RECOMBINANT HUMAN MAST CELL TRYPTASES

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DESCRIPTION (from applicant's abstract): Human mast cell tryptases (trypsin-like serine proteases) are the major protein products of mast cells suggesting that they play an important role in the function of mast cells. Based on published cDNA sequences there are four different human tryptases and the principal investigator hypothesizes that these different mast cell tryptases have different functional characteristics that allow for different functions. The presence of mast cells in almost all tissues indicate that tryptases have significant functions in native an pathological conditions and the general lack of knowledge concerning how these enzymes differ in function provide the rationale for the proposal. The central hypothesis is that the different mast cell tryptases have evolved to serve different functions and that knowledge of the structures and functions of these enzymes is critical to understanding the roles of mast cells in health and disease. In preliminary studies the principal investigator has expressed and purified fully active recombinant human skin tryptase 1 (rHST1) in Pichia pastoris. In Aim 1 site-directed mutagenesis of rHST1 cDNA will be used to express human tryptase beta/2 and tryptase alpha in Pichia pastoris. In Aim 2 Tryptases 1, beta/2 and alpha will be characterized and compared with regard to heparin binding and kinetics on synthetic and natural substrates to define their differences and similarities. In Aim 3 based on the finding that human lung tryptase rapidly and specifically cleaves the single RGD motif in fibrinogen that is bound by alphav-beta-3 integrins, other proteins with RGD sites, including vitronectin and fibronectin, will be examined for cleavage by the three rHTs. This work will provide needed comparative data on tryptases of known sequence and should yield insight as to why humans have such similar, yet different, enzymes in mast cells. Support will allow this laboratory to continue the study of tryptases and the mentoring of undergraduate students, while gaining knowledge that could improve our understanding of the roles of mast cells in diseases, such as asthma, arthritis, cancer, and cardiovascular diseases.