Type 3 lodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme.
Type 3 lodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme.
复制标题
DOI：
10.1172/jci118299
复制
发表时间：
1995-11
期刊：
影响因子：
--
通讯作者：
D. Salvatore;S. Low;M. Berry;A. Maia;John W. Hamey;W. Croteau;Donald;L. Germain;P. Larsen
中科院分区：
文献类型：
--
作者：
D. Salvatore;S. Low;M. Berry;A. Maia;John W. Hamey;W. Croteau;Donald;L. Germain;P. Larsen
3 型碘甲状腺原氨酸脱碘酶 (D3) 催化 T4 和 T3 转化为无活性代谢物。它在胎盘中高度表达，因此可以在整个妊娠期间调节循环胎儿甲状腺激素浓度。我们克隆并表达了 2.1 kb 人胎盘 D3 cDNA，其编码 32 kD 蛋白，T3 5 脱碘的 Km 为 1.2 nM，反向 T3 5' 脱碘的 Km 为 340 nM。该反应需要 DTT，且不受 6n-丙基硫氧嘧啶抑制。我们通过用溴乙酰 [125I]T3 特异性标记蛋白质来定量瞬时表达的 D3。 T3 5' 脱碘的 Kcat/Km 比是反向 T3 5' 脱碘的 1,000 倍以上。人 D3 是一种硒酶，证据如下：(a) 在位置 144 处存在符合读框的 UGA 密码子，(b) 在 D3 cDNA 转染的细胞中合成 32-kD 75Se 标记的蛋白质，以及 (c) 在 mRNA 的 3' 非翻译区中存在其表达所需的硒代半胱氨酸插入序列元件。 D3 硒代半胱氨酸插入序列元件比 1 型脱碘酶或谷胱甘肽过氧化物酶基因中的插入序列元件更有效，表明将硒代半胱氨酸掺入到该酶中具有高度优先性。这种酶从非洲爪蟾蝌蚪到人类的保存意味着在胚胎发育过程中调节甲状腺激素失活的重要作用。
Type 3 iodothyronine deiodinase (D3) catalyzes the conversion of T4 and T3 to inactive metabolites. It is highly expressed in placenta and thus can regulate circulating fetal thyroid hormone concentrations throughout gestation. We have cloned and expressed a 2.1-kb human placental D3 cDNA which encodes a 32-kD protein with a Km of 1.2 nM for 5 deiodination of T3 and 340 nM for 5' deiodination of reverse T3. The reaction requires DTT and is not inhibited by 6n-propylthiouracil. We quantitated transiently expressed D3 by specifically labeling the protein with bromoacetyl [125I]T3. The Kcat/Km ratio for 5 deiodination of T3 was over 1,000-fold that for 5' deiodination of reverse T3. Human D3 is a selenoenzyme as evidenced by (a) the presence of an in frame UGA codon at position 144, (b) the synthesis of a 32-kD 75Se-labeled protein in D3 cDNA transfected cells, and (c) the presence of a selenocysteine insertion sequence element in the 3' untranslated region of the mRNA which is required for its expression. The D3 selenocysteine insertion sequence element is more potent than that in the type 1 deiodinase or glutathione peroxidase gene, suggesting a high priority for selenocysteine incorporation into this enzyme. The conservation of this enzyme from Xenopus laevis tadpoles to humans implies an essential role for regulation of thyroid hormone inactivation during embryological development.