Anchoring fibrils, composed of type VII collagen, are a critical factor in epidermal-dermal adherence. Both genetic and autoimmune disorders targeting type VII collagen result in chronic, debilitating blistering diseases of the skin. Dystrophic Epidermolysis Bullosa (DEB) is due to mutations in COL7A1, the gene that encodes for type VII collagen. It may be inherited in a dominant or recessive (RDEB) pattern and is incurable and often lethal. An acquired form of DEB, epidermolysis bullosa acquisita (EBA), is caused by IgG autoantibodies targeted against type VII collagen which results in perturbed, poorly functioning anchoring fibrils. Our overall goal is to understand the structure and function of type VII collagen and anchoring fibrils as they pertain to epidermal-dermal adherence and DEB. Traditional biochemical methods were unable to provide enough purified type VII collagen to facilitate this work. During our last funding cycle, we resolved this problem using a molecular biology approach and can now make milligram quantities of purified type VII collagen and various domains and sub-domains of the molecule. Using rigorous parameters, we have shown that the recombinant molecule is identical to the authentic molecule. Using 293 cells or RDEB cell lines that cannot make collagen VII, we can transfect wild type COL7A1 or any published DEB mutation into these cells and create type VII collagen molecules that reflect a given DEB gene defect. Likewise, we can create DEB cells that reflect the biology of cells from patients suffering from DEB. We also have an in vivo human skin equivalent model to examine the quantity and quality of anchoring fibrils in designer skin equivalents made from normal skin cells, RDEB wild type cells, gene-corrected cells or designed gene-defective cells. We also developed a new murine model for EBA using euthymic SKH1 hairless mice. In this proposal, using these reagents and methods, we will study the structure and function of wild type and DEB type VII collagen molecules including matrix and cell binding sites, exportation from their cell of origin, resistance to protease, triple helix and anti-parallel dimer formation, disulfide bonding and formation of in vivo anchoring fibrils. We will create RDEB cells harboring 10 COL7A1 mutations known to result in the production of an abnormal type VII collagen alpha chain protein. We will examine the cellular biology of designer DEB cells in terms of morphology, proliferation, matrix attachment and motility. We will examine the biology of our newly developed EBA animal model. By immunoabsorption of anti-type VII collagen antibodies against domains and sub-domains of type VII collagen, we will determine the pathogenic EBA epitopes on type VII collagen.
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