NEXIN-I IN THE REGULATION OF EXTRACELLULAR PROTEASES

NEXIN-I 对细胞外蛋白酶的调节

• 批准号: 3284342
• 负责人: DANIEL J. KNAUER
• 金额: $ 14.67万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284342
• 关键词: affinity chromatography; antibody; antibody formation; autoradiography; binding proteins; cell membrane; chemical binding; crosslink; enzyme complex; fibroblasts; heparin; high performance liquid chromatography; human tissue; laboratory rabbit; membrane proteins; monoclonal antibody; placenta; protease inhibitor; protein biosynthesis; protein sequence; protein structure; radiotracer; receptor; synthetic peptide; tissue /cell culture

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.

蛋白酶Nexin-I(PN-I)是一类具有代表性的细胞分泌蛋白。 统称为蛋白质酶的蛋白质抑制剂 Nexins(PN)。它的分子量为43,000道尔顿，是 与血浆中的蛋白水解酶抑制剂相关但又不同， 肝素辅因子-II(HC-II)和抗凝血酶-III(AT-III)。PN-I 与凝血酶、纤溶酶形成共价连接的络合物， 尿激酶和胰酶，并介导它们的失活，结合， 细胞的内化和降解。因此，Pn-I很可能是 一个重要的监管要素，在许多重要的 涉及这些蛋白酶的生物过程。这个 Pn-I与细胞的相互作用将被研究。感受器 介导PN-I的结合和内化的：蛋白酶 络合物将在分子水平上进行表征，提纯， 就会产生抗它的单抗。这个 分子表征将包括化学交联剂 125I-蛋白酶：人成纤维细胞上与受体的PN-I复合体。 该受体将从人类胎盘膜中提纯 使用标准的生化方法。要么部分提纯 或用同源受体产生单克隆性 抗体。这些抗体将被用来研究动力学 以及对PN-I受体的调节。这将在很大程度上 通过使用代谢标记的组合来实现 然后用35S-蛋氨酸进行免疫沉淀。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。这会导致连杆加速。 PN-I与凝血酶在成纤维细胞上的形成。肝素 将鉴定、纯化和测序Pn-I的结合结构域 使用蛋白质分解消化的组合，然后 反相高效液体的分离 层析法。25个氨基酸或更少的多肽将被 在已鉴定的肝素结合内重叠的合成 将会产生针对它们的PN-I结构域和抗体。这些 抗体将被用来验证生理上的相关性 通过评估其结合肝素的能力来鉴定其肝素结合域 阻断肝素结合和/或增强蛋白水解酶抑制活性。