An ascidian homolog of vertebrate iodothyronine deiodinases
An ascidian homolog of vertebrate iodothyronine deiodinases
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DOI：
10.1210/en.2003-1248
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发表时间：
2004-03-01
期刊：
影响因子：
4.8
通讯作者：
Kuiper, GGJM
中科院分区：
文献类型：
--
作者：
Shepherdley, CA;Klootwijk, W;Kuiper, GGJM
在所有种类的脊椎动物中，激素原T-4到T-3的脱碘是甲状腺激素作用的一个重要活化步骤。在体内实验中，原脊索动物中可能存在碘甲腺原氨酸脱碘酶活性。最近几种海鞘的基因组和转录本的分子克隆允许进一步调查甲状腺相关的海鞘过程。从罗氏盐芥（Halocynthia roretzi，hrDx）克隆的cDNA与脊椎动物的碘甲腺原氨酸脱碘酶基因序列具有显著的同源性（30%的氨基酸同一性），包括在其活性位点存在一个可能编码硒代半胱氨酸（SeC）的读框内UGA密码子。因为不能确定3'非翻译区（UTR）是否含有SeC掺入所必需的SeC插入序列（SECIS）元件，所以产生hrDx编码序列和大鼠脱碘酶SECIS元件的嵌合表达载体，以及含有完整hrDx cDNA的表达载体。用这些载体转染COS、CHO和HEK细胞，并在细胞匀浆中测量脱碘酶活性。外环脱碘酶活性检测使用T-4和反向T-3作为底物，和活性增强的还原辅因子二硫苏糖醇的存在下。在20 - 30 ℃（pH 6-7）温育期间酶活性最佳，并且被金-硫代葡萄糖强烈抑制。盐辛西娅脱碘酶似乎是高米氏常数（K-m）酶（K-m反向T-3，2 μ M;和K-m T-4，4 μ M）。脱碘酶活性完全丧失后，取代SeC残基在推定的催化中心由半胱氨酸或丙氨酸。全长hrDx cDNA的转染产生脱碘酶活性，证实了SECIS元件在3 'UTR中的存在，如SECIS程序所揭示的。总之，我们的研究结果表明，第一次，海鞘碘甲腺原氨酸外环脱碘酶的存在。这就提出了一个假设，即在原索动物中，激素原T-4是通过酶催化外环脱碘而被激活为T-3的。
In all classes of vertebrates, the deiodination of the prohormone T-4 to T-3 represents an essential activation step in thyroid hormone action. The possible presence of iodothyronine deiodinase activity in protochordates has been demonstrated in vivo. Recent molecular cloning of the genomes and transcripts of several ascidian species allows further investigation into thyroid-related processes in ascidians. A cDNA clone from Halocynthia roretzi (hrDx) was found to have significant homology (30% amino acid identity) with the iodothyronine deiodinase gene sequences from vertebrates, including the presence of an in-frame UGA codon that might encode a selenocysteine (SeC) in the active site. Because it was not certain that the 3' untranslated region (UTR) contained a SeC insertion sequence (SECIS) element essential for SeC incorporation, a chimeric expression vector of the hrDx coding sequence and the rat deiodinase SECIS element was produced, as well as an expression vector containing the intact hrDx cDNA. COS, CHO, and HEK cells were transfected with these vectors, and deiodinase activity was measured in cell homogenates. Outer-ring deiodinase activity was detected using both T-4 and reverse T-3 as substrates, and activity was enhanced by the presence of the reductive cofactor dithiothreitol. The enzyme activity was optimal during incubation between 20 and 30 C (pH 6-7) and was strongly inhibited by gold-thioglucose. The Halocynthia deiodinase appears to be a high Michaelis-Menten constant (K-m) enzyme (K-m reverse T-3, 2 muM; and K-m T-4, 4 muM). Deiodinase activity was completely lost upon the substitution of the SeC residue in the putative catalytic center by either cysteine or alanine. Transfection of the full-length hrDx cDNA produced deiodinase activity confirming the presence of a SECIS element in the 3'UTR, as revealed by the SECISearch program. In conclusion, our results show, for the first time, the existence of an ascidian iodothyronine outer-ring deiodinase. This raises the hypothesis that, in protochordates, the prohormone T-4 is activated by enzymatic outer-ring deiodination to T-3.