Phospholipases A2 (PLA2s) hydrolyze the sn-2 ester of glycero-phospholipids to produce a fatty acid and a lysophospholipid. Secreted, 14 kDa PLA2s are involved in a number of inflammatory processes, including: (1) mast cell activation; (2) liberation of arachidonic acid for the biosynthesis of eicosanoids; and (3) allergic reactions. The main goal of this proposal is to better understand the way in which these enzymes bind not only to synthetic vesicles but also to cellular membranes. Site-specific spin labeling of the PLA2 from bee venom and EPR spectroscopy will be used to precisely map the surface of this enzyme that contacts the phospholipid bilayer. Similar studies will be carried out with the phosphatidylinositol-specific phospholipase C from B. cereus. A number of cells involved in the inflammatory response secrete a 14-kDa, non-pancreatic PLA2 that acts on a variety of cellular targets. the enzyme appears to be anchored to the outer surface of cells by binding to heparan sulfate proteoglycan. Attempts will be made to locate the putative heparan sulfate binding site on the surface of the non-pancreatic enzyme by mutation of surface basic residues. the ability of mutants that no longer bind to heparan sulfate to activate cellular targets will then be studied. Bee venom PLA2 is one of the most potent human allergens. Previously, we found that a mutant form of this enzyme that lacks enzymatic activity is much less effective than wild-type enzyme in inducing an IgE and IgG response in mice. It has been proposed that mast cells are a source of interleukin-4, a cytokine that induces the production of IgG and IgE, and we found that enzymatically active bee venom PLA2, but not the inactive mutant, causes mast cells to release interleukin-4 in the absence of stimulation with IgE/antigen. Dr Gelb now wishes to understand these events in more detail as they may shed light on the induction of an immune response and may also lead to the development of more effective strategies for treatment of allergy. The final area is centered around the development of potent inhibitors of 14-kDa secreted phospholipases A2. A combinatorial chemistry approach will be used to prepare a large library of analogs of a lead-compound inhibitor, and novel techniques will be developed for identifying which compounds in the mixture are the most potent inhibitors. We will also study the potent PLA2 inhibitor Thelocin B3 (isolated form a fungus). Attempts will be made to obtain the high resolution crystal structure of the non-pancreatic PLA2 complexed with these inhibitors. This will permit a structure-based approach toward the design of more potent inhibitors.
Showing the most recent 10 out of 23 publications