Coupling of recombinant 5-lipoxygenase and leukotriene A4 hydrolase activities and transcellular metabolism of leukotriene A4 in Sf9 insect cells.
High-level expression of human leukotriene (LT) A4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA4 hydrolase activity in this system is at least 50-fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA4 hydrolase (rvHLTA4h) 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 LTA4 are synthesized in a ratio of 5:1 for 5-H(P)ETE/LT. Coexpression of 5-lipoxygenase and LTA4 hydrolase in these insect cells results in the synthesis of 5-H(P)ETE, LTA4 and in addition LTB4, and the ratio shifts to 2:1 for 5-H(P)ETE/LT. The production of enzymically formed LTB4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA4 hydrolase and 5-lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA4h resulted in export of LTA4 from the rvH5LO cells and transcellular metabolism of LTA4 to LTB4 in the rvHLTA4h Sf9cells.
Mancini
J A
JA
Department of Biochemistry, McGill University, Montreal, Canada.
Evans
J F
JF
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D004851:Epoxide Hydrolases / Q000378:metabolism*; D006801:Humans; D017572:Leukotriene A4 / Q000378:metabolism*; D007975:Leukotriene B4 / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses; D011994:Recombinant Proteins / Q000378:metabolism
D018076:DNA, Complementary; D017572:Leukotriene A4; D011994:Recombinant Proteins; D007975:Leukotriene B4; D001094:Arachidonate 5-Lipoxygenase; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase