Mast cells, basophils, and natural killer cells participate in allergic reactions, host defense mechanisms, and immune surveillance and have been found to contain a unique class of proteoglycans in their secretory granules. The overall goal of this grant proposal will be to study the biochemistry and biology of these intracellular proteoglycans. The transcription, translation, post-translational modification, intracellular transport and sorting, and catabolism of the proteoglycans that reside in mouse bone marrow-derived mast cells, rat serosal mast cells, rat basophilic leukemia-1 cells, and human natural killer (NK) cells will be investigated. Preparative amounts of the proteoglycans from each of these cell types will be obtained in order to determine the structure of the oligosaccharides which are bound to their respective peptide cores, and to raise monoclonal antibodies against each proteoglycan. The monoclonals will be used to study the distribution of these four cell types in vivo, and to study the differentiation of each cell type in vivo and in vitro. Using in vitro culture techniques, the role of intragranular proteoglycans of mast cells, basophils, and NK cells in the regulation of osmotic pressure at the time of granule exocytosis, the concentration and packaging of basic amines and proteases found in the secretory granules, and the inhibition of specific proteases will be investigated. The role of secretory granule proteoglycans in NK cell medited cytotoxicity against tumor cell targets will be studied. The methods to be used in this investigation include culture techniques to obtain large numbers of each of these cell types. Biochemical techniques, including HPLC, gel filtration, ion exchange chromatography, SDS-PAGE, and 500 MHz NMR will be used to purify and analyze each intracellular proteoglycan. Recombinant DNA procedures will be used to study the transcription and translation of the peptide cores of these proteoglycans. Immunocytochemical techniques will be used to localize secretory granule proteoglycans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023483-02
Application #
3135655
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
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
Li, Lixin; Yang, Yi; Stevens, Richard L (2003) RasGRP4 regulates the expression of prostaglandin D2 in human and rat mast cell lines. J Biol Chem 278:4725-9
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
Walsh, Jonathan C; DeKoter, Rodney P; Lee, Hyun Jun et al. (2002) Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity 17:665-76
Li, Y; Li, L; Wadley, R et al. (2001) Mast cells/basophils in the peripheral blood of allergic individuals who are HIV-1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4. Blood 97:3484-90
Huang, C; De Sanctis, G T; O'Brien, P J et al. (2001) Evaluation of the substrate specificity of human mast cell tryptase beta I and demonstration of its importance in bacterial infections of the lung. J Biol Chem 276:26276-84
Wong, G W; Li, L; Madhusudhan, M S et al. (2001) Tryptase 4, a new member of the chromosome 17 family of mouse serine proteases. J Biol Chem 276:20648-58

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