ANCHORING FILAMENT ANALYSIS AND CORRECTION IN EPIDERMOLYSIS BULLOSA

大疱性表皮松解症的锚定丝分析和校正

• 批准号: 6348933
• 负责人: Matt Peter Marinkovich
• 金额: $ 28.26万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6348933
• 关键词: SCID mouse; animal genetic material tag; basal lamina; basement membrane; cell adhesion; cell adhesion molecules; clinical research; cytoskeleton; epidermolysis bullosa; gene mutation; gene therapy; human subject; intercellular connection; intermolecular interaction; keratinocyte; laboratory mouse; laminin; molecular cloning; nonhuman therapy evaluation; protein structure function; site directed mutagenesis; skin transplantation; transplantation immunology

This proposal aims to characterize novel basement membrane zone molecules associated with anchoring filaments and to determine the supramolecular structure and intermolecular interactions of anchoring filament components with lamina dense and hemidesmosomal components. The fundamental hypothesis of this project is that by elelucidating the structure and function of anchoring filaments we will be able to determine the molecular basis of junctional epidermolysis bullosa (JEB) and discover new genes which are affected in this disorder. Preliminary data are resented on three novel anchoring filaments proteins, LAD-1 (mAb 123 antigen), LH39 antigen and laminin 6, which are the three best candidates for new gene mutations in JEB. LAD-1 is a 120 kD upper anchoring filament protein which appears to be involved in insertion of anchoring filaments to the hemidesmosome and is absent in a subset of patients with generalized benign atrophic JEB (GABJEB). LH39 antigen is a lower anchoring filament protein which appears to be involved in insertion of anchoring filaments to the lamina densa and which is absent is a subset of patients with HBEB. Laminin-6 forms a disulfide complex with laminin-5 anchoring filaments, contains a novel a chain and could contain the primary defect in a subset of JEB patients. We propose to determine the entire cDNA sequence of the LH39 antigen and to propose additional experiments to determine its function and potential involvement in JEB. We similarly propose methods to determine the structure of the laminin 6 a chain. The interactions of anchoring filament components with hemidesmosome and lamina densa components will be analyzed by several methods, including solid state, chromatogrphic and centrifugation based ligand binding assays, cell binding assays and an in vitro basement membrane assembly model. Additionally, we propose to characterize the phenotypic features of GABJEB keratinocytes, to tranfect LAD-1 and BP180cDNA to effect phenotypic reversion in these cells and in conjunction with project 3, to study the effects of site directed mutagenesis in JEB keratinocytes using LAD-1, BP180 and laminin-5 cDNA. At the end of the proposed funding period, we hope to have significantly elucidated the basis of dermal-epidermal cohesion across the lamina lucida and to have demonstrated significant new mechanisms involving new candidate genes in JEB, setting the stage for further progress in gene therapy.

该提案旨在表征新型基底膜区域 与锚定丝相关的分子并确定 超分子结构和锚定的分子间相互作用 具有致密层和半桥粒的细丝成分 成分。 该项目的基本假设是 阐明锚定丝的结构和功能，我们将 能够确定交界性表皮松解症的分子基础 bullosa (JEB) 并发现受此影响的新基因 紊乱。 重新发送三种新颖锚定的初步数据 细丝蛋白、LAD-1（mAb 123 抗原）、LH39 抗原和 层粘连蛋白6，这是新基因突变的三个最佳候选者 在杰布。 LAD-1 是一种 120 kD 的上锚丝蛋白， 似乎涉及锚定丝的插入 半桥粒，并且在全身性全身性疾病患者的子集中不存在 良性萎缩性 JEB（GABJEB）。 LH39 抗原是较低的锚定抗原 似乎参与锚定插入的丝蛋白 细丝连接到致密层，其中不存在的是 HBEB 患者。 Laminin-6 与以下物质形成二硫键复合物 laminin-5 锚定丝，包含一个新颖的 a 链，可以 包含 JEB 患者子集的主要缺陷。 我们建议 确定 LH39 抗原的完整 cDNA 序列并 提出额外的实验来确定其功能和潜力 参与 JEB。 我们同样提出了确定方法 层粘连蛋白6a链的结构。 锚定的相互作用 具有半桥粒和致密层的细丝成分 组件将通过多种方法进行分析，包括固态、 基于色谱和离心的配体结合测定、细胞 结合测定和体外基底膜组装模型。 此外，我们建议表征 GABJEB角质形成细胞，转染LAD-1和BP180cDNA以发挥作用 这些细胞中的表型逆转并与项目 3 相结合， 研究定点突变对 JEB 角质形成细胞的影响 使用 LAD-1、BP180 和层粘连蛋白-5 cDNA。 结束时 拟议的资助期限，我们希望已明确阐明 穿过透明层的真皮-表皮凝聚力的基础 已经展示了重要的新机制，涉及新的 JEB中的候选基因，为基因研究的进一步进展奠定了基础 治疗。