Suzuki Shunsuke, Kasai Kentaro, Yamauchi Kiyoshi
Department of Biological Science, Graduate School of Science, Shizuoka University, Shizuoka 422-8529, Japan.
Department of Biological Science, Graduate School of Science, Shizuoka University, Shizuoka 422-8529, Japan; Green Biology Research Division, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
Gen Comp Endocrinol. 2015 Jun-Jul;217-218:43-53. doi: 10.1016/j.ygcen.2015.04.006. Epub 2015 Apr 9.
Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR.
转甲状腺素蛋白（TTR）在脊索动物进化的早期阶段通过基因复制从祖先的5-羟基异尿酸水解酶（HIUHase）分化而来。为了阐明TTR作为细胞外甲状腺激素（TH）结合蛋白是如何参与甲状腺系统的，对重组小鳐鱼（Leucoraja erinacea）TTR的TH结合特性进行了研究。在氨基酸水平上，鳐鱼TTR与其他脊椎动物的TTR具有37%-46%的同源性。由于鳐鱼TTR在N端区域有一个独特的富含组氨酸的片段，因此可以通过镍亲和层析进行纯化。鳐鱼TTR是由14.5kDa亚基组成的46kDa同四聚体，对3,3',5-三碘-L-甲状腺原氨酸（T3）和一些卤代酚的亲和力比对L-甲状腺素高一个数量级。然而，鳐鱼TTR没有HIUHase活性。乙二胺四乙酸（EDTA）处理抑制了[（125）I]T3结合活性，而向经EDTA处理的TTR中添加Zn（2+）以Zn（2+）浓度依赖性方式恢复了[（125）I]T3结合活性。Scatchard分析显示存在两类T3结合位点，解离常数分别为0.24和17nM。然而，1mM EDTA完全消除了高亲和力位点，而剩余的低亲和力位点降低了结合能力。每个TTR的锌含量经定量为4.5-6.3。我们的结果表明，鳐鱼TTR具有紧密的Zn（2+）结合位点，这些位点对于T3结合至少高亲和力位点至关重要。Zn（2+）与N端富含组氨酸的片段结合可能在TTR早期进化过程中TH结合能力的获得或增强中起重要作用。
