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• W2009691812 abstract "Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55–57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5′ sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by ∼30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases. Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55–57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5′ sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by ∼30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases. thioredoxin reductase transforming growth factor α polyacrylamide gel electrophoresis high performance liquid chromatography matrix-assisted laser desorption ionization glutathione peroxidase selenocysteine nonredundant catechol-O-methyltransferase membrane-bound catechol-O-methyltransferase soluble catechol-O-methyltransferase Alzheimer's disease The thioredoxin redox system is one of two major redox systems in animal cells, which, together with the glutathione system, participates in the redox control of a great variety of biological processes involved in cell life and death (1Sies H. Free Radic. Biol. Med. 1999; 27: 916-921Crossref PubMed Scopus (1328) Google Scholar, 2Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 3Halliwell B. Free Radic. Res. 1999; 31: 261-272Crossref PubMed Scopus (734) Google Scholar). Disruption of the gene for thioredoxin 1, a 12-kDa thiol disulfide oxidoreductase, results in embryonic lethality in mice (4Matsui M. Oshima M. Oshima H. Takaku K. Maruyama T. Yodoi J. Taketo M.M. Dev. Biol. 1986; 178: 179-185Crossref Scopus (414) Google Scholar) demonstrating an essential role of the thioredoxin system in the development of mammals. Three distinct thioredoxin reductases (TRs)1 (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar, 6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (146) Google Scholar, 8Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 9Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) are responsible for maintaining thioredoxins in a reduced state and are also capable of reducing a great variety of other protein and nonprotein redox substrates. The cytosolic thioredoxin reductase (TR1), the most characterized of the three enzymes, was known for decades, but only recently it was found to be a selenium-containing protein (11Tamura T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1006-1011Crossref PubMed Scopus (466) Google Scholar). TR1 contains a C-terminal penultimate selenocysteine residue encoded by TGA (12Gladyshev V.N. Jeang K.-T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (405) Google Scholar), and this residue is essential for catalytic activity of the enzyme (13Fujiwara N. Fujii T. Fujii J. Taniguchi N. Biochem. J. 1999; 340: 439-444Crossref PubMed Scopus (54) Google Scholar, 14Lee S.R. Bar-Noy S. Kwon J. Levine R.L. Stadtman T.C. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2521-2526Crossref PubMed Scopus (219) Google Scholar, 15Gasdaska J.R. Harney J.W. Gasdaska P.Y. Powis G. Berry M.J. J. Biol. Chem. 1999; 274: 25379-25385Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). One of the first indications that multiple TR forms occur in mammalian cells was immunoblot analyses of various human TR preparations. An enzyme possessing TR activity that was isolated from a human lung adenocarcinoma cell line NCI-H441 failed to react with antibodies specific for rat liver TR1 (11Tamura T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1006-1011Crossref PubMed Scopus (466) Google Scholar), whereas TR isolated from a human T-cell line JPX9 reacted with these antibodies (12Gladyshev V.N. Jeang K.-T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (405) Google Scholar). Determination of internal peptide sequences of the T-cell TR demonstrated that this protein was TR1 (12Gladyshev V.N. Jeang K.-T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (405) Google Scholar). Unfortunately, the NCI-H441 enzyme was not sequenced, and in retrospect it seems possible, that this TR could have been encoded by a different gene. In addition to a TR form not reacting with anti-TR1 antibodies, subsequent studies identified two forms of TR1 isolated from either NCI-H441 or HeLa cells that showed a positive immunoblot signal with these antibodies (16Liu S.Y. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6138-6141Crossref PubMed Scopus (31) Google Scholar, 17Gorlatov S.N. