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Selenoprotein P can be traced to reports in the early 1970s that demonstrated 75Se incorporation into a plasma protein in the rat (Millar, 1972; Burk, 1973). Later Herrman (1977) showed that this plasma protein was distinct from glutathione peroxidase. It was referred to as selenoprotein P by Tappel’s group because of its plasma location (Motsenbocker and Tappel, 1982) and as 75Se-P by our group because of its incorporation of 75Se (Burk and Gregory, 1982). All workers have now adopted the name of selenoprotein P because it has been shown to be a selenoprotein and it is present in plasma. A further name change will be necessary when its function is established.

Rat liver selenoprotein P contains 10 selenocysteine residues in its primary structure (deduced). It is the only selenoprotein characterized to date that has more than one selenocysteine residue. Selenoprotein P cDNA has been cloned from human liver and heart cDNA libraries and sequenced. The open reading frames are identical and contain a signal peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of the open reading frame of the human cDNA with the rat cDNA reveals a 69% identity of the nucleotide sequence and 72% identity of the deduced amino acid sequence. Two regions in the 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable stem-loop structure similar to the single stem-loop structures reported in 3' untranslated regions of type I iodothyronine 5'-deiodinase and glutathione peroxidase. The stem-loop structure of type I iodothyronine 5'-deiodinase has been shown to be necessary for incorporation of the selenocysteine residue at the UGA codon. Because only two stem-loop structures are present in the 3' untranslated region of selenoprotein P mRNA, it can be concluded that a separate stem-loop structure is not required for each selenocysteine residue.

Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.

The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.

Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport.

Radioreceptor assay technology was used to show the presence in the rat of a receptor that binds selenoprotein P, a selenocysteine-containing rat plasma protein. 75Se-labeled selenoprotein P bound to testis, kidney, and liver membranes. The binding was specific in that increasing amounts of partially-fractionated rat plasma specifically displaced the binding of 75Se-labeled selenoprotein P to testis membrane in a competitive manner. 75Se-labeled selenoprotein P binding was saturable in the presence of increasing amounts of testis membranes. The binding of 75Se-labeled selenoprotein P was optimal at about pH 4.2. Several proteins and blood fractions had little or no significant effect on binding of 75Se-labeled selenoprotein P to testis membranes. All plasma sources tested specifically displaced 75Se-labeled selenoprotein P from testis membrane, indicating that selenoprotein P-related proteins may be widespread in nature. The study indicated that selenoprotein P has a receptor and is involved in selenium transport.

