Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by the production of autoantibodies that react with components of the hemidesmosome, an adhesion-related organelle of basal epidermal keratinocytes. Studies supported by this grant utilized patient autoantibodies as specific probes to facilitate the molecular cloning and characterization of BP180, a major antigen associated with these autoimmune diseases. BP180 was shown to be a type II transmembrane protein with a long C-terminal collagenous domain that projects into the extracellular region beneath the epidermal hemidesmosome. The first specific aim of the current grant application addresses important questions regarding the structure of both the intracellular and extracellular domains of the BP180 protein. In the execution of these studies, high levels of recombinant forms of BP180 will be expressed in cultured mammalian cells using an Epstein Barr Virus-based vector, pCEP4. Using this system, the applicants recently demonstrated that the BP180 ectodomain is capable of forming a homotrimeric complex, with a molecular shape and flexibility properties consistent with their structural model of BP180 in which the ectodomain comprises a series of articulated, rigid rod-like structures. A part of this aim, they plan to define the protein segments that are essential for the assembly of the BP180 trimer, and to investigate the structural consequences of BP180 mutations that are known to be associated with inherited disorders of the basement membrane zone. Questions related to the function of BP180 will be addressed in specific aim 2. To further test their hypothesis that BP180 functions in cell-matrix attachment, they plan to extend their recent studies in which wild type BP180 was transfected into BP180-deficient keratinocytes from patients with generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes and other cultured cells transfected with wild type and mutant BP180 expression constructs will be assayed for alterations in hemidesmosome assembly and/or cell matrix attachment properties. In addition, a variety of approaches will be employed to identify the extracellular BP180 ligand(s) and to determine whether ligand binding is coupled with the transduction of a signal across the membrane.
In specific aim 3, the BP180 ectodomain will be further analyzed as the target of lgG and IgA class autoantibodies associated with several subepithelial blistering diseases such as cicatricial pemphigoid and linear IgA bullous dermatosis.
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