Báo cáo Y học: Characterization of selenoprotein P as a selenium supply protein docx

Characterization of selenoprotein P as a selenium supply protein Yoshiro Saito* and Kazuhiko Takahashi Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 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 selenopro- tein 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. Keywords: selenium; selenoprotein; glutathione peroxidase. Se is an essential micronutrient, and is incorporated into proteins in the form of selenocysteine (Sec) and selenomethi- onine. The term selenoprotein is restricted to Sec-containing proteins [1], and is to be distinguished from proteins that nonspecifically incorporate selenomethionine. Sec is encoded by a UGA codon, formerly known only as a stop codon, in the open reading frame of selenoprotein mRNA that is accompanied by a Sec insertion sequence element in the 3¢-untranslated region in eukaryotes [2]. More than 15 selenoproteins have been foundin animals, andsome of them have been shown to exert biological functions [3]. Four types of glutathione peroxidase (GPx) [4–7], three types of thyroid hormone deiodinase [8–10], three types of thioredoxin reductase (TR) [11–13], selenophosphate synthetase [14] and selenoprotein P (SeP) [15] were identified as enzymes. Other selenoproteins with as yet unidentified functions, such as selenoprotein W [16] and a15-kDa selenoprotein [17], have also been reported. Recently, selenoprotein W was reported as a glutathione-dependent antioxidant in vivo [18]. Some pathological conditions of Se deficiency, such as cancer, coronary heart disease and liver necrosis, are thought to be due to a decrease in selenoprotein levels [19]. It is also well known that Se is essential for cell culture when a serum-free medium is used, but not when a medium containing serum is used [20]. This finding suggests that serum contains some usable form of Se. Identification of the Se supplier is critical to understanding the distribution of Se in the body. There are three serum Se-containing proteins that are regarded as candidates for the Se-supply protein: extracellular GPx (eGPx), SeP and albumin. eGPx, a tetramer containing one Sec per subunit, reduces both hydrogen peroxide and phospholipid hydroperoxide in the presence of glutathione (GSH) and thioredoxin [5,21–23]. SeP is a Se-rich extracellular glycoprotein [24–26]. The sequence of the cDNA predicts that human SeP contains 10 selenocysteines encoded by UGA stop codons in the open reading frame of its mRNA [27]. Several lines of evidence in vivo suggest that SeP is a free radical scavenger [28,29]. Recently, we demonstrated that SeP reduces phospholipid hydroperoxide in the presence of GSH [15]. Albumin may contain Se in the form of selenomethionine, and does not contain the element in stoichiometric amounts [30]. In the present study, we describe the first identification of SeP as a major source of Se for the cells cultured in human serum. We also demonstrate that SeP is more effective as a Se supplier than are any other Se-containing proteins and compounds so far tested. We propose that SeP functions not only as an antioxidative enzyme but also as a Se supplier. EXPERIMENTAL PROCEDURES Chemicals Diisopropyl fluorophosphate was obtained from Kishida Chemical Co., Osaka, Japan; tertiary butyl hydroperoxide and hydrogen peroxide from Nacalai, Kyoto, Japan; GSH, GSH reductase, RPMI-1640 medium, selenocystine, sele- nomethionine, DMEM and Hepes from Sigma-Aldrich Co., St. Louis, MO, USA; recombinant human insulin and human transferrin from Wako, Osaka, Japan; and nickel- nitrilotriacetic acid agarose from Qiagen Inc., Chatsworth, CA, USA. Recombinant human thioredoxin was kindly Correspondence to K. Takahashi, Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo, 060-0812, Japan. Fax: + 81 11 706 4990, Tel.: + 81 11 706 3244, E-mail: kazu@pharm.hokudai.ac.jp Abbreviations: GPx, glutathione peroxidase; cGPx, cellular glutathi- one peroxidase; eGPx, extracellular glutathione peroxidase; GSH, glutathione; PH-GPx, phospholipid hydroperoxide glutathione peroxidase; Sec, selenocysteine; SeP, selenoprotein P; TR, thioredoxin reductase. Enzymes: glutathione peroxidase (EC 1.11.1.9); phospholipid hydro- peroxide glutathione peroxidase (EC 1.11.1.12); glutathione reductase (EC 1.8.1.7); thioredoxin reductase (EC 1.8.1.9). *Present address: Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563–8577, Japan. (Received 10 June 2002, revised 5 September 2002, accepted 8 October 2002) Eur. J. Biochem. 