EXPRESSION CLONING OF A CDNA FOR HUMAN LEUKOTRIENE C-4 SYNTHASE, AN INTEGRAL MEMBRANE-PROTEIN CONJUGATING REDUCED GLUTATHIONE TO LEUKOTRIENE A(4)

Leukotriene (LT) C-4 synthase, an integral microsomal membrane protein, conjugates LTA(4), an epoxide intermediate, to reduced glutathione (GSH) to form a proinflammatory mediator, LTC(4). A sensitive fluorescence-linked immunoassay for LTC(4) was used to screen a KG-1 cDNA expression library for LTC(4) synthase activity after transfection of COS cells and addition of substrate LTA(4). Stepwise resolution of 240,000 colonies in 96 pools led to the identification of individual clones with maximal LTC(4) synthase activity that contained a 694-bp cDNA insert. This insert was composed of a 54-bp 5' nontranslated region, an ATTAAA polyadenylylation signal, and a poly(A)(+) tail. The open reading frame encodes a 16.5-kDa protein with a pI of 11.05. Hybridization with a cDNA probe demonstrated a mRNA transcript of 0.7 kbp in RNAs from human eosinophils and KG-1 cells, which contain LTC(4) synthase. The nucleotide and deduced amino acid sequences of the LTC(4) synthase cDNA show no significant homology to GSH S-transferases but share 31% overall amino acid identity with 5-lipoxygenase activating protein (FLAP). The identity at the N-terminal two-thirds of these two proteins is 44%, with some regions of near identity. Peptide structural analysis of the deduced LTC(4) synthase predicts the presence of three transmembrane domains nearly superimposable on those of FLAP. Moreover, LTC(4) synthase is inhibitable by a FLAP inhibitor, MK-886. Therefore, LTC(4) synthase is distinct from the known GSH S-transferases by nucleotide and consensus amino acid sequences, and its GSH-conjugating function represents a distinct integral membrane protein belonging to a distinct gene family.