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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL063284-01
Application #
2898682
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Wong, G William; Stevens, Richard L (2005) Identification of a subgroup of glycosylphosphatidylinositol-anchored tryptases. Biochem Biophys Res Commun 336:579-84
Yasuda, Shinsuke; Morokawa, Nasa; Wong, G William et al. (2005) Urokinase-type plasminogen activator is a preferred substrate of the human epithelium serine protease tryptase epsilon/PRSS22. Blood 105:3893-901
Abonia, J Pablo; Friend, Daniel S; Austen Jr, William G et al. (2005) Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 174:7285-91
Wong, Guang W; Yasuda, Shinsuke; Morokawa, Nasa et al. (2004) Mouse chromosome 17A3.3 contains 13 genes that encode functional tryptic-like serine proteases with distinct tissue and cell expression patterns. J Biol Chem 279:2438-52
Wong, Guang W; Yang, Yi; Yasuda, Shinsuke et al. (2003) Mouse mast cells express the tryptic protease neuropsin/Prss19. Biochem Biophys Res Commun 303:320-5
Li, Lixin; Yang, Yi; Wong, Guang W et al. (2003) Mast cells in airway hyporesponsive C3H/HeJ mice express a unique isoform of the signaling protein Ras guanine nucleotide releasing protein 4 that is unresponsive to diacylglycerol and phorbol esters. J Immunol 171:390-7
Li, Lixin; Yang, Yi; Stevens, Richard L (2002) Cloning of rat Ras guanine nucleotide releasing protein 4, and evaluation of its expression in rat mast cells and their bone marrow progenitors. Mol Immunol 38:1283-8
Yang, Yi; Li, Lixin; Wong, Guang W et al. (2002) RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demon J Biol Chem 277:25756-74
Qi, Jian Cheng; Stevens, Richard L; Wadley, Robert et al. (2002) IL-16 regulation of human mast cells/basophils and their susceptibility to HIV-1. J Immunol 168:4127-34
Wong, Guang W; Foster, Paul S; Yasuda, Shinsuke et al. (2002) Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase gamma. TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor alpha/signal transducer J Biol Chem 277:41906-15

Showing the most recent 10 out of 19 publications