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• W2102500963 abstract "Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA[Ser]Sec and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function. Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA[Ser]Sec and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function. Selenium is an essential micronutrient in the diet of higher vertebrates, including humans and other mammals. Numerous health benefits have been attributed to this element. For example, evidence suggests that selenium has cancer chemopreventive properties, inhibits viral expression, and delays the progression of AIDS in patients who are positive for the human immunodeficiency virus (HIV+) (reviewed in Ref. 1Hatfield D.L. Selenium: Its Molecular Biology and Role in Human Health. Kluwer Academic Publishers, Norwell, MA2001Crossref Google Scholar). Furthermore, it appears to reduce the risk of heart disease and other cardiovascular and muscle disorders, to slow the aging process, and to have roles in mammalian development, male reproduction, and immune function (1Hatfield D.L. Selenium: Its Molecular Biology and Role in Human Health. Kluwer Academic Publishers, Norwell, MA2001Crossref Google Scholar). Selenoproteins are most certainly responsible for many of the health benefits of selenium. Humans encode 25 selenoproteins in their genome and mice 24, but the functions of only about half of these proteins are now known (2Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1876) Google Scholar). Determining the identity and functions of selenoproteins is essential to understanding the role of selenium in human health. Selenium makes its way into protein as the amino acid selenocysteine (Sec) (reviewed in Ref. 3Hatfield D.L. Gladyshev V.N. Mol. Cell Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (550) Google Scholar). Sec has its own code word, UGA, and its own tRNA designation, Sec tRNA[Ser]Sec. Sec is biosynthesized on its tRNA after the tRNA is aminoacylated initially with serine by seryl-tRNA synthetase. The presence of a stem-loop structure, designated a Sec insertion sequence element, in selenoprotein mRNAs dictates a UGA codon within an open reading frame to function as Sec and not as stop (4Low S.C. Berry M.J. Trends Biochem. Sci. 1996; 21: 203-210Abstract Full Text PDF PubMed Scopus (393) Google Scholar). In addition, much of the Sec protein insertion machinery is unique to this amino acid, and in mammals, there is a specific Sec insertion sequence-binding protein, SBP2 (5Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (314) Google Scholar), and a specific elongation factor, EFsec (6Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Harney J.W. Driscoll D.M. Hatfield D.L. Berry M.J. EMBO Rep. 2000; 1: 158-163Crossref PubMed Scopus (245) Google Scholar, 7Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (245) Google Scholar), required for Sec insertion into protein. Selenoproteins are the only known class of proteins for which expression is determined by the presence of a single tRNA. Thus, manipulating the expression of Sec tRNA[Ser]Sec can perturb the expression of selenoproteins, which in turn provides an important tool in elucidating the biological functions of the various members of this class of proteins and their potential roles in promoting better health. One approach to elucidating the cellular roles of selenoproteins is to knock out the corresponding gene. Indeed, several laboratories have targeted specific selenoproteins for removal from the mouse genome, including glutathione peroxidases 1, 2, and 4 (GPx1 (8Cheng W.H. Ho Y.S. Ross D.A. Valentine B.A. Combs G.F. Lei X.G. J. Nutr. 1997; 127: 1445-1450Crossref PubMed Scopus (131) Google Scholar), GPx2 (9Esworthy R.S. Aranda R. Martin M.G. Doroshow J.H. Binder S.W. Chu F.F. Am. J. Physiol. 2001; 281: G848-G855Crossref PubMed Google Scholar), GPx4 (10Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (556) Google Scholar)), thyroid hormone deiodinase 2 (DIO2) (11Schneider M.J. Fiering S.N. Pallud S.E. Parlow A.F. St. Germain D.L. Galton V.A. Mol. Endocrinol. 