GLYCOSYLTRANSFERASE FUNCTION DURING FERTILIZATION

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。我们的 以前的研究已经确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 通过与其特定的寡糖配体结合在 卵子的细胞外衣。尽管GalTase传统上是 被视为细胞内的生物合成酶，我们现在知道 在细胞表面表达GalTase的同工酶变体 部分原因是一种独特的细胞质序列，它覆盖了 高尔基体滞留-信号，运输GalTase到细胞表面。 此续约申请的目的是继续我们的分析 小鼠受精过程中GalTase功能的变化。到目前为止， 房屋署23479支持在14个同行评审中报告的工作 出版物，其结果可概括如下。 GalTase最初在生殖细胞表面表达是在早期 精子发生，在生殖细胞黏附过程中可能发挥作用 至支持细胞。在附睾体中，可溶性糖苷结合在 精子表面，掩盖了GalTase。这些具有竞争力的糖苷是 在获能过程中从精子表面脱落。从而暴露了 半乳糖苷酶与蛋壳上配体相互作用的结合部位 糖蛋白，ZP3。多价ZP3对GalTase的聚集作用 低聚糖激活异三聚体0-蛋白质级联反应， 导致顶体反应。顶体释放 水解酶。包括N-乙酰氨基葡萄糖苷酶。那 促进精子穿透小带。蛋后 激活，N-乙酰氨基葡萄糖苷酶的全球释放 皮质颗粒破坏ZP3上的GalTase识别位点， 从而导致多精细胞结合受阻。在这次更新中 应用程序，我们希望结合其他函数来处理GalTase函数 可能与之相关的精子蛋白。我们打算：i) 检测ZP3结合对肌动蛋白磷酸化的影响 GalTase胞质结构域，2)鉴定负责 磷酸化精子半乳糖酶，3)检测 磷酸化对0-蛋白活性的影响及氨基酸鉴定 决定G蛋白的细胞质结构域内的残基 激活，4)确定精子蛋白质库中 构成与信号转导有关的复合体 GalTase胞质结构域，5)确定 精子半乳糖苷酶的胞质结构域定义了一类新的 睾丸特异性受体，以及6)检查细胞外 精子半乳糖酶与其他膜复合体的结构域 也可能有助于ZP3结合的蛋白质。

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.