The goals of these studies are to isolate and characterize carboxypeptidase, a newly identified human mast cell secretory granule protease, and to localize and determine the pathobiologic role of mast cell proteolytic enzymes in mast cell mediated skin diseases. Proposed studies will address: 1) purification and physicochemical characterization of human skin mast cell carboxypeptidase; 2) characterization and in vitro reconstitution of the human skin mast cell secretory granule with respect to protease and proteoglycan components; 3) investigation of the biochemical action of purified carboxypeptidase, and enzyme- proteoglycan complexes on purified proteins, peptides, and potential cellular substrates in skin; and 4) examination of the pathobiologic role of human skin mast cell secretory granule proteases in mast cell-associated cutaneous diseases. These studies will add to understanding of the human skin mast cell, and the role of mast cell secretory granule proteases in mediating tissue damage in cutaneous disorders. To accomplish these goals, human skin mast cell carboxypeptidase, identified by an HPLC assay system, will be purified from whole skin extracts by affinity chromatography. A monospecific, polyclonal anticarboxypeptidase antibody will be developed. To understand regulation of carboxypeptidase expression, mast cells from various tissues will be assessed for carboxypeptidase activity by HPLC, and protein by ELISA or RIA, and a human mast cell cDNA library will be made to identify recombinant clones corresponding to carboxypeptidase. To understand their biochemical and biologic activity, enzyme-proteoglycan complexes from dispersed human mast cells or reconstituted from purified components, will be incubated with protein, peptide, and potential cellular substrates such as fibrinogen, laminin, substance P, angiotensin I, and cultured human umbilical vein endothelial cells, respectively. The proteolytic activity of these complexes will be examined in vitro using skin explants. The in situ release, localization and fate of carboxypeptidase will be examined in vitro using skin explants, and in vivo after intradermal injection of human C5a, and in a skin chamber model after challenge with allergens. Finally, the deposition of mast cell granule carboxypeptidase will be examined in vivo in diseases marked by increased vascular permeability, i.e. cold urticaria, and by tissue destruction, i.e. bullous pemphigoid.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR031901-06
Application #
3156142
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1982-07-01
Project End
1993-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143