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Results for “Selenoprotein W”

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At least 19 recordsLinked to original sources

PubMed1999

Purification, characterization, and glutathione binding to selenoprotein W from monkey muscle.

Selenoprotein W was purified from monkey skeletal muscle to investigate its binding of glutathione. The purification was accomplished by concentration of the cytosol with an Amicon cell, gel filtration using Sephadex G-50, cation-exchange chromatography with CM-Sephadex, and reverse-phase high-pressure liquid chromatography using a C-18 Vydac column. Selenoprotein W was monitored during purification by slot blots. These steps resulted in an electrophoretically pure selenoprotein W preparation that was estimated by gel filtration to have molecular weight of about 10 kDa. N-terminal amino acid sequencing was used to confirm that the pure proteins were selenoprotein W. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) revealed that the proteins existed in three masses of 9635 +/- 7, 9371 +/- 11, and 9330 +/- 5 Da. The theoretical mass of the protein predicted from the cDNA sequence is 9330 Da. The 9635-Da form of the protein was shown to contain bound glutathione (306 Da), which could be released by reduction with dithiothreitol at 50 degreesC. The form with a mass of 9371 Da is assumed to result from binding of an unidentified 41-Da moiety to the 9330-Da form of the protein. MALDI peptide mapping with endoproteinase Glu-C suggested that glutathione is bound to the 36th amino acid (cysteine) of selenoprotein W.

Animals↗

PubMed1997

Selenium influences tissue levels of selenoprotein W in sheep.

Because selenium increases the levels of other selenoproteins, the influence of this element on selenoprotein W was examined in wether sheep fed either a low selenium diet (0.02 mg/kg) or the same diet supplemented with 3 mg selenium as selenite per kilogram diet. Muscle biopsies were taken initially and at 3.5, 7.0 and 10.5 wk. The sheep were killed after the last muscle biopsy and samples from nine tissues were taken. Selenoprotein W was determined in tissues by Western blots with a polyclonal antibody against a synthetic peptide based on the protein sequence of the homologous rat selenoprotein W. In supplemented sheep, muscle selenoprotein W was significantly increased over initial levels (P < 0.05) at 7 wk and afterwards, whereas in sheep consuming the low selenium diet, muscle selenoprotein W levels declined significantly (P < 0.05) after 10.5 wk. This selenoprotein was found in various amounts in all tissues examined. The highest levels of selenoprotein W were found in skeletal muscles and heart and the lowest was found in liver. Except for selenoprotein W in brain, the concentrations of selenoprotein W, selenium and glutathione peroxidase activity were significantly higher (P < 0.05) in all tissues from supplemented sheep than in those from unsupplemented sheep. The selenoprotein W levels in brains of the two groups were not significantly different. Thus, selenoprotein W levels in all tissues of sheep except the brain are sensitive to selenium status.

Animals↗

PubMed2002

Selenoprotein W gene regulation by selenium in L8 cells.

The effects of selenium on selenoprotein W gene expression were examined in cultured L8 rat skeletal muscle cells. Selenoprotein W contains selenium as selenocysteine in the primary protein structure and levels of this selenoprotein are affected by selenium. Northern blots indicated that there were no significant changes (P < 0.05) in selenoprotein W mRNA levels during cell proliferation and differentiation. Reduction of selenium concentration in the medium decreased the selenoprotein W mRNA levels. Nuclear run-on experiments with isolated L8 nuclei showed the same rate of selenoprotein W mRNA synthesis in cells cultured in either low selenium or selenium supplemented medium, suggesting that the transcription rate of the selenoprotein W gene is independent of selenium. Measurement of the selenoprotein W mRNA half-life in myoblasts treated with the transcription inhibitor, alpha-amanitin, showed that selenoprotein W mRNA levels decreased over time with an estimated half-life of 57 h for cells grown in low selenium medium. Selenium treatment increased the selenoprotein W mRNA half-life 2-fold. These data suggest that selenium stabilizes selenoprotein W mRNA but has no effect on transcription.

