Mast cells, basophils, and natural killer cells participate in allergic reactions, host defense mechanisms, and immune surveillance and have been found to contain a unique class of proteoglycans in their secretory granules. The overall goal of this grant proposal will be to study the biochemistry and biology of these intracellular proteoglycans. The transcription, translation, post-translational modification, intracellular transport and sorting, and catabolism of the proteoglycans that reside in mouse bone marrow-derived mast cells, rat serosal mast cells, rat basophilic leukemia-1 cells, and human natural killer (NK) cells will be investigated. Preparative amounts of the proteoglycans from each of these cell types will be obtained in order to determine the structure of the oligosaccharides which are bound to their respective peptide cores, and to raise monoclonal antibodies against each proteoglycan. The monoclonals will be used to study the distribution of these four cell types in vivo, and to study the differentiation of each cell type in vivo and in vitro. Using in vitro culture techniques, the role of intragranular proteoglycans of mast cells, basophils, and NK cells in the regulation of osmotic pressure at the time of granule exocytosis, the concentration and packaging of basic amines and proteases found in the secretory granules, and the inhibition of specific proteases will be investigated. The role of secretory granule proteoglycans in NK cell medited cytotoxicity against tumor cell targets will be studied. The methods to be used in this investigation include culture techniques to obtain large numbers of each of these cell types. Biochemical techniques, including HPLC, gel filtration, ion exchange chromatography, SDS-PAGE, and 500 MHz NMR will be used to purify and analyze each intracellular proteoglycan. Recombinant DNA procedures will be used to study the transcription and translation of the peptide cores of these proteoglycans. Immunocytochemical techniques will be used to localize secretory granule proteoglycans.
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