A new extracellular matrix molecule, linkin, has been found in association with a microthread-like filamentous network beneath the basement membrane of all stratified epithelia tested. Direct connections of the microthread- like network can be observed with the lamina densa, collagen fibers, anchoring fibrils and elastic microfibril bundles. The association with collagen fibers appears specific since linkin occurs at regular periodic sites along the fiber.l Linkin can be identified in extracts of human skin as 73-80 kD glycoproteins by Western immunoblots using a mouse monoclonal antibody, B3a. Linkin is synthesized by human skin fibroblasts and secreted into the medium as 80, 87, and 180 kD proteins. As a prelude to the actual purification of linkin, studies will be undertaken to develop reliable assay systems for linkin, determine its solubility properties,l carbohydrate content, maximize tissue extraction and fibroblast synthesis of linkin, and determine the relationship of the immunoprecipitated linkin polypeptides. Purification will be done employing antibody affinity chromatography and/or biophysical methods. Once purified linkin will be further characterized by amino acid analysis and sequencing, peptide mapping and rotary shadowing studies of molecular size and shape. We will also attempt to isolate, clone and sequence the linkin gene from human skin fibroblast cDNA libraries. Self-assembly of linkin will be evaluated biochemically and by rotary shadowing in an attempt to reconstitute the microthread-like filamentous structures seen in vivo. Since multiple interactions are suggested by the connections of linkin-associated microthread-like filaments with other structures, interactions of linkin with other matrix molecules will be examined by double labeling immunoelectron microscopy, rotary shadowing and binding studies. The presence of linkin in early fetal skin development and in wound healing in adult skin suggest a role of linkin in these biological events. We will also examine a possible role of linkin in pathologic processes involving the skin such as lupus erythematosus and the inherited blistering disease, epidermolysis bullosa dystrophica.
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