Animals↗

PubMed2000

Selenoprotein W accumulates primarily in primate skeletal muscle, heart, brain and tongue.

The human selenoprotein W coding region with the selenocysteine codon (TGA) changed to a cysteine codon (TGT) was fused to six histidine codons (at its 3' end), cloned into a prokaryotic expression vector (pTrc99a), and the corresponding mutated selenoprotein W was expressed in bacteria. The protein was purified by Ni-NTA agarose column and reverse phase HPLC. Polyclonal antibodies raised against this protein were used in Western blots to determine tissue distribution of selenoprotein W from rhesus monkeys fed a commercial chow. Selenoprotein W was found in several tissues with highest amounts in skeletal muscle and heart (muscle 6 fold greater than liver) and lowest levels in liver, but selenium concentrations were highest in kidneys (10 fold greater than muscle) and lowest in skeletal muscle. Northern blots using a human selenoprotein W cDNA probe indicated that mRNA levels were highest in monkey skeletal muscle and heart (2-2.5 fold greater than in liver), which is similar to the pattern found with a human multiple tissue Northern blot. However, as in the monkey, selenium concentrations were highest in human kidney and lowest in skeletal muscle and heart. Thus, selenoprotein W protein levels correlated with selenoprotein W mRNA levels but not with tissue selenium concentrations.

Animals↗

PubMed1995

Tissue distribution and influence of selenium status on levels of selenoprotein W.

Rabbits were immunized with two synthetic peptides based on hydrophilic regions of selenoprotein W from rat muscle. The resulting polyclonal antibodies were used in Western blots to determine the compartmentation and tissue distribution of selenoprotein W, and to determine the influence of selenium on the levels of this selenoprotein in rat muscle. Selenoprotein W exists mainly in cytosol, but very small amounts were associated with membranes. Western blots revealed selenoprotein W in muscle, spleen, testis, and brain of rats. Rats were fed diets of either no addition of selenium (0 ppm Se) or additions of 0.1 and 4.0 mg selenium/g (0.1 ppm Se and 4.0 ppm Se) diet for 6 wk. Selenoprotein W was undetectable in skeletal muscle of rats fed the basal diet, detectable in those fed 0.1 ppm selenium in the diet, and much higher in muscle from rats fed 4 ppm selenium diet. In a species comparison, Western blots indicated the presence of selenoprotein W in muscle of rabbits, sheep, and cattle.

Amino Acid Sequence↗

PubMed2000

Selenoprotein W: a review.

Purification of selenoprotein W (Se-W) from rat and monkey muscles was shown to exist in multiple forms: with or without reduced glutathione and/or a 41-Da moiety (identity still unknown). TGA is located at coding position 13 in Se-W complementary DNA (cDNA) from all five species studied (rats, mice, sheep, human and monkey). TGA is also the stop codon in the rodents and sheep cDNA, but TAA is the stop codon in primates. There is an 80% homology of the nucleotide sequence in the coding region among the five species of animals, and the predicted amino acid sequences are 83% identical (rodents identical and primates identical). Se-W levels are highest in muscle, heart and brain from sheep and primates, but very low in rodent hearts. Studies with tissue cultures of muscle and brain cells indicated that selenium influenced Se-W levels. Although the metabolic function of Se-W is unknown, preliminary data suggest that it has an antioxidant function.

Amino Acid Sequence↗

PubMed1997

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(-8) 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.

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PubMed1995

Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium.

