The hemidesmosome is a trans-membrane organelle located at the basement membrane zone (BMZ). Previous studies provide evidence that this structure contains two distinct functional domains: an intracellular domain that interacts with the keratin filaments of basal epidermal cells and an adjoining extracellular domain involved in anchoring the epithelium to the dermis via an association with matrix molecules: i.e., laminin, collagens, fibronectin, etc. The dermal-epidermal linkage is essential for normal tissue integrity and may be involved in other fundamental processes such as cell motility, cell proliferation, and the regulation of epidermal differentiation. A disruption of this linkage appears to be the primary defect in a variety of skin disorders. Bullous pemphigoid (BP) is one such disease that is characterized by vesicles occurring at the BMZ and by the presence of circulating and tissue-bound autoantibodies directed against components of the hemidesmosome. BP autoantibodies bind both intracellular and extracellular domains of the hemidesmosome and recognize primarily two polypeptides, 240 kD and 180 kD. The current research proposal involves the use of BP autoantibodies to identify and characterize the functional domains of hemidesmosomal components using molecular biology techniques. As a first step in this analysis, the laboratory has isolated partial cDNA's corresponding to the 240 kD and 180 kD BP antigens by screening a human keratinocyte cDNA expression library with a BP serum. Lysogen-generated fusion proteins or chemically synthesized peptides will be used to produce antibodies directed against various BP antigen protein domains for use in ultrastructural mapping studies. In vitro binding assays and in vivo gene transfer studies will be implemented to test for the ability of the BP antigens to bind to basal epidermal keratins or to extracellular matrix components. Should such interactions be detected, the binding sites will be further dissected by cDNA deletion analysis and site-directed mutagenesis. The applicant believes that the characterization of the molecular interactions of the BMZ will reveal new and relevant information applicable to the understanding of the dermal-epidermal junction under normal and pathological states.
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