BIOCHEMICAL AND GENOMIC REGULATION OF HUMAN LTC SYNTHASE

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Leukotriene (LT) C4 synthase is the pivotal enzyme involved in conjugating LTA4 with glutathione (GSH) to form LTC4, the parent compound of the cysteinyl leukotrienes. Hence, LTC4 synthase can regulate the biosynthesis of this potent mediator, which contributes to the pathobiology of bronchial asthma through its metabolites, LTD4 and LTE4. The key objective of this project is to define genomic regulation of the LTC4 synthase gene using reporter constructs (Specific Aim 1). Another objective (Specific Aim 2) is to examine regulation of the gene in leukemic cells with and without activation and in non-transformed in vitro-derived eosinophils during maturation. The substrate binding site for LTA4, the membrane topology of the protein and the signal involved in membrane targeting re additional areas for study (Specific Aim 3). Trans-acting factors will be identify through comparison of the identified cis-acting elements with the known factors in a computer data base, and by gel shift and supershift assays. We have identified a 550-bp genomic fragment, composed of 66 bp of the exon 1 and 484 bp of 5' flanking region, that contains a putative initiator element and enhancer elements. Like other TATA-less genes, there is a SP-1 binding site 44 bp 5' of the putative initiator element. These regulatory elements will be further defined by mutagenic analysis of the reporter constructs in transient transfections. Additional regulatory regions will be sought by Dnase I hypersensitivity assays and in vitro footprinting of LTC4 synthase in cells engaged in increased transcript expression. The LTA4 binding site will be defined by site-directed mutagenesis of the wild-type enzyme with kinetic analysis of the mutated recombinant proteins. Anti-peptide antibodies will be used to examine the location of the hydrophilic loops and the N terminus and the C terminus of the enzyme in relation to the outer membrane and perinuclear membrane of the nuclear envelope. The membrane targeting signal of LTC4 synthase will be sought by transfection of cDNAs encoding for LTC4 synthase peptide fragment-green fluorescence protein (GFP) fusion proteins into COS cells followed by fluorescence microscopy examination of the localization of the expressed fluorescence fusion protein.