Rat skeletal muscle selenoprotein W cDNA was isolated and sequenced. The isolation strategy involved design of degenerate PCR primers from reverse translation of a partial peptide sequence. A reverse transcription-coupled PCR product from rat muscle mRNA was used to screen a muscle cDNA library prepared from selenium-supplemented rats. The cDNA sequence confirmed the known protein primary sequence, including a selenocysteine residue encoded by TGA, and identified residues needed to complete the protein sequence. RNA folding algorithms predict a stem-loop structure in the 3' untranslated region of the selenoprotein W mRNA that resembles selenocysteine insertion sequence (SE-CIS) elements identified in other selenocysteine coding cDNAs. Dietary regulation of selenoprotein W mRNA was examined in rat muscle. Dietary selenium at 0.1 ppm as selenite increased muscle mRNA 4-fold relative to a selenium-deficient diet. Higher dietary selenium produced no further increase in mRNA levels.

Amino Acid Sequence↗

PubMed2006

Selenoprotein W during development and oxidative stress.

Selenium is involved in prevention of cancer, heart and muscle diseases, is implicated in immune function, fertility and in delaying the aging process. Selenium deficiency is harmful to brain, heart and skeletal muscles. Selenoprotein W, a member of the selenoprotein family was expressed in developing nervous system, skeletal muscles and heart in mice. Selenoprotein W was highly expressed in proliferating myoblasts and less or not in differentiated myotubes. Selenoprotein W exhibited an immediate response to oxidative stress in proliferating myoblasts, after exposure to hydrogen peroxide, similar to gluteraldehyde-3-phosphate dehydrogenase. We suggest that Selenoprotein W is involved in muscle growth and differentiation by protecting the developing myoblasts from oxidative stress.

Animals↗

PubMed2002

Effect of copper, zinc and cadmium on the promoter of selenoprotein W in glial and myoblast cells.

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404 and 1265 bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured C6 (rat glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265 bp (not 404 bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter.

Animals↗

PubMed2004

Identification of putative transcription factor binding sites in rodent selenoprotein W promoter.

To understand transcriptional regulation of the selenoprotein W (SeW) gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter and antibodies, we observed that specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as to the metal response element (MRE). Although competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore, the SeW gene may be a target for Sp1 whose binding to various regulatory sequences of the SeW promoter may activate or repress the transcription of SeW. The MRE, GRE, AP-1 and LF-A1 sites were also tested but no evidence was obtained for specific binding as indicated by lack of competition with unlabeled probes.

Animals↗

PubMed1997

Conserved features of selenocysteine insertion sequence (SECIS) elements in selenoprotein W cDNAs from five species.

SECIS elements form stem-loop structures in the 3' untranslated regions (UTR) of eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation of selenocysteine at UGA codons, provided the SECIS element lies a sufficient distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS elements that retained important features common to all known SECIS elements. Comparative analysis of these SECIS elements showed that in some regions both predicted secondary structure and nucleotide sequences were conserved, in other areas secondary structure was maintained using different primary sequence, and in still other portions, base pairing was not conserved. The rodent and sheep selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon. Thus, UGA specified both selenocysteine incorporation and termination in a single mRNA. The selenoprotein W SECIS elements contained an additional highly conserved base-paired stem that may prevent inappropriate selenocysteine incorporation at the UGA stop codons.

Amino Acid Sequence↗

PubMed1997

Selenoprotein W cDNAs from five species of animals.

The nucleotide sequences of the open reading frames of cDNAs for selenoprotein W from skeletal muscle of rat, mouse, sheep, rhesus monkey and human are reported. Theoretical translation of the coding sequences indicated highly similar proteins of 88 (mouse and rat) or 87 (human, monkey and sheep) amino acids. In 73 of 88 positions the specified amino acids are identical for all five proteins. TGA encoding selenocysteine is the 13th codon of all the cDNAs. The mouse, rat and sheep open reading frames terminate with TGA but the human and rhesus monkey coding regions terminate with TAA. The encoded amino acid sequences are identical for the rat and mouse proteins, and for the human and monkey proteins. The similarity of the cDNAs continues in the 3' noncoding regions through the putative selenocysteine insertion sequence (SECIS) elements which are required for correct interpretation of the selenocysteine codon. The region between the SECIS elements and the polyadenylation signals showed much lower similarity. The cloned rat gene for selenoprotein W is 5000 bases long, with the 663 bases of the cDNA in six exons. The transcription start site was identified by nuclease protection assay to be 16 bases upstream of the longest cDNA clone. A canonical TATA box occurs 150 bases upstream, but the assay did not indicate the presence of longer mRNAs.