269, 5746–5751 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03298.x provided by Ajinomoto, Co. Inc., Kawasaki, Japan. Human serum albumin and human outdated frozen plasma was kindly donated by the Hokkaido Red Cross Blood Center. Ebselen was kindly provided by Daiichi Pharma- ceutical Co., Ltd, Tokyo, Japan. Other chemicals were of the highest quality commercially available. Preparation of SeP- and eGPx-depleted human serum Six monoclonal antibodies (BD1, BD3, BF2, AE2, AH5 and AA3) against human SeP were immobilized as described previously [31]. After the incubation of anti- human SeP mAbs-Sepharose 4B with human serum for 1 h at 4 °C, the obtained supernatant was used as a SeP- depleted human serum. The depletion of SeP was confirmed by a sandwich enzyme-linked immunosorbent assay for human SeP, as described previously [31]. SeP was undetect- able (< 2%) after this treatment. eGPx-depleted human serum was prepared using anti-human eGPx polyclonal Ig– conjugated Sepharose 4B. The depletion of eGPx was confirmed by coupled enzymatic assay with GSH reductase, as described previously [5]. The enzyme activity of eGPx was undetectable (< 2%) after this treatment. Anti-human TR mAb (KB12)–conjugated Sepharose 4B was used for the control experiment [32]. Se assay Levels of Se in each serum and in the cell were determined according to the fluorometric method of Bayfield and Romalis [33]. Cell culture and cytosol preparation Jurkat E6-1 cells, human T-leukemia (American Tissue Type Collection, Rockville, MD, USA), were maintained in RPMI-1640 medium containing 10% (v/v) fetal bovine serum at 37 °C under an atmosphere of 95% (v/v) air and 5% (v/v) CO 2 . For studies on the effects of selenoprotein- depletion from human serum, the cells were cultured with RPMI-1640 medium containing 5% (v/v) of each human serum. Serum-free medium (RPMI-1640 containing 5 lgÆmL )1 human insulin, 5 lgÆmL )1 human transferrin, 5mgÆmL )1 human serum albumin and 2 l M a-tocopherol) was also used. After culturing for the specified periods, the cells were collected by centrifugation and resuspended in an appropriate volume of 50 m M Tris/HCl, pH 7.4, containing 0.25 M sucrose, 0.1 m M EDTA, 0.7 m M 2-mercaptoethanol and 2 m M diisopropyl fluorophosphate. The cell suspension was sonicated with an Ultra Sonic homogenizer VP-5s (Taitec, Tokyo, Japan), and centrifuged at 105 000 g for 1 h at 4 °C to obtain a cytosolic fraction. GPx enzyme assay GPx activities were examined by following the oxidation of NADPH in the presence of GSH reductase, which catalyzes the reduction of the oxidized GSH formed by GPx [5]. To measure cellular GPx (cGPx) activities, both the sample and reference cuvettes contained 0.1 M Tris/HCl, pH 8.0, 0.2 m M NADPH, 0.5 m M EDTA, 2 m M GSH and 1 U of GSH reductase in a total volume of 1 mL. An aliquot of the cytosolic fraction was added to the sample cuvette only. The reaction mixture was preincubated at 37 °Cfor2min, after which the reaction was started by the addition of 70 nmol tertiary-butyl hydroperoxide to both cuvettes. To measure phospholipid hydroperoxide GPx (PH-GPx) activity, the reaction mixture contained 0.1 M Tris/HCl, pH 8.0, 0.2 m M NADPH, 0.5 m M EDTA, 1 m M NaN 3 , 5m M GSH and 1 U of GSH reductase and 60 nmol 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)- 3-PtdCho hydroperoxide was added [15]. The oxidation of NADPH was followed at 340 nm at 37 °C and activity was expressed as micromoles of NADPH oxidized per minute. TR enzyme assay TR activity was examined by spectrophotometric insulin reduction assay, as described previously with a slight modification [34]. Both sample and reference cuvettes contained 50 m M phophate buffer, pH 7.0, 1 m M EDTA, 0.2 m M NADPH, and 0.8 l M human recombinant thiore- doxin in a total volume of 1 mL. An aliquot of the cytosolic fraction was added to the sample