Mandel S J, Berry M J, Kieffer J D, Harney J W, Warne R L, Larsen P R
Howard Hughes Medical Institute Laboratory, Brigham and Women's Hospital, Boston, Massachusetts.
J Clin Endocrinol Metab. 1992 Oct;75(4):1133-9. doi: 10.1210/jcem.75.4.1400883.
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.
I型5'碘甲状腺原氨酸脱碘酶（5'DI）催化T4脱碘生成具有生物活性的激素T3，且其产量占很大比例。我们最近克隆了大鼠5'DI的互补DNA（cDNA），该cDNA含有稀有氨基酸硒代半胱氨酸，并用其筛选人肝和肾cDNA文库以鉴定人5'DI cDNA克隆。据此，我们构建了编码功能性5'DI的cDNA。2222个碱基对的人5'DI cDNA比人肝、肾和甲状腺Northern印迹中2.4千碱基的杂交带短约200个核苷酸，这是由于缺少5'非翻译序列和多聚A尾。假设第382位的UGA密码子编码硒代半胱氨酸，推导的氨基酸序列编码一个28.7千道尔顿的蛋白质，与人的序列高度同源（相似性88%）。我们在COS - 7细胞中瞬时表达5'DI以确定其编码一种功能性酶并研究其动力学。结果显示rT3的脱碘具有饱和性（Ka 0.52±0.04μmol/L，Vmax 63.2±16.4 pmol min-1 mg-1）。T4和金硫葡糖是rT3脱碘的竞争性抑制剂。6 - n - 丙基硫氧嘧啶（PTU）是rT3脱碘的非竞争性抑制剂（与rT3相比）和竞争性抑制剂（与二硫苏糖醇相比）。6 - n - 丙基硫氧嘧啶抑制T4向T3的转化。用溴乙酰 - 125I - T3对瞬时转染人5'DI cDNA的COS - 7细胞进行标记，显示出一个28千道尔顿的蛋白质。这表明在人以及大鼠信使RNA中，UGA编码硒代半胱氨酸，翻译在核苷酸754至756处的UAA密码子处终止。反式T3和金硫葡糖（100 nmol/L）阻断瞬时表达的人和大鼠5'DI蛋白的溴乙酰 - 125I - T3标记。这些结果表明人5'DI是一种硒蛋白，类似于大鼠的酶。鉴于先前已证明硒原子在该蛋白催化脱碘中起关键作用，我们得出结论，这种微量元素对人类正常甲状腺激素作用至关重要。
