ANCHORING FILAMENT ANALYSIS AND CORRECTION IN EPIDERMOLYSIS BULLOSA

这项建议旨在表征新的基底膜区域 与锚定细丝相关的分子，并确定 锚定的超分子结构和分子间相互作用 具有致密片层和半桥粒的花丝组分 组件。这个项目的基本假设是通过 阐明锚定细丝的结构和功能，我们将 能够确定交界性表皮松解症的分子基础 Bullosa(Jeb)，并发现受其影响的新基因。 无序。对三种新型锚固的初步数据进行了比较 细丝蛋白、LAD-1(单抗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120kD的上链锚定细丝蛋白 似乎参与了将锚定细丝插入到 半桥体，在全身性疾病患者的亚群中缺失 良性萎缩性杰布瘤(GABJEB)。LH39抗原是一种较低的锚定 似乎与锚定插入有关的丝状蛋白 细丝到致密层，而不存在的是 乙肝病毒携带者。层粘连蛋白-6与二硫键形成复合体 层粘连蛋白-5锚定细丝，含有一种新型的链，可以 在部分JEB患者中存在原发缺陷。我们建议 测定LH39抗原的全长cDNAs序列 提出更多实验以确定其功能和潜力 与杰布有关。类似地，我们提出了确定 层粘连蛋白6的结构是一条链。锚定的相互作用 具有半桥粒和致密层的花丝成分 成分将通过几种方法进行分析，包括固态， 基于层析和离心法的配基结合分析 结合实验和体外基底膜组装模型。 此外，我们还建议对表型特征进行表征 GABJEB角质形成细胞对转导LAD-1和BP180cDNA的影响 在这些细胞中的表型逆转，并结合项目3， JEB角质形成细胞定点突变效应的研究 使用LAD-1、BP180和LN-5基因。在结束时， 建议的资助期，我们希望能对 跨透明层的真皮-表皮凝聚力的基础 已经展示了重大的新机制，涉及新的 JEIB中的候选基因，为基因的进一步进展奠定了基础 心理治疗。

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.