ANCHORING FILAMENT ANALYSIS AND CORRECTION IN EPIDERMOLYSIS BULLOSA

该提案旨在描述新型基底膜区的特征 与锚定丝相关的分子，并确定 超分子结构与分子间锚定作用 具有致密板和半桥粒的丝成分 件. 这个项目的基本假设是， 为了阐明锚定丝的结构和功能， 能够确定交界表皮的分子基础 大疱病（JEB），并发现新的基因，这是受影响的， disorder. 初步数据显示，三种新的锚定 丝蛋白、LAD-1（mAb 123抗原）、LH 39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JEB。 LAD-1是一种120 kD的上锚丝蛋白， 似乎参与了锚定丝插入到 半桥粒，并且在全身性骨髓增生异常综合征患者的一个子集中不存在。 良性萎缩性JEB（GABJEB）。 LH 39抗原是一种较低的锚定蛋白， 似乎参与锚定插入的丝蛋白 丝到致密板，这是缺席的是一个子集， HBEB患者 层粘连蛋白-6形成二硫键复合物， 层粘连蛋白-5锚定丝，含有一种新的α链， 包含JEB患者子集中的原发性缺陷。 我们建议 确定LH 39抗原的整个cDNA序列，并 建议进行更多的实验，以确定其功能和潜力 参与JEB。 我们同样提出了方法来确定 层粘连蛋白6A链的结构。 锚定的相互作用 具有半桥粒和致密板的丝成分 将通过几种方法分析组分，包括固态， 基于层析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，我们建议表征的表型特征， GABJEB角质形成细胞，转染LAD-1和BP 180 cDNA， 在这些细胞中的表型逆转，并结合项目3， 研究定点突变在JEB角质形成细胞中的作用 使用LAD-1、BP 180和层粘连蛋白-5cDNA。 结束时 在建议的供资期间，我们希望能够大大阐明 真皮-表皮粘连的基础穿过透明板， 已经展示了重要的新机制， JEB中的候选基因，为基因治疗的进一步进展奠定了基础 疗法

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.