MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION OF MOUSE LEUKOTRIENE C-4 SYNTHASE

Leukotriene C-4 synthase (EC 2.5.1.37) catalyzes the conjugation of re duced glutathione (GSH) with leukotriene A(4) to form the intracellula r parent of the proinflammatory cysteinyl leukotrienes. Human leukotri ene C-4 synthase shares substantial amino acid identity in its consens us N-terminal two-thirds with 5-lipoxygenase-activating protein-and ha s 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 C-4 synthase cDNA using the pol ymerase chain reaction to screen a mouse pcDNA3 expression library wit h oligonucleotide primers based on the translated human leukotriene C- 4 synthase cDNA sequence. Mouse leukotriene C-4 synthase cDNA is 667 b p in length, including the poly(A)-rich tail, and shows 87% similarity with the human cDNA within the open reading frame. The deduced 150-am ino-acid sequence of mouse leukotriene C-4 synthase differs from the h uman enzyme by only 18 amino acids. of which 9 reside at the C terminu s. The potential N-glycosylation site, two protein kinase C phosphoryl ation sites, the two cysteine residues, and the putative inhibitor-bin ding domain (substitutions Thr41-->Ser and Tyr50-->Phe) were conserved in mouse leukotriene C-4 synthase. Northern blot analysis indicated t hat the leukotriene C-4, synthase RNA transcript is widely distributed . The K-m values for leukotriene A(4) methyl ester, leukotriene A(4) f ree acid and GSH were 7.6 mu M, 3.6 mu M and 1.6 mu M, respectively, f or purified human recombinant enzyme, and 10.3 mu M, 2.5 mu M and 1.9 mM, respectively, for purified recombinant mouse enzyme; the correspon ding V-max values were 2.5, 1.3 and 2.7 mu mol . min(-1) . mg(-1) prot ein, respectively, for human enzyme, and 2.3, 1.2 and 2.25 mu mol . mi n(-1) . mg(-1) protein, respectively for mouse enzyme. The 5-lipoxygen ase-activating-protein inhibitor, MK-886, was active against both huma n and mouse recombinant leukotriene C-4 synthase with IC50 values of 3 .1 mu M and 2.7 mu M, respectively. These findings confirm that the le ukotriene C-4 synthases belong to a gene family that includes the 5-li poxygenase-activating protein and suggest that the C-terminal domain o f leukotriene C-4 synthase may not be critical for its conjugation fun ction.