GLYCOSYLTRANSFERASE FUNCTION DURING FERTILIZATION

受精过程中的糖基转移酶功能

• 批准号: 2888952
• 负责人: BARRY D SHUR
• 金额: $ 26.79万
• 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 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2888952
• 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感兴趣 哺乳动物施肥和发育过程中的相互作用。我们的 先前的研究已经确定了一种新型的精子表面受体。 半乳糖基转移酶（Galtase），可促进配子识别 通过与其特异性寡糖配体结合 鸡蛋的细胞外涂料。虽然传统上是galtase 被视为细胞内生物合成酶，我们现在知道 Galtase的同学变体在细胞表面表达 部分是由于独特的细胞质序列覆盖了 高尔基保留信号，将仪表酶运输到细胞表面。 此续签应用的目的是继续我们的分析 小鼠施肥期间的仪酶功能。到目前为止， HD 23479支持了14个同行评审中报告的工作 出版物，其结果可以总结如下。 galtase首先在早期的生殖细胞表面表达 精子发生，在生殖细胞粘附过程中可能起作用 到Sertoli细胞。在附睾中，可溶性糖苷与 精子表面，掩盖仪表酶。这些竞争性糖苷是 在电容期间从精子表面脱落。因此暴露了 Galtase结合位点与其在鸡蛋上的配体相互作用 糖蛋白，ZP3。通过多价ZP3聚合Galtase的聚合 寡糖激活异三聚体0蛋白级联反应， 导致Adrosos反应。 ACROSOME释放 水解酶。包括N-乙酰葡萄糖氨基酶。那 通过Zona促进精子穿透。鸡蛋后 激活，N-乙酰葡萄糖氨基酶从全球释放 皮质颗粒破坏了ZP3上的Galtase识别位点， 因此导致多性体验结合中的块。在此续约中 应用，我们希望根据其他 它可能关联的精子蛋白。我们打算：我） 检查ZP3结合对磷酸化的影响 galtase细胞质结构域，2）识别负责的激酶 磷酸化精子旋构酶，3）检查 0-蛋白激活上的磷酸化并鉴定氨基酸 负责G蛋白的细胞质结构域中的残留物 激活，4）确定精子蛋白的曲目 构成信号转导复合物与 加仑酶细胞质结构域，5）确定是否是否 精子galtase的细胞质结构域定义了一类新的类 睾丸特异性受体，6）检查细胞外是否是否 与其他膜的精子仪酶配合物的结构域 也可能有助于ZP3结合的蛋白质。