One of the reasons why we are able to combat cancer and the numerous pathogens that we come in contact with each day is because our hematopoietic effector cells [including mast cells (MCs), cytotoxic T lymphocytes (CTLs), natural killer cells, basophils, eosinophils, neutrophils, macrophages, and platelets] are able to rapidly release from their intracellular granules large amounts of diverse types of biologically-active molecules during the specific immune or inflammatory reaction. Serine proteases that are enzymatically active at neutral pH predominate in the granules of MCs and CTLs. One of the mysteries in immunology is how effector cells such as MCs and CTLs store high concentrations of enzymatically-active serine proteases in their granules in such a way that these proteases do not undergo autolysis. All granule containing hematopoietic effector cells that participate in immune and inflammatory responses store serglycin proteoglycans (PGs) in their secretory granules. Serglycin PGs possess the same Ser/Gly-rich peptide core to which are attached either heparin or novel forms of chondroitin sulfate (ChS) glycosaminoglycans. Because it is hypothesized that the regulation of the serglycin gene and the post-translational modification of its product is extremely important for the development and function of many of our hematopoietic effector cells, the overall objective of this grant is to use biochemical, immunological, and molecular approaches to deduce the functions of serglycin PGs in MC-mediated immune responses. In Specific Aim 1, a homologous recombination approach will be used to disrupt the serglycin gene and the N-sulfotransferase-2 (NST-2) gene in mice to prevent expression in MCs of all serglycin PGs or only those that contain heparin chains, respectively. The functional consequences on the whole mouse and the bone marrow-derived MCs developed from the transgenic animals will be determined. In the Specific Aim 2, three different types of MC granule proteases will be expressed in insect cells and the interaction of these recombinant enzymes with isolated serglycin PGs will be investigated.