Type VII collagen has been characterized in this laboratory as a unique extracellular matrix macromolecule that forms antiparallel dimers that then aggregate laterally to form structures known as anchoring fibrils. We have postulated that dimer formation is mediated by the amino- terminal non-helical domain (NC-2). The morphology of the anchoring fibrils suggest that they may stabilize the attachment of external epithelia to their underlying stromal matrix. This concept is strengthened by the finding that individuals with the disease of recessive dystropic Epidermolysis Bullosa, characterized by spontaneous separation of the epithelia from the stroma through the plane of the anchoring fibrils, lack both identifiable anchoring fibrils and type VII collagen. One consequence of the antiparallel arrangement of type VII collagen and the subsequent lateral aggregation is that the two ends of the anchoring fibril are identical. These ends, containing the carboxy-termininal non-helical domain (NC-1), interact with the epithelial basement membrane and with structures we described as """"""""anchoring plaques"""""""" that are aggregates of the NC-1 domains, type IV collagen and perhaps other intrinsic basement membrane components. These spatial relationships suggest that the NC-1 domains mediate the formation and stability of the anchoring fibril network. Since disruptions in the anchoring fibril network have severe pathological consequences, it is important to understand the molecular basis for the interactions of anchoring fibrils with anchoring plaques and with basement membranes. We propose to characterize the structure of type VII collagen in sufficient detail to evaluate he relationship of its structure to its anchoring function. We will determine the amino acid sequence of selected portions of the molecule using conventional biochemical methods and recombinent DNA approaches. We will isolate and identify subdomains of NC-1 and evaluate their secondary structure by biophysical techniques. Similarly, we will determine the primary structure of NC-2. Monoclonal antibodies made to synthetic peptides that mimick NC-2 structure will be used to test the hypothesis that NC-2 directs type VII dimerization. We believe that knowledge of the detailed structure of type VII collagen will lead to the formation of an hypothesis of how specific structural regions contribute to the anchoring function of the macromolecule.
