LC/MS/MS identification of 20-hydroxyecdysone in a scorpion ( Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors

• Published in: Insect biochemistry and molecular biology Vol. 41; no. 12; pp. 932 - 937
• Main Authors: Miyashita, Masahiro, Matsushita, Kaori, Nakamura, Shunsuke, Akahane, Sho, Nakagawa, Yoshiaki, Miyagawa, Hisashi
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2011
• Subjects: 20-Hydroxyecdysone; adults; agonists; Animals; Arthropoda; Chromatography, Liquid; ecdysone; Ecdysteroids; ecdysterone; Ecdysterone - isolation & purification; Ecdysterone - metabolism; EcR; Female; hatching; Hemolymph - metabolism; high performance liquid chromatography; hormone receptors; Liocheles australasiae; mass spectrometry; molting; Nymph - chemistry; Nymph - metabolism; nymphs; Poa; ponasterone A; Scorpion; Scorpiones; Scorpions - chemistry; Scorpions - metabolism; Tandem Mass Spectrometry; Tebufenozide; Ecdysteroids; PoA; Scorpion; E; MS; 20E; 20-Hydroxyecdysone; MaA; RXR; Liocheles australasiae; USP; Tebufenozide; EcR; LC; PTTH
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2011.09.002

Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand–receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of [3H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 micromolar, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.