2001; 15: 2137-2148Crossref PubMed Scopus (251) Google Scholar), and selenoprotein P (SelP) 1The abbreviations used are: SelP, selenoprotein P; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GPx, glutathione peroxidase; RPC-5, reverse phase chromatographic column-5. (12Hill K.E. Zhou J. McMahan W.J. Motley A.K. Atkins J.F. Gesteland R.F. Burk R.F. J. Biol. Chem. 2003; 278: 13640-13646Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 13Schomburg L. Schweizer U. Holtmann B. Flohe L. Sendtner M. Kohrle J. Biochem. J. 2003; 370: 397-402Crossref PubMed Scopus (354) Google Scholar). These studies have provided insights into the roles of these selenoproteins in development and cellular metabolism. In a different approach, and to provide alternative models for examining the roles of selenium in health, we initially generated a transgenic mouse carrying a mutant Sec tRNA[Ser]Sec transgene wherein the expressed tRNA lacked a highly modified base, N6-isopentenyladenosine (i6A), at position 37 (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). The i6A-tRNA[Ser]Sec-deficient mice manifested a reduction in selenoproteins that occurred in a protein- and tissue-specific manner. More recently, we generated a conditional knockout of the Sec tRNA[Ser]Sec gene encoding flanking loxP sites whereby the gene is receptive to removal by the Cre recombinase, which may be under the control of promoters targeted for specific organs or tissues (15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). Mice that are homozygous for this floxed allele, designated Trspfl/fl, were crossed to transgenic mice carrying the Cre recombinase under the control of two promoters targeting mammary epithelium. Neither Cre recombinant resulted in complete removal of Trsp. However, one of the promoters, MMTV-Cre, removed about 80% of the Trsp, which resulted in an altered selenoprotein expression in mammary epithelium (15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar) similar to that observed in mouse liver with the mutant transgene (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). However, no apparent phenotypic changes due to selenoprotein deficiency were detected. In the present study, Trsp was selectively removed from liver by mating floxed mice with transgenic mice carrying the Alb-Cre transgene (16Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 17Postic C. Magnuson M.A. Genesis. 2000; 26: 149-150Crossref PubMed Scopus (315) Google Scholar). Characterization of these mice provided important insights into selenoprotein synthesis and transport and identified an essential role of selenoproteins in liver function. Materials—75Selenium (specific activity 1000 Ci/mmol) was obtained from the Research Reactor Facility, University of Missouri, Columbia, MO, and all other reagents were commercial products of the highest grade available. Floxed Trsp (designated Trspfl), strain C57B6, mice have been described (15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar), and heterozygous albumin Cre (designated Alb-Cre+/-), strain C57B6, transgenic mice (16Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 17Postic C. Magnuson M.A. Genesis. 2000; 26: 149-150Crossref PubMed Scopus (315) Google Scholar) were purchased from Jackson Laboratories. Antibodies to SelP were kindly provided by Drs. K. E. Hill and R. F. Burk (Vanderbilt University) and by Drs. U. Schweizer and L. Schomberg (Charité Universitätsmedizin, Berlin, Germany) and those to TR1 were used as described previously (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). The care of animals was in accordance with the National Institutes of Health institutional guidelines under the expert direction of D. L. Sly (SAIC, NCI, National Institutes of Health). Identification of Trsp, Trspfl/fl, and Alb-Cre+/- and Selective Removal of Trsp in Liver—Mice carrying homozygous floxed Trsp (Trspfl/fl) were identified by PCR analysis of tail DNA as described previously (15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar), and the Alb-Cre transgene was identified by PCR analysis of tail DNA with primers 5′-ACCTGAAGATGTTCGCGATTATCT-3′ and 5′-ACCGTCAGTACGTGAGATATCTT-3′, which resulted in a 370 -bp fragment (16Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). Heterozygous floxed Trsp-heterozygous albumin Cre (Trspfl/+-Alb-Cre+/-) mice were generated by mating Trspfl/fl mice with Alb-Cre+/- mice, selecting for the appropriate offspring, and then mating the resulting mice heterozygous for both genes to obtain homozygous floxed Trsp-heterozygous albumin Cre (Trspfl/fll-Alb-Cre+/-), Trspfl/+-Alb-Cre+/-, and Trspfl/fll offspring for Sec tRNA[Ser]Sec, selenoprotein, blood, and pathological analyses. Isolation, Fractionation, and Identification of Specific tRNA Isoforms—Total tRNA was isolated from tissues (18Hatfield D. Matthews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar) and fractionated by RPC-5 chromatography (19Kelmers A.D. Heatherly D.E. Anal. Biochem. 