NEXIN-I IN THE REGULATION OF EXTRACELLULAR PROTEASES

蛋白酶连接蛋白-I（PN-I）是一类细胞分泌的蛋白酶， 蛋白质抑制剂统称为蛋白酶 连接蛋白（PN）。 它的分子量为43，000道尔顿， 与血浆蛋白酶抑制剂相关但不同， 肝素辅因子-II（HC-II）和抗凝血酶-III（AT-III）。 PN-I 与凝血酶，纤溶酶， 尿激酶和胰蛋白酶，并介导它们的失活，结合， 内化和降解。 因此，PN-I很可能是 一个重要的监管因素，在一些重要的 这些蛋白酶参与的生物过程。 的 将检测PN-I与细胞的相互作用。 受体 介导PN-I的结合和内化：蛋白酶 复合物将在分子水平上表征，纯化， 并产生针对它的单克隆抗体。 的 分子表征将包括化学交联 125 I-蛋白酶：PN-I与人成纤维细胞上受体的复合物。 这种受体将从人类胎盘膜中提纯 使用标准的生物化学方法。 要么是部分纯化 或同质受体将用于产生单克隆抗体。 抗体的 这些抗体将被用来研究 和PN-I受体的调节。 这将在很大程度上 通过采用代谢标记和代谢标记的组合来实现 用35 S-甲硫氨酸进行免疫沉淀。 PN-I也 通过细胞表面和细胞外基质与细胞相互作用 糖胺聚糖 这导致了联动加速 PN-I和凝血酶在成纤维细胞上的形成。 肝素 对PN-I的结合结构域进行鉴定、纯化和测序 使用蛋白水解消化，然后 反相高效液相分离 层析 25个氨基酸或更少的肽将是 合成了在已鉴定的肝素结合内重叠的 PN-I的结构域和抗体将针对它们产生。 这些 抗体将被用来验证的生理相关性， 通过评估它们的能力来鉴定肝素结合域， 阻断肝素结合和/或增加蛋白酶抑制活性。

Protease nexin-I(PN-I) is representative of a class of cell-secreted protein inhibitors collectively referred to as the Protease Nexins(PN's). It has a molecular weight of 43,000 Daltons, and is related to, but distinct from the plasma-borne protease inhibitors, heparin cofactor-II(HC-II) and antithrombin-III(AT-III). PN-I forms covalently-linked complexes with thrombin, plasmin, urokinase and trypsin, and mediates their inactivation, binding, internalization and degradation by cells. Thus, PN-I is likely to be a significant regulatory element in a number of important biological processes where these proteases are involved. The interaction of PN-I with cells will be examined. The receptor that mediates the binding and internalization of PN-I:protease complexes will be characterized at the molecular level, purified, and monoclonal antibodies against it will be generated. The molecular characterization will include chemical crosslinking of 125I-Protease:PN-I complexes to receptors on human fibroblasts. The receptor will be purified from human placental membranes using standard biochemical approaches. Either partially purified or homogeneous receptor will be used to generate monoclonal antibodies. These antibodies will be used to study the dynamics and regulation of the PN-I receptor. This will largely be accomplished by employing a combination of metabolic labeling with 35S-methionine followed by immunoprecipitation. PN-I also interacts with cells via cell-surface and extracellular matrix glycosaminoglycans. This leads to an acceleration of linkage formation between PN-I and thrombin on fibroblasts. The heparin binding domain of PN-I will be identified, purified and sequenced using a combination of proteolytic digestion followed by separation on reverse-phase high performance liquid chromatography. Peptides of 25 amino acid or less will be synthesized that overlap within the identified heparin binding domain of PN-I and antibodies will be raised against them. These antibodies will be used to verify the physiological relevance of the identified heparin binding domain by assessing their ability to block heparin binding and/or augment protease inhibitory activity.