cuvette only. The reaction mixture was preincubated at 37 °C for 2 min, after which the reaction was started by the addition of 80 nmol insulin to both cuvettes. The oxidation of NADPH was followed at 340 nm at 37 °C and activity was expressed as micromoles of NADPH oxidized per minute. GSH reductase enzyme assay GSH reductase activities were examined by following the NADPH oxidation in the presence of oxidized glutathione [35]. The assay mixture contained 50 m M phosphate buffer, pH 7.6, 1 m M EDTA, 0.1 m M NADPH, and 1 m M oxidized glutathione. Activity was calculated as micromoles of NADPH oxidized per minute. Purification of SeP and eGPx SeP and eGPx were purified from human plasma using conventional chromatographic methods as described previ- ously [5,15]. RESULTS Immunodepletion of SeP and eGPx from human serum To identify a Se supply protein for the cells cultured in human serum, we first prepared SeP- and eGPx-depleted human serum with immobilized antibodies. The addition of immobilized anti-human SeP and anti-eGPx Igs reduced the Se content to 47 and 81%, respectively (Fig. 1). This suggests that 53 and 19% of serum Se content is derived from SeP and eGPx, respectively. No decrease was observed after treatment with the immobilized control antibody. The residual 28% of Se may be derived mainly from albumin in the form of selenomethionine [30]. Se deficiency in cells cultured with SeP-depleted human serum We then investigated the effect of selenoprotein depletion from serum on the Se-dependent enzymatic activities and Se Ó FEBS 2002 Selenoprotein P as a selenium supply protein (Eur. J. Biochem. 269) 5747 content of cultured Jurkat cells (Fig. 2). When cultured solely in the presence of SeP-depleted serum and not eGPx- depleted or the control serum, the activity of cellular GPx (cGPx), a major Se-dependent enzyme, decreased to 17% that of the control (Fig. 2A). The activity of two other Se-dependent enzymes, phospholipid hydroperoxide GPx (PH-GPx)andTR,alsodecreasedto16and38%, respectively. However, almost no change in the activities of other antioxidant enzymes, such as superoxide dismutase and glutathione reductase, was observed (data not shown). When cultured with SeP-depleted serum, the Se content of the whole cell and the cytosol also decreased to 19 and 35% that of control cells, respectively (Fig. 2B). Next,westudiedthetimecourseofthecGPxactivity(as an indication of Se status) of cells cultured with SeP- depleted serum (Fig. 3). cGPx activity was almost unde- tectable after 4 days. The addition of 270 ngÆmL )1 purified SeP, which corresponded to the SeP concentration of 5% serum, resulted in the complete recovery of cGPx activity within 2 days. Effect of addition of SeP on the cGPx activity of Jurkat cells To compare SeP with other Se-containing proteins or compounds as a Se supplier, we studied the effect of seven reagents on the cGPx activity of the cells (Fig. 4A). SeP was the most effective with a 50% effective dose (ED 50 )of5n M (Se equivalent), followed by eGPx, sodium selenite, seleno- cystine, selenomethionine and albumin. The ED 50 of the former three reagents was 25 n M , and that of the latter two were 300 and 500 n M , respectively. Ebselen had no effect up to 500 n M . To eliminate the effect of serum proteins on the activity of SeP as a Se supplier, a similar experiment was conducted using a serum-free medium. Essentially identical results were obtained using the serum-free medium (Fig. 4B), and SeP was again the most effective. DISCUSSION SeP is an extracellular protein that has been postulated to have an oxidant defense function [28,29]. We recently reported that SeP reduces phospholipid hydroperoxide in the presence of GSH [15]. SeP is also reported to have Fig. 2. Effect of selenoprotein depletion on the selenoenzyme activities and Se content in Jurkat cells. The cells were cultured for 3 days in RPMI-1640 medium containing 5% (v/v) of each human serum. cGPx, PH-GPx, TR activity and Se content were determined, as des- cribed in Experimental procedures. Fig. 3. Effect of SeP on the cGPx activity of Jurkat cells. The cells were cultured in RPMI-1640 medium containing 5% (v/v) of SeP-depleted human serum, and cGPx activity was measured. After 6 days, purified SeP, corresponding to the SeP concentration of 5% serum, was added. Fig. 1. Se concentration in selenoprotein-depleted human serum and the estimation of Se components in human serum. Preparation of each human serum and measurement of Se concentration were conducted as described in Experimental procedures. (A) Se concentrations in selenoprotein-depleted human serum and in the control are shown. These values are the means of six experiments with the error bar in- dicating SD. (B) Se concentrations and percentages in each component were calculated from the data shown in (A). 