1971; 44: 486-495Crossref PubMed Scopus (152) Google Scholar) or by polyacrylamide gel electrophoresis as described (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). Sec tRNA[Ser]Sec and serine tRNASer1 were identified by northern blotting and quantitated as given (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). Labeling of Selenoproteins and GPx1 and TR1 Assays—Mice with genotypes Trspfl/fll-Alb-Cre+/-, Trspfl/+-Alb-Cre+/-, and Trspfl/fll were labeled with 75Se, tissues and organs excised, proteins extracted and electrophoresed, gels stained with Coomassie Blue, and proteins transferred to nylon membranes. The resulting transblots were exposed to a PhosphorImager as described (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar, 15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 20Gladyshev V.N. Stadtman T.C. Hatfield D.L. Jeang K.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 835-839Crossref PubMed Scopus (93) Google Scholar). GPx1 activity was assayed directly, and TR1 activity was assayed after enrichment on ADP-Sepharose as described (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). A major protein differentially expressed between knockout and control livers was identified by N-terminal Edman degradation at the University of Nebraska-Lincoln proteomics core facility. Pathology Evaluation—Mice were sacrificed using CO2 inhalation. Necropsy examination was performed on a subset of mice that were sacrificed or that died spontaneously. The total numbers of mice examined were as follows: Trspf1/f1, scheduled sacrifice, 7 males and 0 females; Trspf1/f1-Alb-Cre+/-, scheduled sacrifice, 4 males and 2 females; and Trspf1/f1-AlbCre+/-, spontaneous death/clinically ill, 3 males and 6 females. A comprehensive set of organs and tissues was collected and fixed in 10% buffered neutral formalin. Tissues were paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin and eosin. The TUNEL assay (Apoptag, Serologicals Corp.) was performed on sections of liver from all mice. Prussian blue stain for iron was performed on samples of necrotic fat. Blood and Selenium Analyses—Blood samples were taken from mice prior to necropsy by cardiac puncture. The serum was obtained by centrifugation and used for determining blood chemistries (run by the Pathology/Histotechnology Laboratory) using standard techniques. The analytes tested were urea nitrogen, total protein, albumin, aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transpeptidase, and total bilirubin. To determine the levels of selenium in the form of low molecular weight selenocompounds or selenoproteins, 300 mg of liver were homogenized in 5 ml of a lysis buffer (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar, 15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 20Gladyshev V.N. Stadtman T.C. Hatfield D.L. Jeang K.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 835-839Crossref PubMed Scopus (93) Google Scholar), and proteins were precipitated in trichloroacetic acid and collected as described previously (21Hames B.D. Rickwood D. Practical Approach Series on CD-ROM. Oxford University Press, 1995-1996Google Scholar). Selenium levels in the extracts, pellets, and trichloroacetic acid supernatants and in all other tissues and organs were determined by the Oscar E. Olsen Biochemistry Laboratories at South Dakota State University. Specific Removal of Trsp from Liver—Alb-Cre has been reported to be highly specific to liver and virtually 100% efficient in hepatocytes when crossed with floxed alleles (16Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). Its complete expression in liver occurred after an initial time lag of only a few weeks (17Postic C. Magnuson M.A. Genesis. 