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8520-8525Crossref PubMed Scopus (85) Google Scholar, 18Gorlatov S.N. Stadtman T.C. Arch. Biochem. Biophys. 1999; 369: 133-142Crossref PubMed Scopus (14) Google Scholar). Yet, these forms differed in catalytic activity, selenium content, and affinity for column matrices. In particular, forms that differed in the ability to bind to a heparin column were extensively characterized (16Liu S.Y. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6138-6141Crossref PubMed Scopus (31) Google Scholar, 17Gorlatov S.N. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8520-8525Crossref PubMed Scopus (85) Google Scholar, 18Gorlatov S.N. Stadtman T.C. Arch. Biochem. Biophys. 1999; 369: 133-142Crossref PubMed Scopus (14) Google Scholar). However, differences in either protein or gene sequences, or in post-translational modifications that were responsible for the observed changes in catalytic and chromatographic properties, were not reported. Recently, two additional thioredoxin reductases, TR2 and TR3, were identified that contained the conserved selenocysteine residue (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar, 6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (146) Google Scholar, 8Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 9Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). These enzymes had also other sequences essential for catalytic activity, including an N-terminal disulfide active center, NADPH- and FAD-binding domains and dimer interface sequences (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar, 6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (146) Google Scholar, 8Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 9Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The three TR enzymes showed >50% sequence identity, although TR1 and TR2 were closely related enzymes, while TR3 was a more evolutionary distant enzyme (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). TR3 (also called TRβ (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar) and TrxR2 (6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar)), like TR1, appeared to be ubiquitously expressed (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar, 6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (146) Google Scholar, 8Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 9Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). This enzyme was described as a mitochondrial thioredoxin reductase because it was shown to contain a mitochondrial signal peptide. In addition, this protein was localized in mitochondria by detecting various transiently expressed, tagged forms of TR3 and by immunoblot assays with antibodies specific for TR3 (5Gasdaska P.Y. Berggren M.M. Berry M.J. Powis G. FEBS Lett. 1999; 442: 105-111Crossref PubMed Scopus (98) Google Scholar, 6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Miranda-Vizuete A. Damdimopoulos A.E. Pedrajas J.R. Gustafsson J.A. Spyrou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (146) Google Scholar, 8Watabe S. Makino Y. Ogawa K. Hiroi T. Yamamoto Y. Takahashi S.Y. Eur. J. Biochem. 1999; 264: 74-84Crossref PubMed Scopus (69) Google Scholar, 9Miranda-Vizuete A. Damdimopoulos A.E. Spyrou G. Biochim. Biophys. Acta. 1999; 1447: 113-118Crossref PubMed Scopus (38) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). TR2 had not been extensively characterized nor its full amino acid sequence reported (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In the present report, we describe the occurrence of multiple forms of TR1 and TR3 and utilize genetic and biochemical techniques to characterize the basis for heterogeneity within animal TR preparations. We find differences in 5′-end cDNA sequences for mammalian TR1, mammalian TR3, and Drosophila TR that resulted from the alternative use of first exons. Male wild type and transforming growth factor α (TGFα)/c-myc double transgenic mice (6–15 months old) were maintained and labeled with 75Se, and their livers and liver tumors were dissected as described previously (19Gladyshev V.N. Factor V.M. Housseau F. Hatfield D.L. Biochem. Biophys. Res. Commun. 