Glycosyltransferase function during fertilization

受精过程中糖基转移酶的功能

• 批准号: 6542488
• 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/6542488
• 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(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年里，我们使用了各种生化和分子方法来表征卵子透明带中一种不寻常的精子表面受体(β-1，4-半乳糖基转移酶I，GATIT)的糖苷配体。除了一些早期的工作外，HD 23479支持了我们对受精过程中步态功能的所有研究。 步态传统上被认为是高尔基复合体中的一种生物合成酶，尽管体细胞表面也有少量的步态。精子的不同寻常之处在于，他们所有的步态都在精子表面表达，在那里它选择性地与卵壳糖蛋白ZP3结合。作为对ZP3诱导的聚集的反应，步态直接或间接地激活百日咳毒素敏感的G蛋白级联，促进顶体反应。我们最近的研究检测了改变步态表达的结果，无论是积极的还是消极的，对异源细胞和小鼠精子都是如此。在这一点上，表达小鼠步态的非洲爪哇卵母细胞与ZP3结合，ZP3或抗步态抗体聚集步态诱导非洲爪哇卵母细胞G蛋白激活和随后的激活。同样，过度表达步态的精子结合更多的ZP3，并经历加速的G蛋白激活和顶体反应。相比之下，步态为零的精子不与ZP3结合，也不会发生ZP3依赖的顶体反应。这些结果清楚地表明，Gall作为ZP3受体发挥作用，并能够激活百日咳毒素敏感的G蛋白级联。 尽管步态缺失的精子不结合ZP3，但它们仍然与透明带结合，尽管它们不会经历随后由透明带诱导的顶体胞吐。这表明非GalT：Non-ZP3受体：配体相互作用介导了最初的精子-小带黏附，GalT-ZP3相互作用参与了随后的顶体胞吐作用。这项提案的具体目标将审查这些新获得认可的精子受体和卵壳配体的候选者。在第一个目标中，我们将检验这样的假设，即负责精子黏附的卵衣配体是卵子衣的ZP3独立的外周相关成分。第二个目标是测试精子蛋白P47是否作为卵子外壳的结合蛋白发挥作用。最后，在第三个目标中，我们将继续分析GALT介导的导致顶体反应的信号转导。