2000; 26: 149-150Crossref PubMed Scopus (315) Google Scholar). We, therefore, examined the status of Trsp in liver of mice with genotypes Trspfl/fl, Trspfl/+-Alb-Cre+/-, and Trspfl/fl-Alb-Cre+/- at 1, 3, and 12 weeks of age using the kidney as a control organ. As shown in Fig. 1A, between 80 and 90% of the gene was removed from the liver of mice about 1 week old (lane 3) and virtually completely removed in older mice (lanes 4 and 5). Trspfl/fl was unaffected in the kidney of Trspfl/fl-Alb-Cre+/- mice (lanes 8–10). The wild type gene (lacking the floxed allele, designated Trsp+) was observed in heterozygous mice encoding Trspfl, Trsp+, and Alb-Cre+/- where Trspfl was removed in liver (Fig. 1A, lane 2) but retained in kidney (lane 7). The small amounts of Trspfl observed in Trspfl/+-Alb-Cre+/- or Trspfl/fl-Alb-Cre+/- mice are likely because of the presence of cell forms other than hepatocytes (see “Discussion”). Total tRNA was isolated from perfused livers and from kidneys of Trspfl/fl, Trspfl/+-Alb-Cre+/-, and Trspfl/fl-Alb-Cre+/- mice. Sec tRNA[Ser]Sec levels were examined by Northern analysis and by RPC-5 chromatography (Fig. 1B). Relative to the amount of serine tRNA1, more than 90% of the Sec tRNA[Ser]Sec had been removed from livers lacking the corresponding gene (Fig. 1B, lane 3), whereas in the kidneys of these mice, the Sec tRNA[Ser]Sec population was unaffected (lane 6), as shown by Northern blot analysis in the upper portion of Fig. 1B. Interestingly, the Sec tRNA[Ser]Sec level was reduced to about 70% of the wild type amount in the liver of Trspfl/+-Alb-Cre+/- mice (see legend to Fig. 1). This reduction in Sec tRNA[Ser]Sec expression was similar to that observed in mice heterozygous for the standard tRNA[Ser]Sec knockout (15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). Virtually all of the Sec tRNA[Ser]Sec appeared to be absent in the liver of mice lacking Trsp in this tissue as compared with Trspfl/fl mice following fractionation of total tRNA from corresponding organs by RPC-5 chromatography, dot blotting on nylon membranes, and hybridization (see graph in lower portion of Fig. 1B). Selenoprotein Expression—The expression of selenoproteins in liver, kidney, plasma, brain and testes of Trspfl/fl, Trspfl/+-Alb-Cre+/-, and Trspfl/fl-Alb-Cre+/- mice was assessed by 75Se labeling of the corresponding mice and by examining the resulting labeled proteins from these tissues following gel electrophoresis. Coomassie Blue-stained gels of total proteins from livers of these mice appeared similar (Fig. 2, lower panels) with the exception of an enriched band in the extract of Trspfl/fl-Alb-Cre+/- mice (designated by an arrow). We extracted this 25-kDa band from gels and sequenced the first 10 residues, PMILGYWNVR. This sequence is identical to that of mouse glutathione S-transferase, demonstrating that the expression of this enzyme is elevated in the liver of selenoprotein-deficient mice. The selenoprotein population was dramatically different in the livers of the three lines of mice. The liver in the Trspfl/fl-Alb-Cre+/- strain lacked virtually any selenoprotein expression. The three major bands observed on the gels of Trspfl/fl and Trspfl/+-Alb-Cre+/- mice were TR1 (57 kDa) and GPx1 (22 kDa), and an uncharacterized selenoprotein at 52 kDa (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar, 15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 20Gladyshev V.N. Stadtman T.C. Hatfield D.L. Jeang K.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 835-839Crossref PubMed Scopus (93) Google Scholar). To verify that the band at 57 kDa was TR1, crude extracts of liver were enriched on ADP-Sepharose columns, which are known to serve as an affinity matrix for this enzyme (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar). The small panel attached to the right of the liver panel shows the Western blot of the corresponding extracts enriched for TR1 from the three mouse lines, demonstrating that the labeled band at 57 kDa was indeed TR1. Interestingly, protein from the three mouse lines, including that from Trspfl/fl-Alb-Cre+/-, appeared to be present in virtually the same amounts. These data suggested that the TR1 polypeptide was efficiently made in the liver knockout mouse, but the open reading frame terminated at the penultimate UGA codon, and thus did not contain Sec (see below). In contrast to the 75Se labeling pattern observed in these three mouse lines in liver, labeling of the corresponding selenoprotein population in kidney was virtually identical. The four major selenoprotein bands in kidney were likely TR1 (57 kDa), an uncharacterized 52-kDa selenoprotein, GPx1 (22 kDa), and GPx 4 (20 kDa) (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar, 15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 20Gladyshev V.N. Stadtman T.C. Hatfield D.L. Jeang K.