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A New Human Selenium-containing Protein

PURIFICATION, CHARACTERIZATION, AND cDNA SEQUENCE*
Open AccessPublished:April 10, 1998DOI:https://doi.org/10.1074/jbc.273.15.8910
      Selenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent75Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting that the T-cell 15-kDa selenoprotein contains a selenocysteine residue encoded by TGA. Post-translational processing of the N-terminal portion of the predicted gene product to give the 15-kDa protein was suggested on the basis of molecular mass, amino acid analysis, and immunoblot assays of the purified protein. The 3′-untranslated region (UTR) of the gene encoding the 15-kDa protein contained a sequence that is very similar to the canonical selenocysteine-inserting sequence element. Computer analysis of transcript map data bases indicated that this gene was located on human chromosome 1. Its coding sequence showed no homology to known protein-encoding genes. The 15-kDa protein gene was expressed as mRNA in a wide range of tissues, with increased levels in the thyroid, parathyroid, and prostate-derived cells as evidenced by searches of partial cDNA sequences in public data bases. Genes corresponding to the 15-kDa selenocysteine-containing protein were found in mice and rats, while the corresponding genes inCaenorhabditis elegans and Brugia malayicontained a cysteine codon in place of TGA. The discovery of a new human selenoprotein provides an additional example of the role of selenium in mammalian systems.
      Selenium has been implicated in immunological function and many other biological processes through various nutritional and biochemical studies (
      • Stadtman T.C.
      ,
      • Hatfield D.L.
      • Gladyshev V.N.
      • Park J.
      • Park S.I.
      • Chittum H.S.
      • Baek H.J.
      • Carlson B.A.
      • Yang E.S.
      • Moustafa M.E.
      • Lee B.J.
      ). This trace element is a natural component of several prokaryotic and eukaryotic proteins. Although selenium occurs in prokaryotic proteins either as a cofactor or as a selenocysteine residue, mammalian selenoproteins identified thus far contain selenium only in the form of selenocysteine, which is the 21st naturally occurring amino acid in protein. A selenocysteine tRNA that decodes UGA has been found in all life kingdoms, suggesting that the use of UGA as a codon for selenocysteine is widespread in nature (
      • Hatfield D.L.
      • Gladyshev V.N.
      • Park J.
      • Park S.I.
      • Chittum H.S.
      • Baek H.J.
      • Carlson B.A.
      • Yang E.S.
      • Moustafa M.E.
      • Lee B.J.
      ). The special conserved stem-loop structures in the 3′-untranslated regions of mammalian selenoprotein mRNAs are essential for recognition of UGA as a codon for selenocysteine, rather than a codon for termination of translation (
      • Low S.
      • Berry M.
      ).
      Of the eleven genes encoding different selenocysteine-containing proteins that have been found thus far in mammals, four encode various glutathione peroxidases (reviewed in Refs.
      • Sunde R.A.
      and
      • Ursini F.
      • Maiorino M.
      • Brigelius-Flohe R.
      • Aumann K.D.
      • Roveri A.
      • Schomburg D.
      • Flohe L.
      ), three encode different thyroid hormone deiodinases (
      • Berry M.J.
      • Banu L.
      • Larsen P.R.
      ,
      • Croteau H.W.
      • Davey J.C.
      • Galton V.A.
      • St. Germain D.L.
      ,
      • St. Germain D.L.
      • Schwartzman R.A.
      • Croteau W.
      • Kanamori A.
      • Wang Z.
      • Brown D.D.
      • Galton V.A.
      ), and others encode thioredoxin reductase (
      • Gasdaska P.Y.
      • Gasdaska J.R.
      • Cochran S.
      • Powis G.
      ), selenophosphate synthetase 2 (SPS2) (
      • 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.
      ), selenoprotein P (
      • Hill K.E.
      • Burk R.F.
      ), and selenoprotein W (
      • Vendeland S.C.
      • Beilstein M.A.
      • Yeh J.-Y.
      • Ream W.
      • Whanger P.D.
      ).
      Selenocysteine is located at the active center and is directly involved, or at least implicated, in the catalytic reactions catalyzed by glutathione peroxidases, thyroid hormone deiodinases, and selenophosphate synthetase 2. Thioredoxin reductase contains selenocysteine (
      • Tamura T.
      • Stadtman T.C.
      ) in a novel C-terminal Gly-Cys-Sec-Gly redox motif (
      • Gladyshev V.N.
      • Jeang K.-T.
      • Stadtman T.C.
      ). This center has been implicated in the peroxidase reaction catalyzed by the enzyme (
      • Gladyshev V.N.
      • Jeang K.-T.
      • Stadtman T.C.
      ) and in a redox interaction with the N-terminal redox disulfide (
      • Arscott L.D.
      • Gromer S.
      • Schirmer R.H.
      • Becker K.
      • Williams C.H.
      ), although further studies are necessary to prove the suggested essential role of selenocysteine in this protein. Selenoprotein P, a protein of unknown function, is unusual in that it contains ten selenocysteine residues. The function of a selenoprotein W also remains unknown.
      In the present study, the isolation of the 15-kDa protein and its gene sequence are described which characterize this protein as a member of a class of mammalian selenoproteins. Although this new human selenoprotein does not have homology to known proteins, genes putatively encoding this protein occur in other mammals as well as in nematodes and plants. Preliminary data on this protein have been presented elsewhere (
      • Gladyshev V.N.
      • Jeang K.-T.
      • Hatfield D.L.
      • Stadtman T.C.
      ).

      Acknowledgments

      The initial part of this work, including the protein purification, was done while V. N. Gladyshev was working in the laboratory of Dr. Thressa Stadtman (Laboratory of Biochemistry, NHLBI, National Institutes of Health). We thank her for support during the course of this work as well as for helpful suggestions during the manuscript writing. We also thank Harvard Microchem for highly professional protein microchemistry services, Dr. Rodney Levine for helpful discussions and MALDI analysis, Dr. Francesca Zappacosta for performing electrospray mass-spectrometry, Dr. Valery Bliskovsky for help with initial computer analyses, and Drs. Valentina Factor and Michael Jensen for helpful discussions and advice.

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