Abstract
It has been proposed that 5-lipoxygenase (5-LO)-activating protein (FLAP) is 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)-hydroxyeicosatetraenoic acid (12(S)-HETE) to 5, 12-diHETE when co-expressed with 5-lipoxygenase. 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 12(S)-HETE and 15(S)-HETE oxygenation by 5-LO is completely inhibitable by the FLAP inhibitor, 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 del 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 inhibitors to FLAP, had no effect on the stimulation of substrate utilization by 5-LO. In contrast to wild type FLAP, the mutant proteins del 37-53, del 106-108, 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 results suggest that the lipid binding site of FLAP overlaps the inhibitor binding site and occupies several regions of the protein not essential for inhibitor 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 general lipid carrier protein for the biosynthesis of multiple oxygenation products of arachidonic acid in addition to its role in leukotriene biosynthesis.
Highlights
Leukotrienes are derived from arachidonic acid through the 5-lipoxygenase pathway
This study focuses on the ability of various mutants of FLAP to support substrate utilization by 5-lipoxygenase when the two proteins are coexpressed using an insect cell baculovirus expression system
Previous work on the co-expression of FLAP and 5-LO in Sf9 insect cells has demonstrated that FLAP stimulates the use of arachidonic acid as a substrate and increases the efficiency with which 5-LO converts 5-H(P)ETE to leukotriene A4 (LTA4) [10]
Summary
Chemicals and Reagents—Restriction endonucleases, DNA polymerase I, Klenow fragment, deoxy-nucleotide triphosphates, and T4 DNA ligase were obtained from Boehringer Mannheim. The recombinant baculovirus was obtained from the supernatant by centrifugation of the Sf9 cells and media at 1000 ϫ g for 5 min. Eicosanoid Synthesis—Sf9 cells were harvested by centrifugation (300 ϫ g for 5 min at room temperature) and washed with Dulbecco’s phosphate-buffered saline (Life Technologies, Inc.). Small aliquots of these cells were retained for immunoblot analysis. Protein Immunoblotting—Sf9 cells expressing the various recombinant proteins were harvested by centrifugation (300 ϫ g for 5 min at room temperature) and washed once with Dulbecco’s phosphate-buffered saline (Life Technologies, Inc.). Immunoblot analysis was performed as described previously [23] using a 1:200 dilution of antisera and 125I-labeled protein A
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