5748 Y. Saito and K. Takahashi (Eur. J. Biochem. 269) Ó FEBS 2002 survival-promoting properties for cultured neurons [36]. Because of its high Se content and extracellular localization, SeP has also been suggested to be a Se supply protein. It is known that serum-free culture media for immune cells and neurons contain insulin (as a growth factor), transferrin (as an iron source) and sodium selenite (as a Se source). Without these compounds, the cells can neither survive nor proliferate. This suggests that some usable form of Se in serum can provide Se to the cells instead of sodium selenite. Using an in vitro cell culture system, we investigated which Se-containing proteins or compounds supplies Se to the cells. Several methods have been employed to determine the proportion of total Se in human serum (or plasma) accounted for by SeP. Using heparin–agarose, SeP accoun- ted for 40% of the Se applied to the column [37]. The SeP in the plasma of Chinese men of varying Se status accounted for 50–60% of the total Se in their plasma [38]. In another approach based on immunoassay, 40–44% of the total Se was reported to be in the form of SeP in healthy US subjects [39]. In our study, we first prepared SeP- and eGPx-depleted human serum. Se analysis shows that SeP and eGPx contains 53% and 19% of the total serum Se content of healthy Japanese human subjects, respectively (Fig. 1B). The proportion of total Se in human serum accounted for by SeP varied between regions and can be expected to reflect differences in Se intake. Thus, our results essentially confirmed the previous findings described above. Jurkat cells that had been cultured in the presence of SeP- depleted serum became Se-deficient in a time-dependent manner. Under these culture conditions, the cells duplicate at 18 h intervals. We speculate that the divided cells contain one half of the Se content, and that the cells became Se-deficient 4 days later. When human peripheral lympho- cytes were incubated with SeP-depleted serum, the cells did not become Se-deficient (Y. Saito and K. Takahashi, unpublished observation). As peripheral lymphocytes do not proliferate, perhaps only proliferating cells (such as hemopoietic cells, spermatocytes and neurons) become Se- deficient when cultured with SeP-depleted serum. The fact that new neurons are continually added to the neocortex of adult macaque monkeys has profound implications for the understanding of the cellular mechanisms of higher cogni- tive functions [40]. However, the addition of purified SeP to Se-deficient cells resulted in the recovery of cellular GPx activity within 2 or 3 days. Next, we compared the Se-supply activity of SeP with two other Se-containing serum proteins, eGPx and albumin. The 50% effective dose (ED 50 ) of SeP, eGPx and albumin was 5, 25 and 500 n M (Se equivalent), respectively. The Se concentration of SeP, eGPx and albumin in 5% human serum was 42, 15 and 22 n M (Se equivalent), respectively. This suggests that SeP mainly supplies Se to cells under physiological conditions. Essentially identical results were obtained in a serum-free medium in the presence of 2 l M a–tocopherol. In a serum- free medium without a-tocopherol, the cells could not survive in a Se-deficient state. This suggests that a–toco- pherol protects Se-deficient cells from cell death by oxidative stress. Actually 5% serum contains 0.9 l M a-tocopherol. Thus, the cells could survive even in a Se-deficient state in the presence of SeP-depleted serum. This speculation is supported by reports that SeP is a suvival-promoting factor for