Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells

Abstract

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3'-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3'-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3'-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.

Fig. 1. Summary of Recombinant GPx-3 Constructs

Recombinant GPx-3 contains a human GPx-3 cDNA driven by a CMV promoter through a V5 epitope and His-tag. The constructs include: a) rGPx-3 with full length 3′-UTR; b) a Cys mutant (Sec73Cys) with a full length 3′-UTR; c) rGPx-3 with a 500 bp 3′-UTR; d) rGPx-3 with a 100 bp SECIS-only 3′-UTR fragment; e) rLacZ control; and f) an empty vector control.

Fig. 2. rGPx-3 mRNA Expression in Transient and Stable Cos7 cells

Reverse transcriptase-PCR detecting rGPx-3 in transiently and stably transfected cell lines.

Fig. 3. Effect of 3′ UTR on rGPx-3 Protein Expression

a) SDS-PAGE and immunoblot of purified rGPx-3 constructs from the media and lysates of transiently transfected Cos7 cells detected by anti-V5 antibody. rLac Z was used as a His-Spin purification control, and actin was used as a loading control. The Western blot is representative of three separate experiments. b) ELISA was used to quantitate protein expression directly for each non-purified secreted rGPx-3 form (p<0.03). Each bar represents the average of three experiments performed in duplicate, and values are given as mean +/− SD.

Fig. 4. Effect of Selenium and Translational Cofactor Treatment on p800 WT rGPx-3 Expression

a) Sodium selenite, seleno-L-methionine, or Se-Methylselenocysteine hydrochloride was added to transiently transfected p800 WT for 48 hr and media examined by His-tag ELISA (p<0.05). Each bar represents the average of three experiments performed in duplicate, and values are given as mean +/− SD. b-e) Addition of 2.5 μg of SBP2, SelD, and tRNA^sec to transiently transfected p800 WT Cos7 cells, after which the media and lysate were examined (after 48 hours) by Western blotting using an anti-V5 antibody; (b, d) and by His-tag ELISA (c, e) with one (b, c) cofactor co-transfection (p<0.05), or two (d, e) cofactor co-transfection (p<0.04). Each bar represents the average of five experiments performed in duplicate, and values are given as mean +/− SD.

Fig. 5. rGPx-3 and Plasma GPx-3 Forms

Western blot of His-Gravi Trap purified rGPx-3 in Cos7 media were run on a SDS-PAGE gel under reducing and non-reducing conditions and compared to partially purified GPx-3 from human plasma. a) rGPx-3 was detected using an anti-V5 antibody; b) GPx-3 from human plasma was detected by using a monoclonal GPx-3 antibody; and c) densitometric ratio of tetrameric-to-monomeric forms of rGPx-3 and partially purified GPx-3 from plasma. The Western blot is representative of three separate experiments. Each bar represents the average densitometry of three experiments, with values given as mean +/− SD.

Fig. 6. Plasma GPx-3 Forms and Effect on GPx-3 Activity

a) rGPx-3 Activity of His-Gravi Trap Purified Cos7 Cell Media. p800 WT rGPx-3 was His-Gravi Trap purified from transiently transfected Cos7 cell media and activity was determined spectrophotometrically and compared to the activity of rGPx-3 Cys. The p800 WT bar represents the average of N=17 experiments and the p800 Cys represents the average of N=8 (p<0.05), with values are given as mean +/− SD. b) In-gel Activity of rGPx-3. rGPx -3 activity was compared to GPx-1 from bovine erythrocytes using GSH and t-BuOOH as substrates. Partially purified GPx-3 from plasma was used as a control. The first panel represents the in-gel assay of the tetrameric of the standard GPx-1 compared to rGPx-3 WT, rGPx-3 Cys, and empty vector. The second panel represents the inverse image of the in-gel assay. The in-gel assay represents three independent experiments. c) In-gel Assay of Partially Purified GPx-3. A 5-fold dilution of the sample was compared to demonstrate a limit of detection with the in-gel assay. The first panel represents the in-gel assay of standard GPx-1 and GPx-3. The second panel represents the inverse image of the in-gel assay. d) rGPx-3 Activity by the Amplex Red Assay. His-Gravi Trap purified and concentrated rGPx-3 WT and rGPx-3 Cys activity using GSH and t-BuOOH (p<0.005). Each bar represents the average of three experiments, with values are given as mean +/− SD. e)Partially purified plasma GPx-3 activity using 2 μM, 20 μM, 200 μM and 2000 μM GSH (p<0.02). Each bar represents the average of three experiments, with values are given as mean +/− SD.