Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus: inhibition by nonsteroidal anti-inflammatory drugs and biosynthesis of 15-hydroxyeicosatetraenoic acid.

摘要: Human prostaglandin G/H synthase (hPGHS)-1 and hPGHS-2, key enzymes in the formation of prostanoids from arachidonic acid, were expressed at high levels COS-7 cells using a T7 RNA polymerase/vaccinia virus expression system. The open reading frame hPGHS-2 cloned into vaccinia without its natural 5' 3' untranslated regions directed only low enzyme activity cells. High-level was achieved by appending region hPGHS-1 to frame, with subsequent hybrid mRNA virus. Enzymatically active recombinant present as glycosylated proteins microsomal fraction prepared infected cells, whereas treated tunicamycin, an inhibitor N-linked glycosylation, enzymatically inactive. major prostanoid products formed microsomes containing either or after incubation acid D2 E2, lower F2 alpha 6-keto-prostaglandin F1 alpha. A range potencies observed for various nonsteroidal anti-inflammatory drugs inhibitors E2 synthesis hPGHS-2. Recombinant both produced 15- 11-hydroxyeicosatetraenoic (HETE) 15-HETE production being stimulated 5-fold preincubation aspirin. Chiral phase performance liquid chromatography analysis showed that aspirin-treated 15(R)-HETE, no detectable 15(S)-HETE.