Coupling of recombinant 5-lipoxygenase and leukotriene A4 hydrolase activities and transcellular metabolism of leukotriene A4 in Sf9 insect cells
Mancini, J.A.; Evans, J.F.
European Journal of Biochemistry 218(2): 477-484
High-level expression of human leukotriene (LT) A-4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA-4 hydrolase activity in this system is at least 50-fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA-4 hydrolase (rvHLTA-4h) was used for coinfection studies with recombinant viral 5-lipoxygenase (rvH5LO). When Sf9 cells expressing 5-lipoxygenase are incubated in the presence of A23187 and arachidonic acid, (5S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid 5-H(P)ETE and LTA-4 are synthesized in a ratio of 5: 1 for 5-H(P)ETE/LT. Coexpression of 5-lipoxygenase and LTA-4 hydrolase in these insect cells results in the synthesis of 5-H(P)ETE, LTA-4 and in addition LTB-4, and the ratio shifts to 2:1 for 5-H(P)ETE/LT. The production of enzymically formed LTB-4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA-4 hydrolase and 5-lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB-4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB-4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA-4h resulted in export of LTA-4 from the rvH5LO cells and transcellular metabolism of LTA-4 to LTB-4 in the rvHLTA-4h Sf9cells.