Regulation of Cellular Interactions by Extracellular Matrix Proteolysis

林赛 9723667 受精后，卵细胞外基质被修饰，以防止精子与卵细胞进一步接触，从而维持受精卵的二倍体状态。使用非洲爪蟾作为模型系统，已经鉴定了多精受精机制的阻断，其涉及由从活化的卵释放的蛋白酶对细胞外基质（卵包膜）糖蛋白的有限蛋白水解。涉及的已知分子是：1）在受精时经历有限蛋白水解的包膜糖蛋白组分（gp 69）; 2）胰凝乳蛋白酶样蛋白酶（卵凝乳酶），其定位于卵的卵周空间（质膜和上覆包膜之间的空间）内的细胞外基质纤维;和3）从卵释放的胰蛋白酶样蛋白酶，其激活卵凝乳酶。在最后一个授权期，卵糜酶和gp 69被克隆，胰蛋白酶样蛋白酶被纯化，它被揭示卵糜酶是不是直接负责gp 69水解作为最初的假设。本研究的具体目标是：（1）纯化和鉴定直接负责gp 69水解的鸡蛋蛋白酶;（2）确定卵糜酶在蛋白酶级联反应中的作用;（3）克隆胰蛋白酶样蛋白酶用于结构/功能研究;（4）免疫定位灭活鸡蛋中的胰蛋白酶样蛋白酶;和（5）确定gp 69水解在精子结合和/或包膜硬化中的功能。这项研究的结果将提供一个更好的了解精卵相互作用的调节，通过细胞外基质的蛋白水解处理，这是一种机制，这是动物发育的基础。

Lindsay 9723667 Following fertilization, the egg extracellular matrix is modified to prevent further sperm contact with the egg, thus maintaining the diploid state of the zygote. Using Xenopus laevis as a model system a block to polyspermy mechanism has been identified which involves the limited proteolysis of extracellular matrix (egg envelope) glycoproteins by proteases released from the activated egg. The known molecules involved are: 1) an envelope glycoprotein component which undergoes limited proteolysis at fertilization (gp69); 2) a chymotrypsin-like protease (ovochymase), which is localized to extracellular matrix fibers within the perivitelline space of the egg (the space between the plasma membrane and the overlying envelope); and 3) a trypsin-like protease released from the egg which activates ovochymase. In the last grant period, ovochymase and gp69 were cloned, the trypsin-like protease was purified, and it was revealed that ovochymase is not directly responsible for gp69 hydrolysis as originally hypothesized. Specific objectives for the proposed research are to: (1) Purify and characterize the egg protease directly responsible for gp69 hydrolysis; (2) Determine the role of ovochymase in the protease cascade; (3) Clone the trypsin-like protease for structure/function studies; (4) Immunolocalize the trypsin-like protease within the inactivated egg; and (5) Determine the function of gp69 hydrolysis in sperm binding and/or envelope hardening. The results of this study will provide an increased understanding of the regulation of sperm-egg interactions through proteolytic processing of the extracellular matrix, a mechanism which is fundamental to animal development.