Dystrophic forms of epidermolysis bullosa (EB) include an acquired autoimmune type of EB called EB and two genetic types, dominant (DDEB) and recessive (RDEB). All of these patients have chronic blistering disease that can be devastating and leave the sequelae of scarring and small epidermal inclusion cysts called milia. All of these disorders are incurable. The blistering is due to a separation at the basement membrane zone (BMZ) between the epidermis and dermis of human skin and is associated with a lack of anchoring fibrils, structures made of a BMZ-specific collagen called type VII collagen and also called """"""""the EBA antigen."""""""" Anchoring fibrils are critical for epidermal/dermal adherence. In EBA, anchoring fibril collagen is the target for autoantibodies, while autoantibodies do not play a role in the genetic dystrophic forms of EB. Therefore, although the etiologies of these three diseases are very different, each disease is characterized by perturbations of anchoring fibrils and involvement of type VII collagen. The principal investigator hypothesizes that in EBA, type VII collagen elicits an autoimmune response, while in the dystrophic, genetic forms of EB, the type VII collagen molecules form structurally unstable anchoring fibrils because of recently described defects in the human gene that encodes type VII collagen. In collaboration with Dr. Uitto's laboratory at the Molecular Biology Institute of Jefferson Medical College, the principal investigator identified the first cDNA encoding for type VII collagen and localized the gene to the short arm of the third chromosome. More recently, the principal investigator has defined the domains of the type VII collagen alpha chain that are the antigenic epitopes for autoantibodies in patients with EBA and bullous SLE. The principal investigator defined four dominant antigenic epitopes in the NC1 amino terminus of the alpha chain. Likewise, the principal investigator has recently defined the fibronectin binding site and the binding sites for type I and type IV collagen. Moreover, the principal investigator has recently established consistent Northern blot analysis and quantitative Western blot analysis to examine the cellular regulation of human keratinocytes for the expression of type VII collagen at the mRNA and protein level. More recently, the principal investigator has used molecular biology methods to stably transfect human 293 epithelial cells with large constructs of the type VII collagen gene and synthesized domains of the type VII collagen alpha chain that are post-translationally modified and glycosylated. The principal investigator is able to synthesize and purify milligram quantities of type VII collagen alpha chain domains. Using these domains as substrates, the principal investigator developed a new ELISA for the immunodiagnosis of EBA and bullous SLE. In the current proposal, the plan is to 1) characterize sub-domains of NC1 that mediate interactions with other extracellular matrix components; 2) produce milligram quantities of full-length type VII collagen alpha chains and study its structure and function; 3) characterize the structure and function of NC2, and 4) determine the antigenic epitopes on eukaryotic recombinant type VII collagen for EBA autoantibodies.
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