Molecular cloning, expression and characterization of mouse leukotriene C4 synthase

Abstract

Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the conjugation of reduced glutathione (GSH) with leukotriene A4 to form the intracellular parent of the proinflammatory cysteinyl leukotrienes. Human leukotriene C4 synthase shares substantial amino acid identity in its consensus N-terminal two-thirds with 5-lipoxygenase-activating protein and has a region (residues 37-58) that exhibits 46% amino acid identity with a domain of this protein (residues 41-62) to which an inhibitor binds. We have now cloned mouse leukotriene C4 synthase cDNA using the polymerase chain reaction to screen a mouse pcDNA3 expression library with oligonucleotide primers based on the translated human leukotriene C4 synthase cDNA sequence. Mouse leukotriene C4 synthase cDNA is 667 bp in length, including the poly(A)-rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150-amino-acid sequence of mouse leukotriene C4 synthase differs from the human by only 18 amino acids, of which 9 reside at the C terminus. The potential N-glycosylation site, two protein kinase C phosphorylation sites, the two cysteine residues, and the putative inhibitor-binding domain (substitutions Thr41 → Ser and Tyr50 → Phe) were conserved in mouse leukotriene C4 synthase. Northern blot analysis indicated that the leukotriene C4 synthase RNA transcript is widely distributed. The K(m) values for leukotriene A4 methyl ester, leukotriene A4 free acid and GSH were 7.6 μM, 3.6 μM and 1.6 mM, respectively, for purified human recombinant enzyme, and 10.3 μM and 1.9 mM, respectively, for purified recombinant mouse enzyme; the corresponding V(max) values were 2.5, 1.3 and 2.7 μmol · min-1 · mg-1 protein, respectively, for human enzyme, and 2.3, 1.2 and 2.2 μmol · min-1 · mg-1 protein, respectively, for mouse enzyme. The 5-lipoxygenase-activating-protein inhibitor, MK-886, was active against bothhuman and mouse recombinant leukotriene C4 synthase with IC50 values of 3.1 μM and 2.7 μM, respectively. These findings confirm that the leukotriene C4 synthases belong to a gene family that includes the 5- lipoxygenase-activating protein and suggest that the C-terminal domain of leukotriene C4 synthase may not be critical for its conjugation function.