1998; 251: 488-493Crossref PubMed Scopus (113) Google Scholar). The coexpression of TGFα and c-Myc results in multiple liver tumor formation by 6 months of age. Three unlabeled wild type mouse livers (4 g) and a single 75Se-labeled wild type liver (0.5 g) were mixed and homogenized in 10 ml of 40 mmTris-HCl, pH 7.5, 100 mm NaCl, 1 mm EDTA, 0.6 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin (buffer A). The homogenate was centrifuged at 18,000 rpm for 20 min, and the supernatant was loaded directly onto a 3-ml ADP-Sepharose (Amersham Pharmacia Biotech) column. The column was washed extensively with 150 mm NaCl and 40 mm Tris-HCl, pH 7.5, and proteins were eluted with 1m NaCl in 40 mm Tris-HCl, pH 7.5. Protein fractions were tested by SDS-PAGE and immunoblot analyses with antibodies specific for TR1 and TR3 (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), pooled, and further analyzed by a two-dimensional gel electrophoresis. TR1 was also isolated from TGFα/c-myc tumors using the same procedure except that 5 unlabeled liver tumors (11.5 g) and one 75Se-labeled liver tumor (3.5 g) were homogenized in 15 ml of buffer A. TR1 was isolated from mouse liver, rat prostate, and a human T-cell line JPX9 according to a three-step procedure that involved DEAE-Sepharose (Amersham Pharmacia Biotech), ADP-Sepharose, and phenyl-HPLC columns (TosoHaas) as described previously (12Gladyshev V.N. Jeang K.-T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (405) Google Scholar). To obtain 75Se-labeled TR1, unlabeled mouse livers were mixed with a single 75Se-labeled wild type liver. Isolated proteins were analyzed by immunoblot assays with antibodies specific for TR1, TR2, and TR3 (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), protein staining with Coomassie Blue and by detecting 75Se with a PhosphorImager (Molecular Dynamics). To further fractionate the enzyme, TR1 preparation was dialyzed against 20 mm Tris-HCl, pH 7.0, and applied onto a heparin-HPLC column (TosoHaas). The proteins were eluted with a gradient of 20–500 mm Tris-HCl, pH 7.0.75Se was detected in fractions with a γ-counter. JPX9 cells were grown on an RPMI 1640 medium in the presence of 10% fetal bovine serum and metabolically labeled with 75Se as described previously (12Gladyshev V.N. Jeang K.-T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (405) Google Scholar). Human A431 cell line was grown as described (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and TRs were purified from sonicated crude extracts on an ADP-Sepharose column as described for mouse enzymes. Two-dimensional gel electrophoresis of 75Se-contining polypeptides was performed as described previously (20Appella E. Arnott D. Sakaguchi K. Wirth P.J. EXS (Exper. Suppl.). 2000; 88: 1-27PubMed Google Scholar). Briefly, 25 μl of each TR preparation (500 μg of total protein), which was partially purified on an ADP-Sepharose column, were resolved in the first dimension on an isoelectric focusing (IEF) gel, followed by separation in the second dimension on a 10% polyacrylamide gel under reducing conditions. The separated polypeptides were electroblotted on polyvinylidene difluoride membranes, the blots stained with Ponceau S, and the75Se-containing proteins detected on the dried membranes by the PhosphorImager analysis. TR samples purified by one-dimensional or two-dimensional gel electrophoresis were transferred onto polyvinylidene difluoride membranes and stained with either Ponceau S or Sulforodamine B. Bands were excised and digested with trypsin as described (21Sutton C.W. Pemberton K.S. Cottrell J.S. Corbett J.M. Wheeler C.H. Dunn M.J. Pappin D.J. Electrophoresis. 1995; 16: 308-316Crossref PubMed Scopus (99) Google Scholar). After digestion, peptides were extracted from the membrane with the addition of 10 μl of formic acid:ethanol (1:1) for 1 h at room temperature. 0.5 μl were sampled directly from the supernatant to the matrix-assisted laser desorption ionization (MALDI) plate, mixed with 0.5 μl of α-cyano-4-hydroxy cinnamic acid matrix (10 mg/ml in acetonitrile/trifluoroacetic acid, 0.1%) and allowed to air-dry before data collection. Spectra were acquired on a Voyager RP mass spectrometer using oxidized bovine insulin β chain as internal standard for calibration (21Sutton C.W. Pemberton K.S. Cottrell J.S. Corbett J.M. Wheeler C.H. Dunn M.J. Pappin D.J. Electrophoresis. 