C1 INHIBITOR GENE AND HEREDITARY ANGIONEUROTIC EDEMA

C1抑制剂（C1INH）是一种丝氨酸蛋白酶抑制剂（serpin），通过C1r、C1s、血浆钾激肽激酶（kallikrein）和因子XII的失活，调节经典补体途径和接触（激肽生成）系统的激活。C1INH缺乏或C1INH蛋白功能失调的杂合性导致遗传性血管性水肿（HAE）。在第一个目标中，将使用基因靶向技术开发HAE的动物模型。我们假设C1NH -/-小鼠无法存活，而C1NH +/-小鼠会发生血管性水肿。为了验证血管水肿是通过接触系统激活介导的这一假设，研究人员将用改变靶蛋白酶特异性的重组C1INH突变体重组缺陷小鼠，并将其与缓激肽受体2 （Bk2R）和C2缺陷小鼠配对。为了验证抑制缓激肽会干扰症状的假设，研究人员用Bk2R拮抗剂治疗HAE小鼠。并对雄激素在血管性水肿中的作用机制进行了分析。为了验证接触系统在脓毒性休克发病机制中发挥作用的假设，我们将测定C1INH +/-小鼠对内毒素休克的易感性。输注具有选择性抑制活性的C1INH蛋白将确定补体和接触系统的作用。第二个目标是针对结构-功能分析。为了验证稳定的复合物形成需要将反应性中心环（RCL）广泛插入β - A片中的假设，将研究RCL突变体和改变β - A片稳定性的突变体。为了验证RCL内外位点决定靶蛋白酶特异性的假设，将测试α - 1抗胰蛋白酶：C1INH嵌合体中的P2突变体、远端环突变体和非RCL突变体。为了验证氨基末端截断的C1INH更有效地抑制表面相关蛋白酶的假设，我们将研究其抑制免疫复合物介导的补体激活、高分子量激肽原（HK）结合的激肽激酶和表面结合因子XIIa失活的能力。为了比较和对比C1INH与其他蛇形蛋白的结构，我们将重组截断的C1INH进行结晶。除了确定C1INH的生物学作用及其功能特征外，这些研究还可能导致改善HAE和其他补体或接触系统激活起作用的疾病的治疗。

C1 inhibitor (C1INH) is a serine proteinase inhibitor (serpin) that regulates activation of the classical complement pathway and the contact (kinin-generating) systems by inactivation of C1r, C1s, plasma kallikrein and factor XII. Heterozygosity for C1INH deficiency or for expression of a dysfunctional C1INH protein results in hereditary angioedema (HAE). In the first Aim, an animal model for HAE will be developed using gene targeting technology. We hypothesize that C1INH-/-mice will not survive and that C1NH +/- mice will develop angioedema. To test the hypothesis that angioedema is mediated via contact system activation, deficient mice will be reconstituted with recombinant C1INH mutants with altered target proteinase specificities, and will be mated with bradykinin receptor 2 (Bk2R) and with C2 deficient mice. To test the hypothesis that inhibition of bradykinin will interfere with symptoms, the HAE mice will the treated with Bk2R antagonists. The mechanism of action of androgens in angioedema also will be analyzed. To test the hypothesis that the contact system plays a role in the pathogenesis of septic shock, the susceptibility of C1INH +/- mice to endotoxin shock will be determined. Infusions of C1INH proteins with selective inhibitory activities will define the roles of the complement and contact systems. The second Aim is directed toward structure-function analyses. To test the hypothesis that stable complex formation requires extensive insertion of the reactive center loop (RCL) into beta sheet A, RCL mutants and mutants that alter the stability of sheet A will be examined. To test the hypothesis that sites within and outside the RCL determine target protease specificity, P2 mutants, distal loop mutants, and non-RCL mutants in alpha1-antitrypsin:C1INH chimeras will be tested. To test the hypothesis that amino terminal-truncated C1INH more efficiently inhibits surface associated proteases, its ability to inhibit immune complex-mediated complement activation, and inactivate high molecular weight kininogen (HK)-bound kallikrein and surface-bound factor XIIa will be examined. To compare and contrast the structure of C1INH with other serpins, recombinant truncated C1INH will be crystallized. In addition to definition of the biologic roles of C1INH and characterization of its function, these studies also may lead to improved therapy of HAE and of other diseases in which activation of the complement or contact systems plays a role.