Selenoprotein expression, including GPx-3 regulation at the translational level, may be influenced by several factors, including a) mRNA stability, which may be affected by mRNA sequence and RNA binding proteins [39
]; b) expression hierarchy of the selenoproteins [40
]; c) the ability of the SECIS element to reinterpret the stop codon for Sec [41
], allowing for the completion of polypeptide translation; and d) the cell or tissue type, and the cell-dependent availability of translational cofactors or selenium uptake mechanisms.
To study the contributors to GPx-3 expression, we relied on transient and/or stable transfection systems. This allowed us to analyze the contributions of the Sec codon, the 3′-UTR, as well as other factors, such as selenium and known translational cofactors, on GPx-3 expression. We found that stable cell lines demonstrated lower expression of rGPx-3 in the media compared to transiently transfected cells, possibly influenced by the number of GPx-3 cDNA that may have integrated into the cellular genome. Owing to this finding, transient cell lines were used for some of our studies.
Human liver (HepG2 [16
], Hep3B [16
], human intestinal epithelial (Caco-2 [16
] and human kidney (Caki-2 [16
], HEK293 [23
] cell lines have previously been shown to secrete endogenous GPx-3 [16
], and GPx-1 mRNA has been detected in HepG2 cells [42
]. The human intestinal epithelialn Caco-2 cell line was also shown to secrete endogenous GPx-3 [17
]. Our study is the first to use Cos7 cells for production of the selenoprotein GPx-3. We have found that Cos7, Caki-2, and HepG2 cells all can support the expression of mature, secreted GPx-3; however, owing to the very low levels of expression in HepG2 and Caki-2 cells, we primarily used Cos7 in our studies. As shown in other studies, conversion of the Sec codon to a Cys codon resulted in increased (i.e., more efficient translation of) protein synthesis [43
Following transfection, we measured both rGPx-3 mRNA and protein. In this way, we could determine whether rGPx-3 expression was affected by facors influencing transcript levels or by translational mechanisms. The lack of difference in mRNA levels and mRNA stability between the p800 WT and p800 Cys constructs suggests that GPx-3 expression is primarily regulated at the translational level in this system. The differences in protein expression between the p800 WT and p800 Cys mutant could be explained by the differences in the selenol and thiol forms of the protein. Selenoprotein synthesis may be limited by translational cofactors in the p800 WT; synthesis of the Sec73Cys mutant, however, does not require any special translational mechanism.
Adequate selenium is important for the formation of Sec on the tRNAsec
. In selenium starved cells, supplementation with inorganic selenium as well as organified forms of selenium, seleno-L-methionine and Se-methylselenocysteine, appears to enhance rGPx-3 protein expression. Seleno-L-methionine is also known to be incorporated in proteins in place of methionine [49
] and must be metabolized to provide selenium exclusively for Sec incorporation into selenoproteins. Se-methylselenocysteine, in contrast, is not directly incorporated into proteins and, theoretically, is available exclusively for Sec incorporation [50
] or can be rapidly excreted lowering the risk of oxidative toxicity associated with inorganic selenium (selenite) use.
The various responses to selenium treatment, particularly at higher concentrations of the selenocompounds, suggest that Cos7 cells may respond differently to different concentrations and different forms. High molar concentrations of sodium selenite may be cytotoxic, as selenite is a potent oxidant at these concentrations. Selenium deficiency is also cytotoxic in culture [51
]. Zhong and colleagues demonstrated that excess sodium selenite induced apoptosis and cell cycle arrest after chronic exposure in human prostate cancer cells [52
]. The same group demonstrated apoptosis by selenomethionine in the same cell lines [53
]. In our cell system, the concentrations of selenium ranged from antioxidant levels to those found to be toxic or pro-oxidant. Each of the selenocompounds used had a unique effect on rGPx-3 production in this system. These findings may relate to differences in cellular uptake or metabolism of each compound.
