STRUCTURE/FUNCTION STUDIES ON FIBRONECTIN/LIGAND BINDING

纤连蛋白/配体结合的结构/功能研究

• 批准号: 2650021
• 负责人: Leslie Ina Gold
• 金额: $ 17.9万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2650021
• 关键词: binding proteins; chemical binding; enzyme linked immunosorbent assay; extracellular matrix; fibrin; fibronectins; heparin; human tissue; immune complex; immune complex diseases; immunocytochemistry; immunoglobulin structure; immunoglobulins; laboratory mouse; ligands; molecular site; protein structure function; wound healing

The objectives of this proposal are to structurally and biochemically define the sites on fibronectin (Fn) that dictate binding to fibrin, heparin, and immunoglobulins (Ig) and employ this information to explore the [patho]physiological role for these ligand-binding interactions in wound repair (fibrin and heparin-binding) and fibrin clot formation, and immune complex (IC) deposition in tissues (fibrin and Ig-biding). We have determined the tertiary structure of the 4th and 5th type 1 structural repeats (4F1.5F1; 93 amino acids) in the N- terminal of Fn, by 1H NMR, and shown that this module pair harbors the fibrin, heparin, and Ig binding sites. A separate fibrin-binding site is in the C-terminal 10th-12th (10F1-12F1) type 1 repeats. We have produced recombinant Fn type 1 modules and functional proteolytic fragments of native Fn for use in the following Specific Aims: Aim 1). To define more precisely the fibrin, heparin, and Ig-binding activity/sites on Fn by solid phase assays (ELISA), by quantitating binding affinity constants and kinetics of binding by real time biospecific interaction analysis, and by implicating interactive residues critical to ligand binding by site directed mutagenesis. This information will be used to explore the [patho]physiological significance of these interaction in vitro and in vivo in the following specific aims: Aim 2). Fn is associated with immune complexes (Ics) in the circulation of patients with a variety of immune complex-related diseases (ICD). Since Fn and fibrin accumulate in the ECM in glomerulonephritis, we hypothesize that the interaction of Fn with Ig and, plasma Fn, as component of Ics, with fibrin, may contribute to the deposition of Ics from the circulation into tissues. To test this hypothesis, cell-generated Fn-containing extracellular matrices (ECMs) will be used as an in vitro model for IC deposition in tissues. Ig- binding (IgBP) and fibrin-binding (FBP) type 1 modules will be tested for their ability to inhibit Ics from biding to ECMs and in preventing IC deposition in normal mice injected with preformed IC's. By immunohistochemical analysis, the co-distribution of Fn, Ics, and fibrin will further support our hypothesis. Aim3). We hypothesize that the interaction of Fn with fibrin is essential for tissue remodeling and specifically in Fn/fibrin matrix assembly and cellular migration on Fn/fibrin wound provisional matrices. To test these hypotheses, FBPs from the N-and C-terminal of Fn will be used in in vitro clot binding and wound repair assays and in vivo, in porcine wound repair. The study of the structure/function relationship of Fn/fibrin and Fn/Ig- binding interactions should elucidate mechanisms of the function of Fn in wound repair and the pathogenesis of ICD.

这项提议的目标是在结构和生化上 定义纤维连接蛋白(FN)上指示与纤维蛋白结合的位点， 肝素和免疫球蛋白(Ig)，并利用这些信息 探索这些配体结合的[病理]生理作用 伤口修复(纤维蛋白和肝素结合)与纤维蛋白的相互作用 组织中血栓的形成和免疫复合物(IC)的沉积(纤维蛋白 和搞笑出价)。我们已经确定了4号的三级结构 和第5个类型1结构重复(4F1.5F1；93个氨基酸)。 Fn末端的核磁共振氢谱，表明该模对含有 纤维蛋白、肝素和免疫球蛋白结合部位。一个单独的纤维蛋白结合部位 是在C端第10-12(10F1-12F1)型重复的。我们有 生产重组FN-1模块和功能性蛋白水解物 用于下列特定目的的本地FN片段：目标1)。 为了更准确地定义纤维蛋白、肝素和Ig结合 固相分析法(EL ISA)测定FN上的活性/位点，通过定量 结合亲和力常数和实时结合动力学 生物特异性相互作用分析，并通过隐含相互作用残基 通过定点突变对配体结合至关重要。这 信息将被用来探索[病态]生理学 这些体外和体内相互作用的意义如下 具体目标：目标2)。FN与免疫复合体(ICs)相关 患者循环中的多种免疫复合体相关 疾病(ICD)。由于FN和纤维蛋白在细胞外基质中积累 肾小球肾炎，我们假设FN与Ig的相互作用 而血浆Fn作为ICS的组成部分，与纤维蛋白一起，可能有助于 循环中的ICS沉积到组织中。为了测试这一点 假设，细胞生成的含有FN的细胞外基质(ECM) 将作为IC在组织中沉积的体外模型。IG- 将测试结合(IGBP)和纤维蛋白结合(FBP)类型1模块 因为他们有能力阻止ICS竞标ECM并防止 注射预制IC的正常小鼠的IC沉积。 免疫组织化学分析FN、ICS和 纤维蛋白将进一步支持我们的假设。目的)。我们假设 纤维连接蛋白与纤维蛋白的相互作用是组织重塑所必需的 特别是在FN/纤维蛋白基质组装和细胞迁移中 纤维连接蛋白/纤维蛋白创面临时基质。为了检验这些假说，FBP 来自FN的N-末端和C-末端将用于体外凝血结合 以及伤口修复试验和活体内，在猪伤口修复。这个 Fn/纤维蛋白和Fn/Ig-结构/功能关系的研究 结合作用应阐明FN的作用机制 在创面修复和ICD发病机制中的作用。