Complement Convertase: Assembly, Function and Regulation

描述(由申请人提供)：补体系统是先天免疫和后天免疫的关键参与者。补体标志着免疫清除或细胞溶解的感染剂，它形成了炎症反应的焦点。此外，补体有助于抗原定位到脾和淋巴结，在那里它降低了特定B细胞激活的门槛。补体也可能是人类疾病中组织损伤的主要原因，如抗体介导的自身免疫、免疫复合物沉积综合征、缺血再灌注损伤和超急性移植物排斥反应。 补体的几乎所有生物学后果都需要对C3的酶促裂解，C3是一种丰富的血清糖蛋白。裂解C3的酶，即C3转换酶，是补体激活途径中的主要参与者，在结构和功能上有两种同源形式，即交替途径(AP)C3转换酶(C3bBb)和经典途径(CP)C3转换酶(C4bC2a)。几种结构上相关的蛋白质，如DAF、CR1、C4BP和因子H，可以抑制不适当的补体激活(例如在自身组织上)。它们的作用是促进活性转换酶不可逆转的解离，这一过程被称为衰变加速。相反，血清蛋白备解素可以部分稳定C3bBb在靶表面。这项工作的长期目标是创造新的补体治疗控制方法。我们的策略是阐明补体激活的关键酶C3转换酶的独特生化特征，并确定有希望的治疗靶点。建立了一种简单的分析AP C3转换酶C3bBb和C3bBbP的组装、稳定和衰变加速的方法：用C3b包被微滴定井，与液相因子B、因子D、二价阳离子孵育，在某些情况下，用ELISA法检测复合体。利用定点突变产生的B因子、DAF和CR1突变株的酶联免疫吸附试验，已经确定了一些可能的活性位点，并提出了几个关键假设。为了验证这些假设，提出了以下具体目标的研究： 1)建立了转化酶组装及其丝氨酸蛋白酶结构域激活的模型； 2)确定备解素如何稳定AP转换酶； 3)阐明了衰变加速的机理。

DESCRIPTION (provided by applicant): The complement system is a critical participant in both innate and acquired immunity. Complement marks infectious agents for immune clearance or cell lysis and it forms a focal point for inflammatory reactions. Moreover, complement facilitates antigen localization to the spleen and lymph nodes, where it lowers the threshold for the activation of specific B cells. Complement can also be a principal cause of tissue damage in human diseases such as antibody-mediated autoimmunity, immune complex deposition syndromes, ischemic reperfusion injury, and hyperacute graft rejection. Nearly all the biological consequences of complement require the enzymatic cleavage of C3, an abundant serum glycoprotein. The enzymes that cleave C3, the C3 convertases, are major players in the complement activation pathways and occur in two structurally and functionally homologous forms, the alternative pathway (AP) C3 convertase (C3bBb) and the classical pathway (CP) C3 convertase (C4bC2a). Several structurally related proteins, DAF, CR1, C4BP, and factor H, inhibit inappropriate complement activation (e.g. on self-tissue). They act to promote the irreversible dissociation of active convertases, a process known as decay acceleration. In contrast, the serum protein properdin can partially stabilize C3bBb on target surfaces. The long-range goal of this work is to create new approaches to the therapeutic control of complement. Our strategy is to elucidate the unique biochemical features of the C3 convertases, the key enzymes in complement activation, and identify promising therapeutic targets. A simple assay was developed for analyzing the assembly, stabilization, and decay acceleration of the AP C3 convertases C3bBb and C3bBbP: Microtiter wells were coated with C3b, incubated with fluid phase factor B, factor D, divalent cation and, in some cases, properdin; complexes were detected by ELISA. Employment of the ELISA-based assay with panels of factor B, DAF and CR1 mutants generated by site-directed mutagenesis has led to the identification of a number of possible active sites and the proposal of several key hypotheses. To test these hypotheses, studies are proposed with the following specific aims: 1) Develop a model for convertase assembly and the activation of its serine protease domain; 2) Determine how properdin stabilizes AP convertases; 3) Elucidate the mechanisms of decay acceleration.