The effects of selenium on mRNA and protein expression may be different in cultured cells than in tissues. Several studies have shown GPx-1 activity and transcript levels are sensitive to selenium deprivation [54
]; however, the decrease in GPx-1 activity did not always coincide with alterations in transcript levels [16
]. Another group found that selenium deficiency reduces serum GPx activity in rats, while kidney GPx-3 mRNA levels remain constant [56
In contrast, Reszka and colleagues showed that changes in GPx-3 mRNA levels in mouse fibrosarcoma cells were affected by culturing cells in low selenite conditions (levels as low as 1–5 ng Se/ml medium) [57
]. These data suggest a low selenium range for optimal selenium expression for these particular cells. Based on our data, it is possible that Cos7 cells regulate selenium availability over a narrow range (levels such as 10–50 ng Se/ml) that affects translation but not mRNA stability. In the studies presented here, we also observed a dose-dependent increase of rGPx-3 expression in Cos7 cells after seleno-L-methionine and Se-methylselenocysteine treatment, suggesting that these compounds are also bioavailable for uptake into Cos7 cells. We are currently exploring the hierarchy of effects of selenium supplementation on GPx-3 and other selenoproteins in cell culture and in vivo
Evidence on the regulation of selenoprotein synthesis is preliminary, and little is known about the efficiency of selenocysteine incorporation [25
]. Additional translational cofactors have recently been shown to be important regulators of selenoprotein synthesis in eukaryotes, such as ribosomal protein L30 and nuclease sensitive binding protein 1 [58
]. Previous experiments in our laboratory showed that GPx-3 expression is affected by SBP2, EFsec, and tRNAsec
]. Data presented here with the Sec translational cofactors demonstrate that SBP2 is important for optimal rGPx-3 expression in Cos7 cells. In contrast, Copeland and colleagues also demonstrated the importance of SBP2 in transfected rat hepatoma cells in vitro
], but no further enhancement was seen in rat hepatoma cells using a luciferase reporter system [60
]. In other studies, tRNAsec
was shown to affect Sec incorporation greatly [61
]. The importance of SBP2 in GPx-3 expression was also shown in studies of human subjects who carry a mutation in SBP2. These individuals had decreased serum GPx-3 activity (13% of normal) and decreased serum selenoprotein P levels [62
], suggesting that SBP2 plays a direct role on GPx-3 and other selenoprotein expression in vivo
. Interestingly, a recent study by Squires and colleagues provided further support for a hierarchy of selenoprotein synthesis. They demonstrated that SBP2 showed preferential binding to certain selenoprotein mRNAs, and may play a key role in preventing nonsense-mediated decay [63
]. The levels of GPx-3 mRNA were too low to be reliably quantified; however, GPx-3 was predicted to be susceptible to nonsense-mediated decay owing to the position of an intron near the UGA (Sec) codon [63
Our laboratory has also previously shown a trend toward increased expression of GPx-3 using SelD and tRNAsec
] in human Caki-2 cells; however, these cofactors did not augment rGPx-3 protein levels when compared to SBP2 co-transfection alone in Cos7 cells. Several factors may influence these differences, including endogenous levels of expression of these factors or other differential features of these model systems.
Regulation of Sec incorporation at a UGA also requires the presence of the SECIS element in the 3′UTR of the transcript. Several studies have demonstrated that a 3′UTR from a selenocysteine gene is sufficient to promote selenocysteine incorporation in a heterologous protein containing a UGA in the open reading frame [64
]. These and other studies have defined sequence and structural characteristics of the stem-loop structure that are required for a functional SECIS element [66
]. For the rat type I deoidinase [67
], rat thioredoxin reductase [68
], and phospholipid hydroperoxide glutathione peroxidase [69
], minimal functional SECIS elements of 45, 59, and 82 nt have been defined. We found, however, that a 100 nt piece overlapping the entire stem-loop-SECIS element of the human GPx-3 was insufficient for UGA-read-through in our cell system.
