GENETIC ENGINEERING OF FOURTH COMPLEMENT COMPONENT

拟议计划的长期目标是在氨基会议上了解 酸水平如何补充成分C4，这在两者中都发挥着核心作用 补体的激活和调节，发挥其众多功能。 特别是，我们希望利用分子生物学的工具来检查 C4中的结构变化如何影响补体活性和 宿主防御的整体功能。对这些的理解 关系对于解释 C4多态形式的意义，它也可能提供洞察力 探讨观察到的C4缺乏与免疫复合体疾病的关系 以及C4基因与主要组织相容性复合体的关系。 将使用定点突变和重组DNA技术来 对C4的变种进行基因工程。这些变体将通过以下方式获得 将最近分离的全长C4cDNA克隆到真核细胞中 表达载体，通过体外操作改变天然序列， 并将由表达载体携带的改变后的cDNA导入到 哺乳动物细胞系在成熟细胞株中的表达 分泌的蛋白质。具体计划包括建造和表征 小鼠C4变异体的设计(A)以精确定位结构缺陷(S) 一种无功能的C4亚型，性限制蛋白(SLP)；(B)评估 糖基化和硫酸化在C4功能中的作用；(C)鉴定 负责调节体内蛋白质反应性的氨基酸 C4中的硫酯；和(D)确定氨基酸的替代(S) 一种天然多态形式的低溶血活性的原因 小鼠C4。 待评估的工程蛋白的性质包括：(A) 溶血活性；(B)对C1S切割的敏感性；(C)存在 内部硫酯；(D)对亲核剂和杂色体的敏感性；(E) 活性硫酯与羟基和氨基的相对反应性 (F)在因子I存在的情况下对切割的敏感性 C4结合蛋白(C4-BP)；和(G)激活后结合 致敏细胞、C2a、C4-BP和补体受体CR1。

The long-term goal of the proposed program is to understand at the amino acid level how complement component C4, which plays a central role in both complement activation and regulation, carries out its numerous functions. In particular, we wish to exploit the tools of molecular biology to examine in depth how structural variations in C4 affect complement activity and the overall functioning of host defenses. An understanding of these relationships will be particularly valuable for interpreting the significance of polymorphic forms of C4 and it may also provide insights into the observed associations of C4 deficiency with immune complex disease and of the C4 gene with the major histocompatibility complex. Site-specific mutagenesis and recombinant DNA technology will be used to genetically engineer variants of C4. These variants will be obtained by placing recently isolated full-length C4 cDNAs into an eucaryotic cDNA expression vector, altering the native sequence by in vitro manipulation, and introducing the altered cDNA, carried by the expression vector, into a mammalian cell line capable of expressing the transfected cDNA as a mature secreted protein. Specific plans include construction and characterization of murine C4 variants designed (a) to pinpoint the structural defect(s) in a non-functional C4 isotype, sex-limited protein (Slp); (b) to assess the roles of glycosylation and sulfation in C4 function; (c) to identify the amino acids responsible for modulating the reactivity of the internal thiolester in C4; and (d) to identify the amino acid replacement(s) responsible for the low hemolytic activity of a natural polymorphic form of murine C4. Properties of the engineered proteins to be evaluated include: (a) hemolytic activity; (b) susceptibility to cleavage by C1s; (c) presence of an internal thiolester; (d) sensitivity to nucleophiles and chaotropes; (e) relative reactivity of the activated thiolester with hydroxyl and amino groups; (f) susceptibility to cleavage by Factor I in the presence of C4-binding protein (C4-bp); and (g) the ability to bind, after activation, to sensitized cells, to C2a, to C4-bp, and to complement receptor CR1.