Department of Biosphere Sciences, School of Human Sciences, Kobe College 4-1, Okadayama, Nishinomiya-shi, Hyogo 662-8505, Japan.
Aquat Toxicol. 2011 Oct;105(3-4):708-16. doi: 10.1016/j.aquatox.2011.09.008. Epub 2011 Sep 17.
A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [(3)H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K(d))=2.14 nM. Competitive binding assays showed that the IC(50) values for ponasterone A, muristerone A, 20-hydroxyecdysone, and α-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC(50) values for two dibenzoylhydrazine ligands (tebufenozide and chromafenozide) were >1.0 × 10(5)nM. The intra- and inter-assay coefficient of variation values for the IC(50) values of 20-hydroxyecdysone were 14.7% (n=5) and 16.1% (n=8), respectively. Our results indicate that the binding assay with a mixture of abEcRdef and abUSPdef can be used to screen compounds with a broad range of binding affinities for crustacean EcRs.
人们已经做出了全球性的努力，以建立可以识别内分泌干扰化学品 (EDCs) 对无脊椎动物影响的筛选和测试方法。我们研究的目的是开发一种用于糠虾（Americamysis bahia）蜕皮激素受体（EcR）的体外受体结合测定法。我们克隆了糠虾 EcR cDNA（2888 个核苷酸）和 ultraspiracle（USP）cDNA（2116 个核苷酸），并确定它们分别编码长度为 570 和 410 个氨基酸的预测蛋白。这些蛋白的推导氨基酸序列在 EcR 中与其他节肢动物共享 36-71%的同源性，在 USP 中共享 44-65%的同源性。系统发育分析表明，糠虾 EcR 与另一种糠虾 Neomysis integer 的 EcR 一起被归类为一个独立的簇，并且该簇很早就从甲壳类动物的其他分类群中分化出来。然后，我们在大肠杆菌中表达了糠虾 EcR（abEcRdef）和 USP（abUSPdef）的配体结合结构域（DEF 区）作为谷胱甘肽 S-转移酶（GST）融合肽。通过亲和层析纯化融合肽并去除 GST 标签后，我们将肽进行配体-受体结合测定。[(3)H]-ponasterone A 单独与 abEcRdef 或 abUSPdef 肽不结合，但与 abEcRdef/abUSPdef 混合物强烈结合，解离常数（K(d)）为 2.14 nM。竞争性结合测定表明，ponasterone A、muristerone A、20-羟基蜕皮酮和α-蜕皮酮的 IC(50)值分别为 1.2、1.9、35 和 1200 nM，而两种二苯甲酰肼配体（tebufenozide 和 chromafenozide）的 IC(50)值均大于 1.0×10(5)nM。20-羟基蜕皮酮的 IC(50)值的内和间分析变异系数值分别为 14.7%（n=5）和 16.1%（n=8）。我们的结果表明，使用 abEcRdef 和 abUSPdef 的混合物进行结合测定可以用于筛选对甲壳类动物 EcR 具有广泛结合亲和力的化合物。
