Cellular oxygenation of 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid by 5-lipoxygenase is stimulated by 5-lipoxygenase-activating protein

It has been proposed that 5-lipoxygenase (5-LO)-activating protein (FLAP) i s an arachidonate transfer protein for leukotriene biosynthesis, Using the Spodoptera frugiperda (Sf9) insect cells, we demonstrate that FLAP causes a large stimulation (190-fold) of the conversion of 12(S)-hydroxyeicosatetra enoic acid (12(S)-HETE) to 5,12-diHETE when co-expressed with 5-lipoxygenas e. We also demonstrate that FLAP can stimulate (2-2.5-fold) the oxygenation of 15(S)-HETE by 5-LO to 5,15-diHETE, The stimulation of both 15(S)-HETE a nd 15(S)-HETE oxygenation by 5-LO is completely inhibitable by the FLAP inh ibitor, MK-886. In order to determine which residues of FLAP are important for 12(S)-HETE and arachidonic acid utilization by 5-LO, various mutants of FLAP were co-expressed with 5-LO in Sf9 cells. The FLAP deletion mutants d el 37-53, del 52-58, del 106-108, and del 148-161 and the point mutant D62N were analyzed. The D62N mutation, which reduces the binding of indole inhi bitors to FLAP, had no effect on the stimulation of substrate utilization b y 5-LO. In contrast to wild type FLAP, the mutant proteins del 37-53, del 1 06-108, and del 148-161 failed to stimulate 12(S)HETE and arachidonic acid utilization by 5-LO. Only one of the latter three mutations (del 37-53) has been shown to abolish the binding of indole inhibitors to FLAP. These resu lts suggest that the lipid binding site of FLAP overlaps the inhibitor bind ing site and occupies several regions of the protein not essential for inhi bitor binding. Because FLAP can stimulate the utilization of 12(S)-HETE, 15 (S)-HETE, and arachidonic acid by 5-LO, FLAP may also function as a more ge neral lipid carrier protein for the biosynthesis of multiple oxygenation pr oducts of arachidonic acid in addition to its role in leukotriene biosynthe sis.