Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.
Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3'-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.
Fujiwara
N
N
Department of Biochemistry and Molecular Biology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Fujii
T
T
Fujii
J
J
Taniguchi
N
N
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005979:Glutathione Peroxidase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D012643:Selenium / Q000172:deficiency; D017279:Selenocysteine / Q000378:metabolism*; D017384:Sequence Deletion; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium