GENETIC ENGINEERING OF FOURTH COMPLEMENT COMPONENT

第四补体成分的基因工程

• 批准号: 3291705
• 负责人: Ronald T Ogata
• 金额: $ 20.87万
• 依托单位: MEDICAL BIOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-12-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291705
• 关键词: chemical structure function; complement; complement pathway; complement receptor; complementary DNA; genetic manipulation; glycosylation; guinea pigs; laboratory mouse; protein engineering; protein sequence; tissue /cell culture

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.

拟议计划的长期目标是了解氨基 酸水平如何补充成分C4，它在两者中起着核心作用 补体激活和调节，执行其许多功能。 特别是，我们希望利用分子生物学的工具来检查 深入了解C4的结构变化如何影响补体活性， 宿主防御的整体功能。 了解这些 关系将是特别有价值的解释 C4的多态性形式的重要性，它也可以提供见解 观察到的C4缺乏症与免疫复合物疾病之间的联系 和C4基因与主要组织相容性复合体。 定点突变和重组DNA技术将用于 基因工程变异的C4 这些变体将通过以下方式获得： 将最近分离的全长C4 cDNA放入真核cDNA中， 表达载体，通过体外操作改变天然序列， 以及将由表达载体携带的改变的cDNA引入到 能够将转染的cDNA表达为成熟的 分泌蛋白 具体计划包括建设和表征 小鼠C4变异体的设计（a），以查明结构缺陷（S）， 非功能性C4同种型性别限制蛋白（Slp）;（B）评估 糖基化和硫酸化在C4功能中的作用;（c）鉴定 氨基酸负责调节内部的反应性， C4中的硫酯;和（d）鉴定氨基酸置换 负责天然多晶型的溶血活性低， 鼠C4。 待评估的工程化蛋白质的性质包括： 溶血活性;（B）对C1裂解的敏感性;（c）存在 （d）对亲核试剂和离液剂的敏感性;（e） 活化硫酯与羟基和氨基的相对反应性 （f）在以下存在下对因子I切割的敏感性： C4结合蛋白（C4-bp）;和（g）在活化后， 致敏细胞、C2 a、C4-bp和补体受体CR 1。