The micronutrient selenium is associated with several proteins which have different biological roles. In particular glutathione peroxidase (GSH-Px) and phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) are involved in the cell antioxidant system [I]. These selenoproteins contain the element selenium as Se-cysteine (Se-cys) which is incorporated into proteins by recognition of the stop-codon UGA as a codon for Secys [2]. When the selenium supply is limiting, it is known that the activity and concentration of these selenoproteins is reduced [3] and that the control of protein synthesis is accompanied by alteration in mRNA abundances [4,5]. In addition reduced availability of selenium leads to termination of translation due to the recognition of the UGA codon as a normal stop codon [6]. Previous studies on selenoprotein gene expression in rat liver have shown that the levels of selenoprotein mRNAs do not necessarily follow the changes in enzyme activity [7]. This suggests there is a differential regulation of the different mRNAs involving a possible control at a transcriptional or posttranscriptional level. Nuclear run-off assays on isolated liver nuclei showed Se-deficiency to have no affect on transcription of the two selenoproteins, indicating the importance of a post-transcriptional control which may involve regulation of mRNA stability [7]. The aim of the present work was to develop a cell culture model system which would allow the study of the effect of selenium supply on mRNA stability, particularly the regulation of GSH-Px and PHGSH-Px gene expression. Rat hepatoma H4 cells (H4 I1 EC3) were grown in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum until they reached confluence. They were then maintained in serum free medium to which was added either insulin ( 5 pg/ml) and transferrin ( 5 pg/ml) (selenium-deficient cells) or insulin, transfernin and selenium as sodium selenite (7 ng/ml) (selenium-replete cells). GSH-Px and PHGSH-Px enzyme activities were measured by standard methods [8,9] on cells washed and suspended in phosphate buffered saline (pH 7.4). Cells were incubated with 0.1% peroxide-free Triton X-100, 0.3 mM NADPH, 5 mM reduced glutathione and 0.7 U/ml glutathione reductase. The oxidation of NADPH upon addition of H202 for GSH-Px and phospholipid hydroperoxide (PC-OOH) for PHGSHPx to the sample cuvette was followed spectrophotometrically at 340 nm. During culture in selenium-deficient medium there was a 80 % reduction in GSH-Px activity after 2-4 days and this reached 92 % of that found in H4 selenium-replete cells after 8 days. When the selenium-deficient H4 cells were subsequently placed in medium containing 7 ng/ml sodium selenite an increase in GSH-Px activity occured, reaching the original GSH-Px activity at 8 days (Fig. 1). The mean activity of GSH-Px in selenium-replete cells was 49.8 mu/ mg protein. Similar results were obtained for PHGSH-Px although, after 8 days, PHGSH-Px activity was reduced by only 50 % compared to that in selenium-replete cells. The greater effect of selenium deficiency on GSH-Px activity compared to PHGSH-Px activity is comparable to that found in liver from animals fed either a Se-deficient or adequate diet for six weeks [7]. The present results demonstrate that the rat hepatoma H4 cell line responds to selenium depletion and repletion with decreased and then restored levels of GSH-Px and PHGSH-Px enzymatic activity. As in the rat [7], in selenium-deficient H4 cells the enzyme changes were paralled by a reduction in GSH-Px mRNA level but not that of PHGSH-Px mRNA. The changes in both enzyme and mRNA levels were similar to those observed in rat liver during selenium-deficiency, suggesting that the H4 cells provide a good model system to study the regulation of GSH-Px and PHGSH-Px gene expression. GSH-Px activity during seleniumdepletion and selenium-repletion

Selenoproteins constitute a family of enzymes involved in the management and regulation of reactive oxygen species, signaling molecules that are also affiliated with molecular damage and disease. The family contains two intrinsically disordered membrane proteins: selenoprotein S (SelS) and selenoprotein K (SelK). Both of these bitopic membrane proteins reside in the endoplasmic reticulum and contain the highly reactive amino acid selenocysteine, The precise function of SelK and SelS is presently still unknown, however it has been shown that in vivo they participate in anti-oxidant defense. In addition they are members of the Endoplasmic Reticulum Associated Protein Degradation (ERAD) pathway, which is responsible for dislocation of misfolded proteins from the ER for degradation in the cytoplasm. We have shown that SelK and SelS belong to the very small group of intrinsically disordered proteins that exhibit enzymatic functions. We demonstrate that SelS is an efficient reductase capable of reducing disulfide bonds, while SelK has a weak but nevertheless relevant lipid peroxidase activity. The latter is particular noteworthy because lipid hydroperoxides are accessible to only a limited set of peroxidases, but play a critical role in membrane health.

Selenoprotein synthesis requires the co-translational recoding of a UGA Sec codon. This process involves an RNA structural element, called Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2). Several selenopro-tein mRNAs undergo unusual cap hypermethylation by the trimethylguanosine synthase 1 (Tgs1), which is recruited by the ubiquitous Survival of MotoNeu-rons (SMN) protein. SMN, the protein involved in spinal muscular atrophy, is part of a chaperone complex that collaborates with the methylosome for RNP assembly. Here, we analyze the role of individual SMN and methylosome components in selenoprotein mRNP assembly and translation. We show that SBP2 interacts directly with four proteins of the SMN complex and the methylosome core proteins. Nevertheless, SBP2 is not a methylation substrate of the methylosome. We found that both SMN and methylosome complexes are required for efficient translation of the selenoprotein GPx1 in vivo. We establish that the steady-state level of several selenoprotein mRNAs, major regulators of oxidative stress damage in neurons, is specifically reduced in the spinal cord of SMN-deficient mice and that cap hypermethylation of GPx1 mRNA is affected. Altogether we identified a new function of the SMN complex and the methylosome in selenoprotein mRNP assembly and expression.

Background Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA[Ser]Sec. Results To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA[Ser]Sec gene specifically deleted in myeloid cells. These mutant mice were devoid of the "selenoproteome" in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. Conclusion Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.