascade. Despite the fact that GaIT-null sperm do not bind ZP3, they still bind to the zona pellucida, although they do not undergo subsequent zona-induced acrosomal exocytosis. This implies that non-GalT:non-ZP3 receptor:ligand interactions mediate initial sperm-zona adhesion, and that GalT-ZP3 interactions participate in subsequent acrosomal exocytosis. The specific Aims of this proposal will examine candidates for these newly appreciated sperm receptors and egg coat ligands. In the first Aim, we will test the hypothesis that the egg coat ligand responsible for sperm adhesion is a ZP3-independent, peripherally associated component of the egg coat. The second Aim will test whether the sperm protein p47 functions as a binding protein for the egg coat. Finally, in the third Aim, we will continue our analysis of GalT-mediated signal transduction leading to the acrosome reaction.

描述（由申请人提供）：我的实验室对哺乳动物受精过程中细胞相互作用的分子基础感兴趣。在过去的20年中，我们使用了多种生化和分子方法来表征异常的精子表面受体（β-1,4-甲基乳糖基转移酶I，步态），用于卵Zona pellucida中的糖苷配体。除了一些早期工作外，HD 23479支持我们在受精过程中所有步态功能的研究。 传统上，步态被视为高尔基体复合物中一种生物合成酶，尽管在体细胞的表面上也出现了少量。精子是不寻常的，因为它们的所有步态均在精子表面上表达，在那里它有选择地结合了卵外胶糖蛋白zp3。为了响应ZP3诱导的聚集，步态直接或间接地激活百日咳毒素敏感的G蛋白级联反应，从而导致了ACROSOM反应。我们最近的研究已经检查了在异源细胞和小鼠精子上改变步态表达的后果。在这方面，表达鼠步态的爪蟾卵母细胞结合ZP3，ZP3或抗基因抗体的步态聚集会诱导G蛋白的激活，并随后激活Xenopus卵母细胞。同样，过表达步态的精子结合了更多的ZP3并经历加速的G蛋白激活和ACROSOM反应。相比之下，步态为null的精子不结合ZP3，并且不经历ZP3依赖性的Acrosom反应。这些结果清楚地表明，胆囊充当ZP3受体，并且能够激活百日咳毒素敏感的G蛋白级联反应。 尽管步态无效的精子不结合ZP3，但它们仍然与Zona pellucida结合，尽管它们没有随后会导致Zona诱发的脊髓胞菌病。这意味着非galt：非ZP3受体：配体相互作用介导了初始的精子 - Zona粘附，而Galt-Zp3相互作用参与了随后的杂色胞吐作用。该提案的具体目的将检查这些新欣赏的精子受体和鸡蛋外配体的候选人。在第一个目的中，我们将测试以下假设：卵外壳配体负责精子粘附是卵外涂层的ZP3无关，外围相关的成分。第二个目标将测试精子蛋白p47是否起卵外涂层的结合蛋白。最后，在第三个目标中，我们将继续对GALT介导的信号转导的分析，从而导致Adrosos反应。