GLYCOSYLTRANSFERASE FUNCTION DURING FERTILIZATION

受精过程中的糖基转移酶功能

• 批准号: 6182226
• 负责人: BARRY D SHUR
• 金额: $ 27.43万
• 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 2002-05-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182226
• 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

My laboratory is interested in the molecular basis of cellular interactions during mammalian fertilization and development. Our previous studies have identified a novel sperm surface receptor. galactosyltransferase(GalTase), that facilitates gamete recognition by binding to its specific oligosaccharide ligand on the extracellular coat of the egg. Although GalTase is traditionally viewed as an intracellular biosynthetic enzyme, we now know that an isozymic variant of GalTase is expressed on the cell surface due, in part, to a unique cytoplasmic sequence that overrides the Golgi retention- signal, transporting GalTase to the cell surface. The purpose of this renewal application is to continue our analysis of GalTase function during fertilization in the mouse. Thus far, HD 23479 has supported the work reported in 14 peer-reviewed publications, the results of which can be summarized as follows. GalTase is first expressed on the germ cell surface during early spermatogenesis, where it may function during germ cell adhesion to Sertoli cells. In the epididymis, soluble glycosides bind to the sperm surface, masking GalTase. These competitive glycosides are shed from the sperm surface during capacitation. thus exposing the GalTase binding site for interaction with its ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a heterotrimeric 0-protein cascade, leading to the acrosome reaction. The acrosome releases hydrolytic enzymes. including N-acetylglucosaminidase. that facilitates sperm penetration through the zona. After egg activation, the global release of N-acetylglucosaminidase from the cortical granules destroys the GalTase recognition site on ZP3, thus leading to the block in polyspermic binding. In this renewal application, we wish to address GalTase function in light of other sperm proteins with which it may associate. We intend to: I) examine the effect of ZP3 binding on the phosphorylation of the GalTase cytoplasmic domain, 2) identify the kinase responsible for phosphorylating sperm GalTase, 3) examine the effects of phosphorylation on 0-protein activation and identify amino acid residues within the cytoplasmic domain responsible for G-protein activation, 4) identify the repertoire of sperm proteins that constitute the signal transduction complex in association with the GalTase cytoplasmic domain, 5) determine whether the cytoplasmic domain of sperm GalTase defines a novel class of testis-specific receptors, and 6) examine whether the extracellular domain of sperm GalTase complexes with other membrane proteins that may also contribute to ZP3 binding.

我的实验室对细胞的分子基础很感兴趣 哺乳动物受精和发育过程中的相互作用。我们 先前的研究已经鉴定了一种新的精子表面受体。 半乳糖基转移酶（GalTase），促进配子识别 通过结合到其特定的寡糖配体上， 卵的细胞外膜。虽然GalTase传统上 作为一种细胞内的生物合成酶，我们现在知道， GalTase的同工酶变体在细胞表面表达 部分是由于一种独特的细胞质序列， 高尔基体滞留-信号，将GalTase运输到细胞表面。 这次更新申请的目的是继续我们的分析 在小鼠受精过程中的GalTase功能。到目前为止， HD 23479支持了14个同行评审报告中报告的工作 出版物，其结果可以总结如下。 GalTase首先表达于生殖细胞表面， 精子发生，可能在生殖细胞粘附过程中发挥作用 支持细胞在附睾中，可溶性糖苷结合到 精子表面，掩蔽半乳糖苷酶。这些竞争性糖苷是 在获能过程中从精子表面脱落。因此暴露 鸡蛋壳上半乳糖转移酶与其配体相互作用的结合位点 糖蛋白ZP 3。多价ZP 3对GalTase的聚集作用 寡糖激活异源三聚体0-蛋白质级联， 导致顶体反应。顶体释放 水解酶包括N-乙酰氨基葡糖苷酶。的 促进精子穿透尿道。卵后 激活，N-乙酰氨基葡萄糖苷酶从细胞中的整体释放， 皮质颗粒破坏了ZP 3上的GalTase识别位点， 从而导致多精结合的阻断。在这次更新中， 应用程序，我们希望根据其他方法来解决GalTase功能 与之相关的精子蛋白。我们打算：（一） 检查ZP 3结合对磷酸化的影响， GalTase胞质结构域，2）鉴定负责 磷酸化精子GalTase，3）检查 磷酸化对0-蛋白活化和鉴定氨基酸 负责G蛋白的胞质结构域内的残基 激活，4）鉴定精子蛋白的所有组成部分， 构成与所述细胞结合的信号转导复合物。 GalTase胞质结构域，5）确定是否 精子GalTase的胞质结构域定义了一类新的 睾丸特异性受体，和6）检查细胞外是否 精子GalTase复合物与其他膜的结构域 也可能有助于ZP 3结合的蛋白质。