cultured central neurons [36]. In the study in which SeP was identified as a survival-promoting factor, the culture medium contained transferrin, insulin, albumin but not sodium selenite. Our observations indicate that SeP functions as a Se supplier for the proliferating cells. Little is known about the function of a group of serum proteins concerned in the supply of substances of small molecular weight. The most studied of these is transferrin, which is concerned in the distribution of iron from the intestinal absorption sites to the various tissues requiring iron [41]. Albumin is believed to supply many low-molecular-mass substances such as metals, amino acids and fatty acids [42]. Transferrin and albumin noncovalently bind iron and many low-molecular-mass substances, respectively. As Se is covalently bound in SeP, the supply mechanism is presumed to require digestion of SeP by proteases and peptidases, and the breakdown of selenocysteine for release of its Se, and must be proved in the future. A serum-free medium containing Se (sodium selenite) was used to culture a variety of cells, neurons and hemopoietic (especially immune) cells. Without Se, the cells could neither survive nor proliferate. SeP is taken up in greater amounts Fig. 4. Effect of the addition of Se-containing compounds on the cGPx activity of Jurkat cells in serum or serum-free medium. In the presence of variable amounts of Se-containing compounds, the cells were cultured with SeP-depleted serum (A) or serum-free medium (B), as described in Experimental procedures. After 3 days, the cells were collected and cGPx activity was measured. Open circles, SeP (185 nmol SeÆmg )1 of protein); closed circles, eGPx; (53 nmol SeÆmg )1 of protein); open squares, sodium selenite; closed squares, selenocystine; open triangles, selenomethionine; closed triangles, human serum albumin (8.3 pmol SeÆmg )1 of protein); open circles with broken line, ebselen. Ó FEBS 2002 Selenoprotein P as a selenium supply protein (Eur. J. Biochem. 269) 5749 by the brain but not by other organs in Se-deficient animals [43], suggesting a critical function of this selenoprotein in this organ. It has survival-promoting properties for cultured neurons [36] and its mRNA is present in the brain [44]. Furthermore, it is reported that astrocytes and cerebellar granule cells secrete SeP [45]. A recent finding that new neurons are continually added to the neocortex suggests that SeP secreted from astrocytes and cerebellar granule cells may supply Se to proliferating neurons. Numerous studies suggest that a deficiency of Se is accompanied by a loss of immunocompetence, probably not unconnected with the fact that Se is normally found in significant amounts in immune tissues such as the spleen, and lymph nodes. Both cell-mediated immunity and B-cell function can be impaired [46]. Supplementation with Se has marked immunostimu- lant effects, including an enhancement of proliferation of activated T cells [47]. In the present study, we identify SeP as a Se supply protein to proliferating T lymphoma cells. The essential roles of Se in brain and immune functions described above and our in vitro studies strongly suggest that SeP supplies Se to the proliferating brain and hemopoietic cells. Thus, we propose that SeP functions not only as an antioxidative enzyme but also functions as a Se supplier. ACKNOWLEDGEMENTS This work was supported in a party by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Hokkaido Red Cross Blood Center for providing human plasma. Y. Saito is the recipient of a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. REFERENCES 1. Stadtman, T.C. (1996) Selenocysteine. Annu. Rev. Biochem. 65, 83–100. 2. Hatfield, D.L. & Gladyshev, V.N. (2002) How selenium has altered our understanding of the genetic code. Mol. Cell. Biol. 22, 3565–3576. 3. Behne, D. & Kyriakopoulos, A. (2001) Mammalian selenium- containing proteins. Annu. Rev. Nutr. 21, 453–473. 4. Flohe, L., Gunzler, W.A. & Schock, H.H. (1973) Glutathione peroxidase: aselenoenzyme. FEBS Lett. 32, 132–134. 5. Takahashi, K., Avissar, N., Whitin, J. & Cohen, H. (1987) Puri- fication and characterization of human plasma glutathione per- oxidase: a selenoglycoprotein distinct from the known cellular enzyme. Arch. Biochem. Biophys. 