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 835-839Crossref PubMed Scopus (93) Google Scholar). GPx1 and TR1, which are major selenoproteins expressed in the liver, often exhibit changes in expression that are opposite to each other (14Moustafa M.E. Carlson B.A. El-Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (115) Google Scholar, 15Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 27Gladyshev V.N. Factor V.M. Housseau F. Hatfield D. Biochem. Biophys. Res. Commun. 1998; 251: 488-493Crossref PubMed Scopus (113) Google Scholar). To confirm the decreased expression of these two selenoproteins in Trsp knockout liver, we assayed GPx1 and TR1 activities in mouse lines carrying the Trspfl/fl and Trspfl/fl-Alb-Cre+/- genotypes. Consistent with the 75Se labeling assays, the liver of Trspfl/fl-Alb-Cre+/- mice had less than 5% of the GPx1 activity of that found in the floxed control mouse, whereas the activities in kidney were virtually identical in these two mouse lines (data not shown). TR1 activity was similarly reduced in the liver of the Trspfl/fl-Alb-Cre+/- mice (data not shown), but TR1 levels were virtually unchanged (see Fig. 2 and its legend), providing further evidence that the protein is terminated at the penultimate UGA Sec codon. Two major selenoproteins, SelP and GPx3, have been described in plasma (Ref. 22Burk R.F. Hill K.E. Motley A.K. Biofactors. 2001; 14: 107-114Crossref PubMed Scopus (151) Google Scholar and references therein). SelP has been reported to be synthesized in the liver and transported to plasma (23Kato T. Read R. Rozga J. Burk R.F. Am. J. Physiol. 1992; 262: G854-G858Crossref PubMed Google Scholar, 24Saijoh K. Saito N. Lee M.J. Fujii M. Kobayashi T. Sumino K. Mol. Brain Res. 1995; 30: 301-311Crossref PubMed Scopus (50) Google Scholar) and to be imported into the brain and testes (25Yang X. Hill K.E. Maguire M.J. Burk R.F. Biochim. Biophys. Acta. 2000; 1474: 390-396Crossref PubMed Scopus (39) Google Scholar), whereas the kidney-proximal tubules are the major source of plasma GPx3 (26Avvisar N. Ornt D.B. Yagil Y. Horowitz S. Watkins R.H. Kerl E.A. Takahashi K. Palmer I.S. Cohen H.J. Am. J. Physiol. 1994; 266: C367-C375Crossref PubMed Google Scholar). Brain has been reported to express SelP mRNA, but not testes, suggesting that SelP is also synthesized in brain but not testes (25Yang X. Hill K.E. Maguire M.J. Burk R.F. Biochim. Biophys. Acta. 2000; 1474: 390-396Crossref PubMed Scopus (39) Google Scholar). The SelP level was reduced about 75% in plasma in Trspfl/fl-Alb-Cre+/- mice as shown in Fig. 2, “Plasma” panel. To verify that the major labeled band observed in plasma was SelP, a membrane containing the 75Se-labeled liver extracts from these three mouse lines was exposed to antibodies for SelP (Fig. 2, see small panel (insert) attached to the right of the Plasma panel). The Western blot shows that the first and second lanes (Fig. 2) with plasma of Trspfl/fl and Trspfl/+-Alb-Cre+/- mice yielded a positive signal with SelP antibodies, but that the lane with plasma from Trspfl/fl-Alb-Cre+/- mice did not respond to SelP antibodies. These results demonstrate that the major 75Se-labeled band in plasma is SelP (see also Ref. 22Burk R.F. Hill K.E. Motley A.K. Biofactors. 2001; 14: 107-114Crossref PubMed Scopus (151) Google Scholar). It should also be noted that SelP antibodies do not detect SelP in tissues. 2K. E. Hill and R. F. Burk, personal communication. In contrast, the Coomassie Blue-stained gels of total proteins from plasma of the three mouse lines appeared to be similar. The 75Se-selenoprotein labeling pattern observed in brain was similar in the different genetic backgrounds of each animal examined, whereas in testes a band that migrates at the expected position of SelP was reduced. The labeling pattern of GPx3 in plasma suggests that its level may not be affected, providing further evidence that its presence in this tissue is the result of its synthesis in kidney (26Avvisar N. Ornt D.B. Yagil Y. Horowitz S. Watkins R.H. Kerl E.A. Takahashi K. Palmer I.S. Cohen H.J. Am. J. Physiol. 1994; 266: C367-C375Crossref P" @default.
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