Human skin keratinocytes are affixed to the underlying dermal matrix by a complex attachment mechanism, many elements of which are unique to external eipthelia. These include the hemidesmosomes which mediate attachment of keratin filaments to the basolateral plasma membrane, anchoring filaments that appear to bridge the hemidesmosomes to the lamina densa of the basement membrane, and the anchoring fibril network, that attaches the lamina densa to the subjacent dermis by physical entrapment of dermal fibrous elements. Failure of this attachment complex results in several forms of the blistering diseases jointly termed Epidermolysis Bullosa (EB). While the phenomena of dermal-epidermal separation is well documented, the molecular events leading of these separations are not unambiguously known. During the past three years of funding, this grant has supported studies that identified a large and complex C-terminal globular domain of type VII collagen (NC-1) and indicated involvement of this domain in a new model of the anchoring fibril network. The model is based upon structural characterization of type VII collagen and ultrastructural immunolocalization of NC-1. The model predicts interactions of NC-1 with itself, with type IV collagen and with other intrinsic basement membrane components both with the lamina densa and within structures we have termed """"""""anchoring plaques"""""""". This application proposes continuation of these studies to: (1) investigate the possible interactions of NC-1 with itself and with domains of type IV collagen and other basement membrane proteins using solid phase interaction assays and biophysical techniques; (2) to attempt to isolate additional NC-1 binding proteins by affinity chromatography and to characterize their structures; (3) to begin identification of the structural components of hemidesmosomes by first producing monoclonal antibodies to membrane fractions enriched for them and use of these antibodies for immunoisolation and structural characterization of them; and (4) to assess the ability of cultured EB keratinocytes to synthesize type VII collagen using cDNA hybridization and immunoblotting techniques, and to evaluate the defective anchoring fibril networks of individuals with recessive dystrophic EB relative to the above described model, using electron microscope immunohistochemistry.
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