Regulation of Cellular Interactions by Extracellular Matrix Proteolysis

受精后，卵细胞外基质被修饰以防止精子进一步与卵细胞接触，从而维持受精卵的二倍体状态。以非洲爪蟾为模型系统，确定了多精障碍机制，该机制涉及激活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。已知参与的分子有：1)包膜糖蛋白组分，在受精时进行有限的蛋白水解（gp69）；2)一种凝乳胰蛋白酶样蛋白酶（卵泡酶），定位于卵周间隙（质膜与上覆包膜之间的间隙）内的细胞外基质纤维；3)一种类似胰蛋白酶的蛋白酶从卵子中释放出来，可以激活卵泡酶。在上一个资助期，我们克隆了卵泡酶和gp69，并纯化了胰蛋白酶样蛋白酶，结果表明卵泡酶并不像最初假设的那样直接参与gp69的水解。该研究的具体目标是：(1)纯化和表征直接负责gp69水解的鸡蛋蛋白酶；(2)确定卵泡酶在蛋白酶级联中的作用；(3)克隆胰蛋白酶样蛋白酶进行结构/功能研究；(4)在失活的鸡蛋中免疫定位胰蛋白酶样蛋白酶；(5)确定gp69水解在精子结合和/或包膜硬化中的作用。这项研究的结果将提供对通过细胞外基质的蛋白水解加工调节精子-卵子相互作用的更多理解，这是动物发育的基础机制。

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.