DISRUPTION AND EXPRESSION OF MAST CELL PROTEASE GENES

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DESCRIPTION (adapted from applicant's abstract): Mouse mast cells (MCs) express varied combinations of at least 12 serine proteases (designated mouse MC protease (mMCP) 1 to 10, cathepsin G, and granzyme B) and an exopeptidase (designated mouse MC carboxypeptidase A (mMC-CPA) that are enzymatically active at neutral pH. Although these granule proteases and their human homologues have been invaluable for identifying distinct populations of MCs in tissues and for understanding MC development, the biological substrates for most of the proteases have yet to be determined. Presumably, the number and type of proteases each MC expresses are related to the number and type of proteins it must degrade or activate in a particular tissue environment. Although many of the MCPs are highly homologous with one another, each protease has a unique set of amino acids in its substrate-binding cleft. The cloning of the cDNAs and genes that encode the varied mouse MC proteases and their human homologues now allows the use of complementary approaches to address their function and metabolism. In Specific Aim 1, the chromosome 17 complex, where the mouse MC tryptase genes reside, will be mapped and sequenced to identify the remaining protease genes in the complex. Transgenic mice will be generated that have certain of these tryptase genes disrupted and the consequences of ablating such genes on the development and function of the MCs in the heart, lung, uterus, and other organs will be assessed. For example, functional effects will be assessed in the lung by determining whether or not MC cell activation in a particular tryptase-null mouse primes the airways for augmented response to a specific agonist. In Specific Aim 2, recombinant mouse and human tryptases will be generated to evaluate their substrate specificities, and to determine the mechanisms by which they are inactivated and metabolized in vivo. Finally, the recombinant tryptases will be used to obtain low molecular weight inhibitors that are specific for each protease.