Kuiper George G J M, Klootwijk Willem, Morvan Dubois Ghislaine, Destree Olivier, Darras Veerle M, Van der Geyten Serge, Demeneix Barbara, Visser Theo J
Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
Endocrinology. 2006 Jul;147(7):3519-29. doi: 10.1210/en.2005-0711. Epub 2006 Apr 6.
In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T(3) in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T(4) or reverse T(3) (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K(m)) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T(4) (K(m) 0.5 microm) and rT3 (K(m) 0.5 microm) and inner-ring deiodinase activity with T(4) (K(m) 0.4 microm). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC(50) > 1 mm) but was sensitive to gold thioglucose (IC(50) 0.1 mum) and iodoacetate (IC(50) 10 microm). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T(4) and rT3 (K(m) about 4 microm) and was highly sensitive to 6-PTU (IC(50) 2 microm). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.
在诸如林蛙属和非洲爪蟾属的青蛙中，若没有功能性甲状腺，变态发育就不会发生。先前的研究表明，青蛙的协调发育需要组织和阶段依赖性的II型和III型碘甲状腺原氨酸脱碘酶表达模式，以便在变态发育的适当阶段在组织中获得所需水平的细胞内T(3)。到目前为止，在蝌蚪中尚未检测到I型碘甲状腺原氨酸脱碘酶（D1），其定义为具有微摩尔范围内的米氏常数（K(m)）值且对6-丙基-2-硫尿嘧啶（6-PTU）敏感的T(4)或反式T(3)（rT3）外环脱碘酶（ORD）活性。我们从脑组织中获得了一个非洲爪蟾D1 cDNA克隆。该克隆的完整序列（1.1 kb，包括聚腺苷酸尾）编码一个由252个氨基酸残基组成的开放阅读框，与其他脊椎动物的D1酶具有高度同源性。核心催化中心包括一个由UGA编码的硒代半胱氨酸残基，3'非翻译区（约300 nt）包含一个硒代半胱氨酸插入序列元件。用含有全长cDNA的表达载体转染细胞导致匀浆中产生显著的脱碘酶活性。该酶对T(4)（K(m) 0.5微摩尔）和rT3（K(m) 0.5微摩尔）表现出ORD活性，对T(4)（K(m) 0.4微摩尔）表现出内环脱碘酶活性。重组非洲爪蟾D1对6-PTU的抑制基本不敏感（IC(50) > 1 mM），但对金硫葡萄糖（IC(50) 0.1微摩尔）和碘乙酸（IC(50) 10微摩尔）敏感。由于非洲爪蟾D1中硒代半胱氨酸下游第2位的残基是脯氨酸，而在所有克隆的对PTU敏感的D1酶中是丝氨酸，我们制备了非洲爪蟾D1的Pro132Ser突变体。突变酶对T(4)和rT3表现出显著增强的ORD活性（K(m)约4微摩尔），并且对6-PTU高度敏感（IC(50) 2微摩尔）。在非洲爪蟾的肝脏、肾脏、脑和肠道中几乎检测不到天然D1活性，但在幼蛙脑以及成年肝脏和肾脏中观察到显著的D1 mRNA表达。这些结果表明非洲爪蟾组织中存在一种对6-PTU不敏感的D1酶，但其在蝌蚪变态发育过程中的作用仍有待确定。
