Identification of the two-phase mechanism of arachidonic acid regulating inflammatory prostaglandin E2 biosynthesis by targeting COX-2 and mPGES-1

Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE₂) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE₂ biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE₂ synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE₂ production. AA demonstrated two phase activities to regulate inflammatory PGE₂ production. In the first phase (<2 μM), AA was a COX-2 substrate and converted to increasing production of PGE₂. In the second phase with a further increased AA level (2-10 μM), AA bound to mPGES-1 and inhibited the PGE₂ production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH₂ as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40-100% binding to [¹⁴C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.