1995; 16: 308-316Crossref PubMed Scopus (99) Google Scholar, 22Zappacosta F. Borrego F. Brooks A.G. Parker K.C. Coligan J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6313-6318Crossref PubMed Scopus (94) Google Scholar). Mass spectrometric analysis of native or alkylated human TR proteins was performed on a API300 triple quadrupole mass spectrometer equipped with a Micro-IonSpray source. Prior to MS analysis proteins were purified by RP-HPLC on a narrow-bore Vydac C4 column (150 × 2.1 mm, 5 μm) using a linear gradient of 0–70% acetonitrile containing 0.1% trifluoroacetic acid and a flow rate of 250 μl/min. Samples were manually collected and directly injected into the mass spectrometer ion source by infusion at 1 μl/min. Reduction and alkylation of TRs was conducted in denaturing conditions (6 m guanidine HCl, pH 8.0) using iodoacetamide as an alkylating agent. Mouse cDNA clones (clone ID 607552, accession number AI662374, Stratagene mouse skin cDNA library; and clone ID 2064717, accession number AI789478, Sugano mouse kidney-mkia cDNA library) were purchased from Research Genetics and their insert sequences experimentally determined. The two clones overlapped for 510 nucleotides and the combined cDNA sequence was 3626 nucleotides long. SDS-PAGE, immunoblot, and isoelectrofocusing analyses were performed with electrophoretic supplies and gels from Novex. Immunoblot membranes were developed with a SuperSignal detection kit (Pierce) or an ECL detection kit (Amersham Pharmacia Biotech) using previously developed antibodies specific for TR1, TR2, and TR3 (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). We have previously demonstrated strict isozyme specificity of these antibodies (10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Nucleotide sequences were analyzed with BLAST programs (23Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar). Exons were predicted with a NetGene2 program. Mitochondrial signal peptides were predicted with the PSORT II or SignalP programs. To determine whether mouse TR1 occurs in multiple forms and if the distribution of these forms differs between normal and malignant cells, we partially isolated TRs from a pool of75Se-labeled wild type mouse livers and from a pool of75Se-labeled liver tumors developed in TGFα/c-myc double transgenic mice. To minimize potential losses of certain TR forms due to different elution profiles of these forms on columns commonly used in TR isolation procedures, we utilized only a single step, an affinity ADP-Sepharose chromatography, to purify TRs. When 75Se-labeled protein extracts obtained from either normal livers or liver tumors were fractionated on an ADP-Sepharose column, TR1, seen as a 75Se-labeled band (Fig.1 A, lanes 3 and 6), and TR3, which is masked by TR1 due to its lower abundance (6Lee S.R. Kim J.R. Kwon K.S. Yoon H.W. Levine R.L. Ginsburg A. Rhee S.G. J. Biol. Chem. 1999; 274: 4722-4734Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 10Sun Q.-A. Wu Y. Zappacosta F. Jeang K.-T. Lee B.J. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 1999; 274: 24522-24530Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), were separated from the remainder of 75Se-labeled proteins (Fig. 1 A). Two other major 75Se-labeled proteins that did not bind to the column were glutathione peroxidase 1 (GPx1) and glutathione peroxidase 4 (GPx4) (19Gladyshev V.N. Factor V.M. Housseau F. Hatfield D.L. Biochem. Biophys. Res. Commun. 1998; 251: 488-493Crossref PubMed Scopus (113) Google Scholar). Since we used identical procedures for fractionation of normal and tumor samples, comparison of GPx1 and other selenoprotein bands in normal (Fig. 1 A, lane 1) and tumor (Fig. 1 A, lane 4) samples suggested that the levels of GPx1 were decreased in tumors. A similar decrease was previously observed when normal livers from TGFα/c-myc mice were compared with liver tumors from the same mice (19Gladyshev V.N. Factor V.M. Housseau F. Hatfield D.L. Biochem. Biophys. Res. Commun. 1998; 251: 488-493Crossref PubMed Scopus (113) Google Scholar). On the other hand, the latter studies found that expression of TR1 was slightly increased in liver tumors relative to surrounding normal livers in TGFα/c-myc mice (19Gladyshev V.N. Factor V.M. Housseau F. Hatfield D.L. Biochem. Biophys. Res. Commun. 1998; 251: 488-493Crossref PubMed Scopus (113) Google Scholar). Fig. 1 B shows immunoblot analyses of mouse TR1 and TR3 enriched from normal wild type and transgenic malignant livers on an ADP-Sepharose column. Proteins isolated from these two sources migrated similarly on SDS-PAGE. TR1 had a molecular mass of ∼57 kDa, whereas TR3 migrated as an ∼55-kDa protein. Although TR1 is more abundant than TR3 in liver (16Liu S.Y. