MOLECULAR-CLONING OF THE GENE FOR MOUSE LEUKOTRIENE-C-4 SYNTHASE

Leukotriene C-4 (LTC4) synthase (LTC4S), an integral membrane protein, catalyzes the conjugation of leukotriene A(4) with reduced glutathion e to form LTC4, the biosynthetic parent of the additional cysteinyl le ukotriene metabolites. An XmnI-digested fragment of a P1 clone from a 129 mouse ES library contained the full-length gene of 2.01 kb for mou se LTC4S. The mouse LTC4S gene is comprised of 5 exons of 122, 100, 71 , 82 and 241 nucleotides, with intron sizes that range from 76 nucleot ides to 937 nucleotides. The intron/exon boundaries are identical to t hose of the human genes for LTC4S and 5-lipoxygenase-activating protei n (FLAP). Primer extension demonstrated a single transcription-initiat ion site 64 bp 5' of the ATG translation-start site. Nucleotide sequen cing of 1.2 kb of the 5' flanking region revealed multiple putative si tes for activating protein-2, CCAAT/enhancer-binding protein, and poly oma virus enhancer-3. Fluorescent in situ hybridization mapped the mou se LTC4S gene to mouse chromosome 11, in a region containing the genes for interleukin 13 and granulocyte/macrophage-colony-stimulating fact or, and orthologous to the chromosomal location of 5q35 for the human LTC4S gene. Thus, the mouse LTC4S gene is similar in size, intron/exon organization and chromosomal localization to the human LTC4S gene. Re cent mutagenic analysis of the conjugation function of human LTC4S has identified R51 and Y93 as critical for acid and base catalysis of LTA (4) and reduced glutathione, respectively. A comparison across species for proteins that possess LTC4S activity reveals conservation of both of these residues, whereas R51 is absent in the FLAP molecules. Thus, within the glutathione S-transferase superfamily of genes, alignment of specific residues allows the separation of LTC4S family members fro m their most structurally similar counterparts, the FLAP molecules.