Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.
The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.
Mandel
S J
SJ
Howard Hughes Medical Institute Laboratory, Brigham and Women's Hospital, Boston, Massachusetts.
Berry
M J
MJ
Kieffer
J D
JD
Harney
J W
JW
Warne
R L
RL
Larsen
P R
PR
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D015698:Genomic Library; D006801:Humans; D066298:In Vitro Techniques; D007453:Iodide Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D017279:Selenocysteine / Q000235:genetics*; D013961:Thyroid Gland / Q000201:enzymology; D014162:Transfection
D000345:Affinity Labels; D011994:Recombinant Proteins; D017279:Selenocysteine; D004247:DNA; D007453:Iodide Peroxidase