The formation of the tetrameric and monomeric rGPx-3 forms under non-reduced conditions suggests a potential limitation with the synthesis, processing, and formation of the tetramer that may affect its net enzymatic activity. GPx-3 is thought to exist primarily as a tetramer in plasma. It is possible that dimeric and monomeric forms may contribute to GPx-3 activity; however, the tetramer is considered the active form of the enzyme [36
]. In purifying rGPx-3 from media, we found that a greater proportion was monomeric rather than tetrameric compared with the purified plasma form. It is not clear whether this difference is due to the expression system (V5/His-tag, the low levels of GPx-3 produced) or the purification process. Alternatively, the cell type may not have provided all the necessary factors for optimal translational conditions and tetramer assembly. Another possibility is that in vivo
plasma kinetics may differ for the monomeric and tetrameric forms such that the monomeric form is preferentially removed from the circulation. While a barely detectable monomeric form still existed under non-reducing conditions in partially purified GPx-3 from plasma, rGPx-3 in our system was principally monomeric. A putative GPx-3 crystal structure suggest that two asymmetric units form a tetramer [36
]; GPx-4 is the only known GPx family member that functions as monomer. It would be interesting to understand further if and how quaternary structure influences activity; data presented here, however, suggest that specific activity is not influenced significantly by quaternary structure, as specific activity of the rGPx-3 was similar to that of partially purified human plasma GPx-3.
Spectrophotometric and Amplex Red rGPx-3 activity measurements show differences in catalytic activities between a Sec-containing and Cys-containing GPx-3 active site likely as a consequence of the Sec representing the more catalytically active form based on differences in its pKa 5.2 for a selenol and compared with the pKa of a thiol (8.5) [70
]. Based on the Amplex Red assay, partially purified plasma GPx-3 reduced t
-BuOOH with only 2 mM GSH and not at physiological extracellular GSH concentrations. One study has shown purified GPx-3 may utilize other substrates such as TR, Trx, and Grx [38
] in the physiological range. In our hands, neither TR or TRx or Grx had any effect as reducing substrates.
We determined the kcat
of a purified GPx-3 to be approximately 1.8 × 108
using GSH as a substrate, suggesting a catalytically efficient enzyme. Previous kinetic assays suggest plasma GPx-3 has a 10-fold weaker (i.e., lower) affinity for the substrate (Km
of 5.3) [37
] than for GSH than cellular GPx-1 due to two less GSH binding sites [36
], with a specific activity of approximately 20–26 U/mg of protein [32
], suggesting extracellular GSH concentrations cannot provide the necessary reducing equivalents for plasma GPx-3 activity. Theoretically, GPx-3’s secretion into plasma and other extracellular fluids suggests it may function as an antioxidant in the extracellular environment. Thus far, GPx-3 has only been demonstrated to act as a peroxidase in lung lining fluid where the concentration of GSH is significantly higher than in plasma [71
]; however, the concentration of GPx-3 is higher in plasma than in epithelial lining fluid [72
It has been suggested that GPx-3 may not function as a glutathione peroxidase, but as a TRx peroxidase since it reacted with a higher affinity using reducing equivalents from the thioredoxin system [38
] in in vitro
assays. The crystal structure of GPx-3 shows a thioredoxin fold [36
] similar to other enzymes like Trx and Grx [73
] and, yet, GPx-3 may be less reactive toward H2
than GPx-1 owing to the fact that GPx-3 has two less GSH binding sites than GPx-1 [36
]. Trx has been suggested to be a more effective electron donor for SelP [74
]. Since TR, Trx, and Grx are found in the extracellular compartment and protein disulfide isomerase, TR, and Trx are also found on the cell surface [75
], it may be possible that the major extracellular selenoproteins, GPx-3 and SelP, work with these or other unknown reducing equivalents found on or near the cell membrane that may have effluxed from the intracellular compartment [79
] to produce effective peroxidase activity in that microenvironment. Future studies are needed to address this issue.