The specific aim of this project is to define the molecular structure of gelsolin in order to better understand its role in the cell and in the blood. Cytoplasmic gelsolin is a cytoskeletal protein of a variety of human tissues, which in vitro is capable of shortening actin filaments in a calcium dependent manner. It was first found in leukocytes and is thought to provide the means by which the actin filament structure is altered in response to micromolar calcium fluxes intracellularly, thereby allowing movement and secretion. A plasma variant of gelsolin is closely related to cytoplasmic gelsolin in structure and function in vitro. The role of plasma gelsolin is uncertain but it has been shown by an ELISA assay to be present in all humans studied. It binds fibrin and fibronectin, and may be important in coagulation and/or clearing of actin filaments from the circulation released by cell death. Plasma levels of gelsolin appear to be reduced in the Adult Respiratory Distress Syndrome but no other disease examined. A human hepatoma derived cell line, Hep G2, specifically makes both forms of gelsolin. The simultaneous production of a cytoskeletal and closely related secreted protein is unique in mammalian cells.
The aims of this project are as follows. The molecular structure of gelsolin will be determined by a combination of protein structural analysis and gene technology. The primary amino acid sequence will be determined by partially sequencing purified intact gelsolin and peptide fragments of gelsolin, and gelsolin cDNA clones will be isolated and sequenced. Sites of actin-binding and Ca+2 binding within the molecule will be determined by studying proteolytically-derived fragments for these activities. The gelsolin cDNA clones will be used to identify genomic clones to determine the gelsolin gene structure with a view to determining how regulation is exerted over synthesis and movement of each form of gelsolin within the cell. The gelsolin cDNA clone will also be used to assess the tissue distribution of gelsolin and the response of gelsolin synthesis in various tissues to factors such as steroids, cellular differentiation, and transforming agents. The gelsolin cDNA clones will permit comparison of gelsolin with homologous actin-binding proteins including villin.
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