MOLECULAR ANALYSIS OF ENDOTOXIN RECEPTOR FUNCTION

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Septic shock is a highly lethal syndrome triggered by bacterial toxins. A major leap in our understanding of the pathogenesis of septic shock has been the discovery of LPS receptor protein on cells and in the plasma. CD14 is a 55 kDa glycophosphatidylinositol-linked protein surface expressed on the surface of macrophages and polymorphonuclear leucocytes. It also circulates in the plasma. Both membrane and soluble CD14 are critical parts of the LPS recognition system in human beings. Macrophages are stimulated via CD14 to make cytokines, enzymes and a variety of other responses to LPS. Other stimulatory bacterial products, such as peptidoglycan and lipoprotein from Gram-positive bacteria and lipoarabinomannan from mycobacteria also stimulate responses via CD14. Soluble CD14 complexed with LPS stimulates endothelial cells, smooth muscle cells, astrocytes and mesangial cells to express ICAM, ELAM, E- selectin, to make cytokines and nitrous oxide. Over the past five years, we have described some of the critical regions within the N-terminal half of CD14. CD14 is not homologous to other LPS binding proteins. Our hypothesis is that determining the specific structural features of CD14 which encode the various functions of CD14 will yield novel and meaningful information about LPS-protein interactions and the ensuing activation of cells. It is our goal to define the structural features of CD14 which determine its functions. The specific aims of this proposal are: 1. To determine the structural regions of CD14 which enable CD14 to bind lipopolysaccharides, interact with LBP, initiate cellular activation of cells expressing mCD14, enable internalization of LPS-LBP complexes, activate cells via the sCD14-LPS-dependent pathway; 2. To test whether the structural features of CD14 which recognize LPS are also involved in recognizing the surface molecules of Gram-positive microorganisms 3. To determine the three dimensional structure of CD14 by multidimensional NMR and X-ray crystallography we believe that better understanding of the structural domains of CD14 may allow design of new therapeutic strategies for the treatment of septic shock. Since septic shock currently has a mortality rate of 50%, and is a common occurrence in hospitalized patients, effective therapy would be a major medical advance.