Structure/function of 105-KD basement membrane protein

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DESCRIPTION: (Verbatim) Skin basement membrane zone (BML) serves as an interface barrier and as an adherent connection between the outer layer of skin, the epidermis, and inner 'layer of skin, the dermis. Some of the BMZ components, such as 8P230, BP180 (type XVII collagen) were discovered as a result of being targeted by an autoimmune reaction. Using sera from patients with autoimmune blistering diseases and autoantibodies to BMZ components, the cDNAs encoding BMZ components, such as BP18O and type VII collagen, were determined and became very valuable in biomedical research. The autoantibody-delineated nucleotide sequences allow synthesis of large quantities of recombinant proteins, which in turn significantly facilitates the understanding of pathogenesis of blistering process and the normal connecting function of BMZ components. We now have the opportunity to discover and understand another previously unknown BMZ component, termed p105, also identified as a result of an autoimmune reaction. This novel autoimmune blistering skin disease is characterized by extensive blisters and erosions clinically, subepidermal separation with neutrophilic infiltration histologically, in vivo lgG deposition at BMZ and circulating lgG binding to the dermal side of salt-split skin substrate immunopathologically, in vivo lgG deposition and circulating lgG binding to the lower lamina lucida ultrastructural!y, and lgG autoantibodies recognizing a 1O5-kDa epidermal protein immunochemically. This p105 has been further characterized for its distinction from the HO5-kDa laminin-5 -y2 chain by immunochemical methods, its isoelectric point by two-dimensional gel electrophoresis and immunoblotting, its ionic strength by Mono 0 anion-exchange column chromatography, and its N-terminal amino acid sequence by protein sequencing. Furthermore, a monoclonal antibody has been generated against this p105 protein. Having cloned two cDNAs encoding BMZ components, the principal investigator has gained experience in molecular cloning and is now ready to propose the following works to study the structure and function of this novel BMZ component p105. In this proposal, we aim to molecularly clone the human p105 cDNA sequence, to express the human p105 recombinant protein, to study the in vitro functions of human p105 protein, to molecular clone the mouse p105 cDNA, and to passive transfer of anti-mouse p105 antibodies to new born mice. Understanding of the structure and function of this new BMZ component p105 will shed light on the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering skin diseases, gestational development, epidermal cancer metastasis, and cutaneous wound healing.