Molecular cloning, characterization and mRNA expression analysis of a novel selenoprotein: avian selenoprotein W from chicken

In this study, a novel avian selenoprotein W (SelW) gene was cloned from chicken cerebral tissue. The complete nucleotide sequence of the gene contained a 258 bp open reading frame encoding 85 amino acids. Bioinformatics approaches identified the chicken SelW protein is characterized by a β-α-β-β-β-α secondary structure pattern, wherein β1 and β2 are parallel strands forming a classical β1-α1-β2 motif, which is also observed in thioredoxin-like fold proteins. The protein has a candidate CXXU redox motif which is located in the loop (residues 10-13) between β1 and α1. The 3D structural similarity between mouse and chicken SelW protein suggests that the proteins may exhibit similar functions. Additionally, a selenocysteine insertion sequence (SECIS) element was found in the 3'-untranslated region of the SelW mRNA. The SECIS element was classified as form II. Moreover, we analyzed the mRNA expression of SelW genes in 36 different tissues of 60-day-old chickens; the expression of SelW was detected in all tissues, indicating that SelW is expressed widely in chicken tissue. Hence, we suggest that SelW might play an important role in the biochemical functions of Se in birds.     

Dietary selenium regulation of transcript abundance of selenoprotein N and selenoprotein W in chicken muscle tissues

Selenium (Se), selenoprotein N (SelN) and selenoprotein W (SelW) play a crucial role in muscle disorders. Se status highly regulates selenoprotein mRNA levels. However, few attempts have been performed on the effect of dietary Se supplementation on muscle SelN and SelW mRNA levels in birds. To investigate the effects of Se on the regulation of SelN and SelW mRNA levels in muscle tissues, one-day-old male chickens were fed either a commercial diet or a Se-supplemented diet containing 1.0, 2.0, 3.0 or 5.0 mg/kg sodium selenite for 90 days. Muscle tissues (breast, flight, thigh, shank and cardiac muscles) were collected and examined for Se content and mRNA levels of SelN and SelW. Moreover, Selenophosphate synthetase-1 (SPS-1) and selenocysteine-synthase (SecS) mRNA levels were analyzed. Significant increases in SelN mRNA levels were obtained in breast, thigh and shank muscles treated with Se, with maximal effects at 3.0 mg Se/kg diet, but 2.0 mg Se/kg diet resulted in peak levels of Sel N mRNA in flight muscles. Changes in SelW mRNA abundance in thigh and shank muscles increased in response to Se supply. After reaching a maximal level, higher Se supplementation led to a reduction in both SelN and SelW mRNAs. However, SelN and SelW mRNA levels displayed a different expression pattern in different skeletal and cardiac muscles. Thus, it suggested that skeletal and cardiac muscles SelN and SelW mRNA levels were highly regulated by Se supplementation and different muscle tissues showed differential sensitivity. Moreover, Se supplementation also regulated the levels of SPS1 and SecS mRNAs. The mRNA levels of SPS1 and SecS were enhanced in the Se supplemented groups. These data indicate that Se regulates the expression of SelN and SelW gene and affect the mRNA levels of SecS and SPS1.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

Effects of Se-depletion on glutathione peroxidase and selenoprotein W gene expression in the colon

Selenium (Se)-containing proteins have important roles in protecting cells from oxidative damage. This work investigated the effects of Se-depletion on the expression of the genes encoding selenoproteins in colonic mucosa from rats fed diets of different Se content and in human intestinal Caco-2 cells grown in Se-adequate or Se-depleted culture medium. Se-depletion produced statistically significant (P<0.05) falls in glutathione peroxidase (GPX) 1 mRNA (60-83%) and selenoprotein W mRNA (73%) levels, a small but significant fall in GPX4 mRNA (17-25%) but no significant change in GPX2. The data show that SelW expression in the colon is highly sensitive to Se-depletion.     

Gene expression of selenoproteins can be regulated by selenoprotein K silencing in chicken myoblasts

The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72 h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis. The results showed that the designed siRNA effectively inhibited Selk expression (decreased by 20, 29 and 43 % on 24, 48 and 72 h, respectively) and the mRNA expression levels of the 23 selenoproteins were influenced by silencing Selk differently (P < 0.05). Time-dependent pattern of mRNA expression after siRNA treatment in three groups were found similar: one group including Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Sepw1, Selh, Sepp1, Selo and Sepx1, another group including Sepn1, Sels, Selt, Selm and Sep15 and other group including Dio2 and Dio3. The results of correlation analysis showed that Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio3, Sepn1, Sels, Sepw1, Selt, Selh, Sep15, Seli and Selu had a positive correlation with Selk, while Dio2 and Sepp1 had a negative correlation with Selk. PCA data also indicated that Txnrd1, Txnrd2, Dio2, Selpb, Sepp1and Selo may play special roles in response to Selk silencing. In summary, these results indicated that different selenoproteins possess and exhibits distinct responses to silencing of Selk in chicken myoblasts.     

