DISRUPTION AND EXPRESSION OF MAST CELL PROTEASE GENES

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Granule proteases, which represent the major protein constituents of mast cells, have been used to identify distinct phenotypic populations in tissues. However, little is known about their functions, how they are activated, or how they are metabolized inside and outside of the mast cell. As a result of cytokine/factor-dependent regulation, mouse mast cells express varied combinations of at least seven serine proteases [designated mouse mast cell protease (mMCP) 1 to 7] and an exopeptidase [designated mouse mast cell carboxypeptidase A (mMC-CPA)] that are enzymatically active at neutral pH. Presumably, the number and type of proteases each mouse mast cell expresses are related to the number and type of proteins these effector cells must degrade or activate in their different tissue environments. Although many of the mMCPs are highly homologous with one another, each protease has its own unique set of amino acids in its substrate-binding cleft. While discrete functions seem likely, the specific substrates of each mast cell protease within and across superfamilies remain to be elucidated. The cloning of the cDNAs and genes that encode these mouse mast cell granule proteases now allows the use of two complementary approaches in this Project to address the metabolism and function of the mast cell's secretory granule proteases. In Specific Aim 1, the chromosome 14 complex where the mast cell chymase (mMCP-1 to mMCP-5) genes reside will be mapped. Transgenic mice will be generated that have the mMC-CPA, mMCP-7, mMCP-2, and possibly other chymase genes disrupted. The consequences of ablating such genes on the development of mast cells in the transgenic animals will be assessed by morphometric and immunohistochemical analyses. Functional effects will be assessed by determining whether or not mast cell activation in a particular protease-null, transgenic animal primes the airways for augmented response to a specific agonist as does a wild type animal. In Specific Aim 2, "pro" and "mature" mouse mast cell proteases will be expressed in insect cells and rat basophilic leukemia cells. These recombinant proteases will be purified and then used to assess how mast cell proteases are enzymatically activated in the granule, to examine their substrate specificities in vitro, and to determine the mechanisms by which they are metabolized in vitro and in vivo.