| Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. A deficiency in GPx-3 is associated with an increased risk of stroke and cardiovascular disease. Expression of GPx-3 and other selenoproteins involves the recognition of a UGA codon as a site for selenocysteine incorporation and requires specific signals in the 3' untranslated region (UTR), including a selenocysteine insertion sequence (SECIS) element. Owing to its low expression and intrinsic enzymatic activity, we sought to ascertain regulatory determinants involved in GPx-3 expression. Recombinant GPx-3 (rGPX-3) constructs with various lengths of the 3' UTR along with a Sec73Cys mutant were generated. We also examined the activity of GPx-3 using a redox-sensitive fluorescence based enzymatic endpoint assay. Our results demonstrate that the wild type and Sec73Cys mutant are detectable in the cell media of transiently transfected Cos7 cells, as well as in the media of stable transfectants; however, the Sec73Cys mutant expressed at higher levels than the wild type rGPx-3. We also found that a minimal 3'UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression, whereas other forms of the 3'UTR did not significantly affect rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 (SBP2), a translational cofactor, were also found to increase wild type rGPx-3 expression. Electrophoretic analysis of native plasma GPx-3 showed that it exists primarily as a tetramer, whereas the majority of rGPx-3 is monomeric. The specific enzymatic activity of the wild type Sec-containing rGPx-3 was significantly higher than the Sec73Cys rGPx-3. These results demonstrate a role for the 3' UTR, adequate selenium stores, and translational cofactors in regulating protein translation, quaternary assembly, and activity of rGPx-3. |