Deletion analysis of the rodent selenoprotein W promoter

To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 404 base pair truncations of genomic DNA lying immediately upstream of the SeW coding sequence were cloned into a luciferase reporter vector (pGL3-Basic from Promega, Madison, WI, USA). 3656 and 406 base pair mouse SeW promoter constructs were also compared. SeW promoter activity was assayed in two rat cell lines: L8 muscle cells and C6 brain cells. The SeW promoter was 2-7 times more active (p<0.01) than SV40 promoter. Promoter activity of constructs of the SeW promoter ranging from 200 base pairs to 51 base pairs gradually decreased to zero in brain cells, but fell precipitously to zero in muscle cells. Some truncations stimulated promoter activity, suggesting the full-length promoter may contain binding sites for factors that suppress SeW expression.     

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels

BackgroundSelenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition.Methods3′ and 5′ RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins.ResultsThe seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish.ConclusionTaken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.     

Selective production of rat mutant selenoprotein W with and without bound glutathione

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.     

Different distributions of selenoprotein W and thioredoxin during postnatal brain development and embryogenesis

Whereas the levels of other selenoproteins in the brain decrease when selenium is deficient, the level of selenoprotein W (Se-W) is maintained, suggesting that it has a critical role in the brain. Previously, we reported that Se-W is a GSH-dependent antioxidant [Jeong et al. (2002)]. In this study, the expression of Se-W and thioredoxin (Trx) in the brain and during embrynic development was analyzed by an in situ hybridization technique. Se-W mRNA was highly expressed in the cortex, dentate gyrus, and hippocampus of postnatal rat brains, and in the spinal cord and brain of developing embryos. In contrast, Trx mRNA was highly expressed in the cerebellum, olfactory bulb, and dentate gyrus of postnatal rat brains, and in the liver, telencephalon, and back muscle of developing embryos. Thus these two antioxidant proteins have different and non-overlapping expression patterns. The distribution of Se-W suggests that it plays an important role as an antioxidant in the developing brain and embryo.     

Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death

It has been reported that selenoprotein W (SelW) mRNA is highly expressed in the developing central nerve system of rats, and its expression is maintained until the early postnatal stage. We here found that SelW protein significantly increased in mouse brains of postnatal day 8 and 20 relative to embryonic day 15. This was accompanied by increased expression of SOD1 and SOD2. When the expression of SelW in primary cultured cells derived from embryonic cerebral cortex was knocked down with small interfering RNAs (siRNAs), SelW siRNA-transfected neuronal cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. TUNEL assays revealed that H₂O₂-induced apoptotic cell death occurred at a higher frequency in the siRNA-transfected cells than in the control cells. Taken together, our findings suggest that SelW plays an important role in protection of neurons from oxidative stress during neuronal development.     

Characterization of selenoprotein P as a selenium supply protein.

Selenium (Se) is well known to be essential for cell culture when using a serum-free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se-supplier, T-lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se-containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se-dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P-depleted serum. Compared with other Se-containing proteins, the addition of purified selenoprotein P to the selenoprotein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se-supply protein, delivering Se to the cells.     

Effects of selenium and serum on selenoprotein W in cultured L8 muscle cells

When rat L8 muscle cells were cultured to examine the effects of serum and selenium concentration on selenoprotein W levels and glutathione peroxidase (GPX) activities, no significant differences (P > 0.05) were found in selenoprotein W levels and GPX activities during differentiation. With three different forms of selenium, selenoprotein W levels and GPX activities were shown to increase in L8 myotubes cultured in media with these selenocompounds. Selenite was utilized more efficiently than selenocysteine for both selenoprotein W and GPX activity, but selenium as selenomethionine was less available. Both the protein content and mRNA levels for selenoprotein W were affected by the selenium content of the media. Northern blot data indicated that the expression of selenoprotein W mRNA increased significantly when L8 myotubes were cultured with selenium (P > 0.05). L8 myotubes cultured in 10% calf serum (CS) versus 2% CS with or without addition of 10 m selenium indicated that the increase of selenoprotein W level in L8 myotubes cultured with higher serum concentration (10% CS) is due to the higher selenium concentration in media rather than serum itself.