256, 677–686. 6. Ursini,F.,Maiorino,M.&Gregolin,C.(1985)Theselenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim. Biophys. Acta 839, 62–70. 7. Chu, F.F., Doroshow, J.H. & Esworthy, R.S. (1993) Expression, characterization, and tissue distribution of a new cellular selenium- dependent glutathione peroxidase, GSHPx-GI. J. Biol. Chem. 268, 2571–2576. 8. Behne, D., Kyriakopoulos, A., Meinhold, H. & Kohrle, J. (1990) Identification of type I iodothyronine 5¢-deiodinase as a seleno- enzyme. Biochem. Biophys. Res. Commun. 173, 1143–1149. 9. Croteau, W., Davey, J.C., Galton, V.A. & St Germain, D.L. (1996) Cloning of the mammalian type II iodothyronine deiodinase. A selenoprotein differentially expressed and regulated in human and rat brain and other tissues. J. Clin. Invest 1998, 405–417. 10. Croteau, W., Whittemore, S.L., Schneider, M.J. & St Germain, D.L. (1995) Cloning and expression of a cDNA for a mammalian type III iodothyroninedeiodinase. J. Biol. Chem. 270, 16569– 1657511. 11. Tamura, T. & Stadtman, T.C. (1996) A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity. Proc. Natl Acad. Sci. USA 93, 1006–1011. 12. Gasdaska, P.Y., Berggren, M.M., Berry, M.J. & Powis, G. (1999) Cloning, sequencing and functional expression of a novel human thioredoxin reductase. FEBS Lett. 442, 105–111. 13. Sun, Q.A., Wu, Y., Zappacosta, F., Jeang, K.T., Lee, B.J., Hatfield, D.L. & Gladyshev, V.N. (1999) Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases. J. Biol. Chem. 274, 24522–24530. 14. Guimaraes, M.J., Peterson, D., Vicari, A., Cocks, B.G., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Ferrick, D.A., Kastelein, R.A., Bazan, J.F. & Zlotnik, A. (1996) Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism? Proc. Natl Acad. Sci. USA 93, 15086–15091. 15. Saito, Y., Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E. & Takahashi, K. (1999) Selenoprotein P in human plasma as an extracellular phospholipid hydroperoxide glu- tathione peroxidase. Isolation and enzymatic characterization of human selenoprotein P. J. Biol. Chem. 274, 2866–2871. 16. Vendeland, S.C., Beilstein, M.A., Chen, C.L., Jensen, O.N., Barofsky, E. & Whanger, P.D. (1993) Purification and properties of selenoprotein W from rat muscle. J. Biol. Chem. 268, 17103– 17107. 17. Gladyshev, V.N., Jeang, K.T., Wootton, J.C. & Hatfield, D.L. (1998) A new human selenium-containing protein. Purification, characterization, and cDNA sequence. J. Biol. Chem. 273, 8910– 8915. 18. Jeong, D., Kim, T.S., Chung, Y.W., Lee, B.J. & Kim, I.Y. (2002) SelenoproteinW is a glutathione-dependent antioxidant in vivo. FEBS Lett. 517, 225–228. 19. Allan, C.B., Lacourciere, G.M. & Stadtman, T.C. (1999) Responsiveness of selenoproteins to dietary selenium. Annu. Rev. Nutr. 19, 1–16. 20. McKeehan, W.L., Hamilton, W.G. & Ham, R.G. (1976) Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Natl Acad. Sci. U.S.A. 73, 2023–2027. 21. Takahashi, K., Akasaka, M., Yamamoto, Y., Kobayashi, C., Mizoguchi, J. & Koyama, J. (1990) Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences. J. Biochem. 108, 145–148. 22. Yamamoto, Y. & Takahashi, K. (1993) Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxides. Arch. Biochem. Biophys. 305, 541–545. 23. Takebe, G., Yarimizu, J., Saito, Y., Hayashi, T., Nakamura, H., Yodoi, J., Nagasawa, S. & Takahashi, K. (2002) A com- parative study on the hydroperoxide and thiol specificity of the glutathione peroxidase family and selenoprotein P. J. Biol. Chem. 277, 41254–41258. 24. Burk, R.F. & Hill, K.E. (1994) Selenoprotein P. A selenium-rich extracellular glycoprotein. J. Nutr. 124, 1891–1897. 25. Mostert, V. (2000) Selenoprotein P: properties, functions, and regulation. Arch. Biochem. Biophys. 376, 433–438. 26. Saito, Y. & Takahashi, K. (2000) Selenoprotein P: Its structure and functions. J. Health Sci. 46, 409–413. 27. Hill, K.E., Lloyd, R.S., Yang, J.G., Read, R. & Burk, R.F. (1991) The cDNA forrat selenoprotein P contains 10 TGA codons in the open reading frame. J. Biol. Chem. 266, 10050–10053. 