GENETIC ENGINEERING OF SHORT CONSENSUS REPEAT ELEMENTS

短一致重复元件的基因工程

• 批准号: 3291707
• 负责人: Ronald T Ogata
• 金额: $ 25.84万
• 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 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291707
• 关键词: SDS polyacrylamide gel electrophoresis; chemical binding; complement; complement pathway; genetic manipulation; immunoprecipitation; laboratory mouse; molecular cloning; mutant; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; transfection

The short consensus or complement repeat (SCR) is a modular protein structural unit that is usually encoded in distinct gene exons. SCRs have recently become the focus of substantial interest because they have been found in a wide range of apparently unrelated proteins including a component of the blood coagulation system, a vaccinia virus-encoded protein, a lymphokine receptor, 3 cellular adhesion receptors, and at least 10 complement proteins. SCRs range from 60-70 amino acids in length, they contain 4 or 6 conserved cysteines, and have a consensus sequence involving about 40% of the residues. They are likely to be independent structural units, similar in concept to immunoglobulin domains, consisting of a basic framework structure onto which sequence variations related to specific functions are superimposed. The function(s) of SCRs are in many cases unknown but their wide distribution suggests that they fulfill crucial structural and functional roles. They have been most extensively studied in the complement proteins. In most and perhaps all of these cases, their primary function is to bind to complement components C3 and C4, and perhaps secondarily to provide a linear, extended structure. Binding of SCR-containing proteins to both C3 and C4 are essential for activation and regulation of the two complement pathways. The binding of SCR-containing proteins to C3 and C4 is specific; with distinct SCRs binding to either C3 or C4. This grant proposes to use genetic engineering methods to dissect the interaction between SCRs and complement components C3 and C4. In particular we plan to identify amino acids within SCRs, and within C3 and C4 which are necessary for binding, and for binding specificity. As models of this interaction, we will examine the binding of murine C4 to murine C4-binding protein, and of murine C3 to murine factor H. The long-term goal of the program proposed is to understand at the amino acid sequence level the rules governing the binding specificities of SCRs and the structural features of their protein targets. This understanding should provide clues to the specificities and functions of these structural motifs in other (especially non- complement) proteins, and may provide a more unified understanding of the interactions and evolutionary relatedness of both soluble and membrane-bound proteins in the bloodstream. The ability to alter the specificities or reactivities of these proteins is likely to have implications for the eventual development of therapeutic agents.

短共有序列或互补重复序列（SCR）是一种模块化蛋白质 通常由不同基因外显子编码的结构单位。 SCRs 最近已经成为人们关注的焦点，因为它们 在许多明显无关的蛋白质中发现 包括血液凝固系统的一个组成部分， 病毒编码蛋白，淋巴因子受体，3细胞粘附 受体和至少10种补体蛋白。 SCR范围为60-70 在长度上，它们含有4或6个保守的半胱氨酸，并且 具有涉及约40%残基的共有序列。 他们 可能是独立的结构单元，在概念上类似于 免疫球蛋白结构域，由基本框架结构组成， 与特定功能相关的序列变异 叠加的 SCR的功能在许多情况下是未知的，但是它们的广泛应用是不确定的。 分布表明，它们实现了关键的结构和 功能角色。 这些问题在过去几年中得到了最广泛的研究， 补体蛋白 在大多数甚至所有这些情况下， 主要功能是结合补体成分C3和C4， 也许其次是提供线性的、延伸的结构。 结合 C3和C4的SCR蛋白质是必不可少的 两种补体途径的激活和调节。 的结合 对C3和C4具有特异性; 与C3或C4结合的SCR。 这项资助计划使用基因工程方法来解剖 SCR与补体成分C3和C4之间的相互作用。 在 特别是，我们计划鉴定SCR内的氨基酸，以及C3内的氨基酸。 和C4，它们是结合和结合特异性所必需的。 作为这种相互作用的模型，我们将研究鼠 C4与鼠C4结合蛋白的结合，以及鼠C3与鼠H因子的结合。 该计划的长期目标是了解 氨基酸序列水平的规则， SCRs的特异性及其蛋白质的结构特征 目标的 这种理解应该提供线索， 以及这些结构基序在其他（特别是非 补体）蛋白质，并可能提供更统一的理解， 可溶性和可溶性蛋白质的相互作用和进化相关性 血液中的膜结合蛋白质。 能够改变 这些蛋白质的特异性或反应性可能具有 对治疗剂的最终开发的影响。