STRUCTURE-FUNCTION OF HUMAN SERPIN REGULATORY DOMAINS

人抗凝血酶III和人蛋白酶Nexin I是 大丝氨酸蛋白酶抑制物(SERPIN)家族。目前的研究是 重点阐述了两个重要的基因操作 这些SERPIN中的调节域。肝素结合区介导 肝素对两者的抗蛋白酶活性的激活，以及 建议负责细胞外基质的定位。这个 肝素活性的丧失在基因上与几个 血栓性疾病涉及血管紧张素转换酶III。这一领域已经被生化 并免疫定位于D螺旋及其邻近区域 ATIII和PN1。一系列定点突变体，靶向赖氨酸和 精氨酸残基将在杆状病毒中构建和表达，以 确定肝素结合和激活所必需的残留物，以及 肝素结合在细胞外基质中的作用 本地化。另一个感兴趣的区域是清除受体。 结合结构域。这个结构域在生物化学上定位于 与SERPIN密切相关的α-1-抗胰蛋白酶的羧基末端区域。 将使用两种方法来确定受体结合域是否 ATIII和PN1的位置相似。两国之间的关系 ATIII和PN1清除受体，以及α-受体。 1-在肝细胞中发现的抗胰蛋白酶，也将被研究。在……里面 第一种方法，羧基中的定点缺失突变体 ATIII和PN1的末端区域将被构建并在 杆状病毒评估ATIII和ATIII这些区域的潜在作用 PNL清除受体结合。作为一种补充方法，合成 代表ATIII完整氨基酸序列的多肽文库 和PN1，将通过免疫和受体结合试验进行筛选 来独立地确定这个地点。这些研究的最终目标是 开始Clear受体的鉴定和纯化(S) 调节ATIII摄取和降解的物质：蛋白酶和 PN1：蛋白酶复合体。

Human antithrombin III and human protease nexin I are members of the large serine protease inhibitor (SERPIN) family. The present studies are focused on the elucidation and genetic manipulation of two important regulatory domains in these SERPINS. The heparin binding domain mediates the activation of the anti-protease activities of both by heparin, and proposedly is responsible for extracellular matrix localization. The loss of heparin activation has been genetically linked to several thrombotic disorders involving ATIII. This domain has been biochemically and immunologically localized to the D helix and adjacent regions in both ATIII and PN1. A series of site-directed mutants, targeting lysine and arginine residues, will be constructed and expressed in baculovirus to determine the essential residues for heparin binding and activation, and to evaluate the role of heparin binding in extracellular matrix localization. The other region of interest is the clearance receptor binding domain. This domain has been biochemically localized to the carboxy terminal region of a closely related SERPIN, alpha-1-antitrypsin. Two approaches will be used to determine if the receptor binding domains of ATIII and PN1 are similarly located. The relationship between the ATIII and PN1 clearance receptors, and the clearance receptor for alpha- 1 -antitrypsin identified in liver cells, will also be investigated. In the first approach, site-directed deletion mutants in the carboxy terminal regions of ATIII and PN1 will be constructed and expressed in baculovirus to evaluate the potential role of these regions of ATIII and PNl clearance receptor binding. As a complementary approach, synthetic peptide libraries representing the entire amino acid sequences of ATIII and PN1, will be screened immunologically and by receptor binding assays to identify this site independently. The final goal of these studies is to begin a characterization and purification of the clearance receptor(s) that mediate the uptake and degradation of ATIII:Protease and PN1:Protease complexes.