ANCHORING FILAMENT ANALYSIS AND CORRECTION IN EPIDERMOLYSIS BULLOSA

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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.