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6138-6141Crossref PubMed Scopus (31) Google Scholar), the ratio of these enzymes was approximately the same in wild type and tumor samples (Fig. 2 B), suggesting that expression of TR3, like that of TR1 (but in contrast to GPx1), was unchanged or perhaps somewhat elevated during malignant transformation in TGFα/c-myc mice.Figure 2Two-dimensional gel electrophoresis of mouse TR preparations. Wild type liver and TGFα/c-myc liver tumor TR preparations were subjected to two-dimensional gel electrophoresis analysis, followed by detection of 75Se by PhosphorImager analysis (A-D) and staining of proteins with Ponceau S (E and F). Panels A andB show full two-dimensional gels, whereas panels C-F show enlarged areas that correspond to 75Se signals on panels A and B. Panels A, C, andE show TR1 isolated from wild type livers, and panels B, D, and F, from liver tumors.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to the mouse samples, we analyzed TR1 isolated from rat prostate by a standard three-column procedure (Fig. 1 B, lane 3), and a TR preparation isolated from a human epidermoid A431 cell line using a single ADP-Sepharose column (Fig. 1 B, lane 4). Interestingly, although the major form of human TR1 migrated as a 57-kDa band, human TR3 was present as a mixture of 55–57-kDa species. While mouse TR preparations isolated from normal and tumor samples migrated as single species and exhibited similar electrophoretic properties on SDS-PAGE gels (Fig. 1 B), this electrophoretic technique is often insufficient to resolve minor differences within preparations. Therefore, to further test protein preparations shown in Fig. 1 B, lanes 1 and 2, we separately analyzed normal and tumor TRs by two-dimensional gel electrophoresis. Comparison of 75Se and protein profiles on two-dimensional gels for normal and tumor samples suggested that all signals shown in Fig. 2corresponded to TR1. TR3 was not detected on two-dimensional gels because it was present in lower levels than TR1 and exhibited a different isoelectric point. The two-dimensional gel electrophoresis analysis (Fig. 2) revealed dramatic heterogeneity within TR1 preparations from normal and tumor samples. TR1 bands were separated on two-dimensional gels according to both charge and mass. Nine representative bands from two-dimensional gels for normal and tumor samples (Fig. 2, E andF) were digested with trypsin. Tryptic digests were analyzed by MALDI time-of-flight mass-spectrometry (MALDI-TOF MS). Experimentally determined peptide masses matched to the peptide masses predicted from the tryptic digestion of a deduced mouse TR1 sequence (see details of mouse TR1 sequences below) and covered >50% of the protein sequence (cysteine-containing and N-terminal peptides were not detected). No post-translational modifications within TR1 that could potentially contribute to different mobilities of TR1 forms on two-dimensional gels were detected. In addition, no significant differences in tryptic peptide maps were found between normal and tumor samples. In addition to multiple TR1 forms that were resolved by two-dimensional gel electrophoresis (Fig. 2) but migrated as a single 57-kDa band on SDS-PAGE gels (Fig. 1 B), we detected TR1 forms that significantly differed from the 57-kDa isoforms. We noted that longer exposure of 75Se signals detected with a PhosphorImager and analysis of anti-TR1 immunoblot signals often produced two additional bands, ∼67 and ∼110 kDa. These bands partially co-purified with the 57-kDa forms and were present as minor forms in apparently homogeneous enzyme preparations. The 67- and 110-kDa species did not cross-react with anti-TR2 and anti-TR3 antibodies. Migration properties of the 110-kDa minor band corresponded to that of the TR1 homodimer, although the biochemical basis for the possible dimer formation under these conditions is not clear. The possible origin of the 67-kDa band is discussed below. We further tested if the extensive heterogeneity observed within mouse TR1 preparations, occurs in human TR1 preparations. For this purpose, we selected a human T-cell line, J" @default.
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