Endotoxins (gram-negative bacterial lipopolysaccharides, or LPS) play a major role in the pathogenesis of invasive gram-negative bacterial diseases; the inflammatory reaction elicited by these molecules may produce great morbidity and even death. Little is known about host mechanisms for recognizing and modulating the toxic signal information in LPS. During the past decade we have purified, characterized, and cloned a human leukocyte enzyme, acyloxyacyl hydrolase (AOAH), that selectively removes certain acyl chains from the lipid A region of lipopolysaccharides, thereby detoxifying the LPS. Although LPS detoxification is the likely function of the enzyme in animals, recent findings suggest alternative roles such as LPS signal transduction and phospholipid turnover. We now would like to build on our previous work to determine the biological role(s0 of acyloxyacyl hydrolase in human neutrophils and macrophages, and to lay groundwork for subsequent in vivo uses of the enzyme. The proposed research will (1) use site-directed mutagenesis to determine structure function relationships for AOAH and to produce mutated enzyme to use in the subsequent studies, (2) determine the functions of AOAH in human macrophages and optimize the uptake of exogenous AOAH by these cells, (30 determine the fate and LPS-deacylating ability of exogenous recombinant AOAH in mice, (4) localize AOAH in neutrophils and macrophages and evaluate its intracellular movement in response to various stimuli, and (5) clone rabbit AOAH CDNA to use for in vivo studies of AOAH synthesis and regulation. These studies should greatly clarify the roles played by this novel enzyme and provide a solid scientific foundation for its possible uses in the prevention and therapy of gram-negative bacterial diseases (endotoxic shock). Useful new information will also be generated about cellular uptake mechanisms and the intracellular fate of LPS, a bacterial toxin that is encountered continuously by animals, including man.
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