Studies on Leukotriene Biosynthesis and Signal Transduction.

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PAF and leukotrienes are lipid mediators involved in inflammatory/allergic response as well as various cardiovascular and immunological disorders. We have carried out experiments on biosyntheses and signal transduction of these lipid mediators.1. Isolation of PAF and leukotriene receptors.By expression cloning using Xenopus oocyte system, we isolated a cDNA of PAF receptor as the first successful example of receptor cloning of lipid mediators. A human homologue was also obtained. They are composed of 342 amino acid residues with putative seven transmembrane spanning domains. These receptors were expressed in COS cells or CHO cells, and cellular signalings are extensively studied. It was shown that PAF receptor couples to phospholipases, adenylate cyclase, and MAP kinase via different types of G- proteins. Leukotriene receptors (B4, and D4) were solubilized from guinea pig leukocyte and lung membrane, respectively in an active form. They activate phospholipase A_2 and C via Gi proteins. … More Molecular cloning of these receptors by two strategies (protein purification and expression cloning) is currently ongoing.2. Leukotriene A_4 hydrolase. a bifunctional enzyme.Leukotriene B_4, a potent chemotactic factor, is produced by an enzyme termed leukotriene A4 hydrolase. By cDNA cloning and elucidation of the primary structure of the enzyme have shown that the enzyme is highly homologous to various types of metalloproteinases. In fact, it was shown that the enzyme contains a zinc ion, which is essential for both intrinsic aminopeptidase activity and leukotriene A_4 hydrolase. To determine the active site of the enzyme, site-directed mutagenesis experiments were performed. The mutation of His-296, or Glu-315 yielded loss of zinc ion with concomitant disappearance of both enzyme activities. However, by changing Glu-297 to other amino acids. two enzyme activities were clearly separated. The mechanism of incorporation of water molecule as well as the physiological significance of bifunctional role of enzyme remain unclear. Less