The ability to introduce novel antimicrobial proteins into the phagolysosomes of granulocytes or macrophages may have important therapeutic implications but is currently limited by our rudimentary understanding of posttranslational processing and targeting of proteins destined for phagolysosomes. Because of their abundance and small size, neutrophil defensins present a compelling model for the exploration of posttranslational processing of granule proteins and their packaging into granules. Pulse-chase labelling, subcellular fractionation, and ultrastructural immunolocalization studies will be undertaken to determine the subcellular location of the proteolytic processing steps of human defensin precursors in HL-60 cell line and human bone marrow cells. We will characterize the endopeptidases that are responsible for defensin maturation. We will ascertain whether defensin precursors undergo posttranslational modification and whether proteoglycans play an obligatory role in defensin processing and packaging. We will search for specific carrier proteins involved in the intracellular sorting of defensins to azurophil granules. We will express a small exogenous (rabbit) defensin cDNA in HL-60 cells, and by PCR mutagenesis explore the structural requirements for the correct processing and packaging of defensin protein. We will use retroviral vectors to express exogenous defensins in phagocytic cell lines and then evaluate their posttranslational processing, subcellular targeting and effects of defensins on the microbicidal activity of the modified phagocytes. Overall, the studies will shed light on basic mechanisms of synthesis and subcellular trafficking of the major polypeptide constituent of azurophil granules. In the longer term, these studies may lead to therapeutic strategies for targeting exogenous microbicidal proteins into the cytoplasmic granules of human or animal neutrophils, monocytes and macrophages.
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