Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.
Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.
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DOI：
10.1210/jcem.75.4.1400883
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发表时间：
1992-10
期刊：
影响因子：
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通讯作者：
S. Mandel;M. Berry;J. Kieffer;J. Harney;R. Warne;P. Larsen
中科院分区：
文献类型：
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作者：
S. Mandel;M. Berry;J. Kieffer;J. Harney;R. Warne;P. Larsen
I型5'碘甲状腺原氨酸脱碘酶（5' DI）催化T4脱碘为生物活性激素T3，并占其产生的重要部分。我们最近克隆了大鼠5' DI的互补DNA（cDNA），其中含有稀有氨基酸硒代半胱氨酸，并利用其筛选人类肝脏和肾脏cDNA库以鉴定人类5' DI cDNA克隆。由此，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、肾和甲状腺的北方印迹中的2.4-腺苷酸酶杂交带短约200个核苷酸，因为缺少5'非翻译序列和聚腺苷酸尾。推导的氨基酸序列编码28.7千道尔顿的蛋白质，假设UGA密码子在382位编码硒代半胱氨酸，并且与大鼠高度同源（88%相似性）。我们在COS-7细胞中瞬时表达了5' DI，以确定其编码功能酶并研究其动力学。这些显示rT 3的饱和脱碘（Ka 0.52 +/- 0.04 mumol/L和Vmax 63.2 +/- 16.4 pmol min-1 mg-1）。T4和金硫葡萄糖是rT 3脱碘的竞争性抑制剂。6-n-丙基硫氧嘧啶（PTU）是rT 3脱碘的非竞争性抑制剂（与rT 3）和竞争性抑制剂（与二硫苏糖醇）。6-正丙基硫氧嘧啶抑制T4向T3转化。用溴乙酰基-125 I-T3标记瞬时转染人5' DI cDNA的COS-7细胞，证明了28千道尔顿的蛋白质。这表明在人类以及大鼠信使RNA中，UGA编码硒代半胱氨酸，翻译终止于核苷酸754至756处的UAA密码子。反向T3和硫代葡萄糖金（100 nmol/L）阻断瞬时表达的人和大鼠5' DI蛋白的溴乙酰基-125 I-T3标记。这些结果表明，人的5' DI是一种硒蛋白，类似于大鼠的酶。鉴于先前证明的关键作用，硒原子在催化脱碘的这种蛋白质，我们得出结论，这种微量元素是必不可少的正常甲状腺激素的行动在人。
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.