28. Burk, R.F., Hill, K.E., Awad, J.A., Morrow, J.D., Kato, T., Cockell, K.A. & Lyons, P.R. (1995) Pathogenesis of diquat- induced liver necrosis inselenium-deficient rats: assessment of the roles of lipid peroxidation and selenoprotein P. Hepatology 21, 561–569. 5750 Y. Saito and K. Takahashi (Eur. J. Biochem. 269) Ó FEBS 2002 29. Burk, R.F., Hill, K.E., Awad, J.A., Morrow, J.D. & Lyons, P.R. (1995) Liver and kidney necrosis in selenium-deficient rats de- pleted of glutathione. Laboratory Invest. 72, 723–730. 30. Deagen, J.T., Beilstein, M.A. & Whanger, P.D. (1991) Chemical forms of selenium in selenium containing proteins from human plasma. J. Inorg. Biochem. 41, 261–268. 31. Saito, Y., Watanabe, Y., Saito, E., Honjoh, T. & Takahashi, K. (2001) Production and application of monoclonal antibodies to human selenoprotein P. J. Health Sci. 2247, 346–352. 32. Yarimizu, J., Nakamura, H., Yodoi, J. & Takahashi, K. (2000) Efficiency of selenocysteine incorporation in human thioredoxin reductase. Antioxid. Redoxsignal. 2, 643–651. 33. Bayfield, R.F. & Romalis, L.F. (1985) pH Control in the fluoro- metric assay for selenium with 2,3- diaminonaphthalene. Anal. Biochem. 144, 569–576. 34. Holmgren, A. & Bjornstedt, M. (1995) Thioredoxin and thior- edoxin reductase. Methods Enzymol. 252, 199–208. 35. Luthman, M. & Holmgren, A. (1982) Rat liver thioredoxin and thioredoxin reductase: purification and characterization. Bio- chemistry 21, 6628–6633. 36. Yan, J. & Barrett, J.N. (1998) Purification from bovine serum of a.23survival-promoting factor for cultured central neurons and its identification as selenoprotein-P. J. Neurosci. 18, 8682–8691. 37. Akesson,B.&Martensson,B.(1991)Chromatographyofsele- noproteins in human serum using matrix-bound heparin. Int. J. Vitam. Nutr. Res. 61, 72–76. 38. Deagen, J.T., Butler, J.A., Zachara, B.A. & Whanger, P.D. (1993) Determinationof the distribution of selenium between glutathione peroxidase, selenoprotein P, and albumin in plasma. Anal. Bio- chem. 208, 176–181. 39. Hill, K.E., Xia, Y., Akesson, B., Boeglin, M.E. & Burk, R.F. (1996) Selenoprotein P concentration in plasma is an index of selenium status inselenium-deficient and selenium-supplemented Chinese subjects. J. Nutr. 126, 138–145. 40. Gould, E., Reeves, A.J., Graziano, M.S. & Gross, C.G. (1999) Neurogenesis in the neocortex of adult primates. Science 286, 548– 552. 41. Qian, Z.M. & Tang, P.L. (1995) Mechanisms of iron uptake by mammalian cells. Biochim. Biophys. Acta 1269, 205–214. 42. Curry, S., Brick, P. & Franks, N.P. (1999) Fatty acid binding to human serum albumin: new insights from crystallographic studies. Biochim. Biophys. Acta 1441, 131–140. 43. Burk, R.F., Hill, K.E., Read, R. & Bellew, T. (1991) Response of ratselenoprotein P to selenium administration and fate of its selenium. Am. J. Physiol. 261, E26–E30. 44. Saijoh, K., Saito, N., Lee, M.J., Fujii, M., Kobayashi, T. & Sumino, K. (1995) 24Molecular cloning of cDNA encoding a bovine selenoprotein P-like protein containing 12 selenocysteines and a (His-Pro) rich domain insertion, and its regional expression. Mol. Brain Res. 30, 301–311. 45. Yang, X., Hill, K.E., Maguire, M.J. & Burk, R.F. (2000) Synthesis and secretion of selenoprotein P by cultured rat astrocytes. Bio- chim. Biophys. Acta 1474, 390–396. 46. McKenzie, R.C., Rafferty, T.S. & Beckett, G.J. (1998) Selenium: an essential element for immune function. Immunol. Today 19, 342–345. 47. Kiremidjian-Schumacher,L.,Roy,M.,Wishe,H.I.,Cohen,M.W. & Stotzky, G. (1994) Supplementation with selenium and human immune cell functions. II. Effect on cytotoxic lymphocytes and natural killer cells. Biol. Trace Elem. Res. 41, 115–127. Ó FEBS 2002 Selenoprotein P as a selenium supply protein (Eur. J. Biochem. 269) 5751 . Characterization of selenoprotein P as a selenium supply protein Yoshiro Saito* and Kazuhiko Takahashi Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido. Proc. Natl Acad. Sci. USA 93, 15086–15091. 15. Saito, Y. , Hayashi, T., Tanaka, A. , Watanabe, Y. , Suzuki, M., Saito, E. & Takahashi, K. (1999) Selenoprotein P in human plasma as an extracellular. human fibroblasts. Proc. Natl Acad. Sci. U.S .A. 73, 2023–2027. 21. Takahashi, K., Akasaka, M., Yamamoto, Y. , Kobayashi, C., Mizoguchi, J. & Koyama, J. (1990) Primary structure of human plasma