Amino Acid Sequence↗

PubMed1996

Selenoprotein W of rat muscle binds glutathione and an unknown small molecular weight moiety.

When purified from rat muscle, selenoprotein W is fractionated into four forms distinguished by slightly different chromatographic behavior. Precise masses of the four forms were determined by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. The mass distribution of the forms (9549, 9592, 9853, and 9898 d) suggests that they occur through derivatization of the lowest mass form with two moieties of approximate masses 44 and 305 d. The apparent 305 d moiety was demonstrated to be glutathione (307 d) by reductive release from the 9853 d protein form with 1000-fold excess of dithiothreitol at 50 degrees C. Milder conditions failed to remove the glutathione. The reduction produced nearly stoichiometric amounts of free glutathione as determined by HPLC of a fluorometric derivative. HPLC retention of the protein changed to match that of the 9549 d form, and a change of mass to 9550 d was observed by MALDI mass spectrometry. The identity of the 44 d moiety is unknown. The presence of glutathione in isolated selenoprotein W may suggest its involvement in the metabolism of this tripeptide.

Animals↗

PubMed2002

Selenoprotein W is a glutathione-dependent antioxidant in vivo.

The function of selenoprotein W (Se-W) was investigated by cloning the corresponding cDNA from mouse brain and expressing it in CHO cells and H1299 human lung cancer cells. Overexpression of Se-W markedly reduced the sensitivity of both cell lines to H2O2 cytotoxicity. The intracellular peroxide concentration of the transfected cells was lower than that of the parental cells in the absence or presence of extracellular H2O2. The resistance to oxidative stress conferred by Se-W was dependent on glutathione. Expression of Se-W mutants in which selenocysteine-13 or cysteine-37 was replaced by serine did not confer resistance to H2O2, implicating these residues in the antioxidant activity of Se-W in vivo.

Animals↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2003

Gene structure and tissue expression of human selenoprotein W, SEPW1, and identification of a retroprocessed pseudogene, SEPW1P.

We have determined that the human SEPW1 (selenoprotein W) gene maps to chromosome 19q13.3, spans approximately 6.3 kb and comprises six exons, in contrast to the previously published five exons. The gene lacks canonical TATA and CAAT boxes, but has numerous Sp1 consensus binding sites upstream of multiple transcription start sites. SEPW1 is expressed in all of the 22 tissues assayed, and shows highest expression in skeletal muscle and heart. Additionally, we have also identified a retroprocessed SEPW1 pseudogene, SEPW1P, which maps to chromosome 1p34-35.

Amino Acid Sequence↗

PubMed2001

Selenoprotein W in overexpressed and underexpressed rat glial cells in culture.

Selenium deficiency results in undetectable levels of selenoprotein W (SeW) in muscle but has very little effect upon its content in the brain and thus rat glial cells were studied. Previous work showed that glutathione (GSH) is bound to SeW and this study was undertaken to elucidate its possible antioxidant functions. Full length cDNA of SeW was cloned to inducible LacSwitch expression vector and stably transfected in C6 rat glial cells. After induction, SeW and its mRNA were expressed 22- and 11-fold higher respectively than control. The cDNA coding region of SeW was cloned to the vector in the antisense direction and stably transfected in C6 cells for underexpression of the protein. After induction, SeW expression was reduced to 20% of the control cells. Glutathione peroxidase activity and GSH levels were not significantly different between induced and control cells. There was a greater survival rate of overexpressed than control cells when incubated with 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH), suggesting SeW possibly has an antioxidant function.

Animals↗