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

Abstract

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 [(3)H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 μM, 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.

Phytochemical Analysis of Pfaffia glomerata Inflorescences by LC-ESI-MS/MS

“…In addition, the protonation of C25-OH followed by loss of water is favored due to the formation of a stable tertiary carbonium ion, which is also produced from the C14-OH [22,24]. Other characteristic fragment ions at m/z 371 and at m/z 165 were observed in the ESI-MS/MS spectrum obtained for peak 2 , as also reported in previous studies [23,26,27]. The fragment ion with m/z 371 was produced from cleavage between C-23 and C-24 in the side chain, followed by the loss of two molecules of water, as described by Destrez et al .…”

The Ecdysteroidome of Drosophila: influence of diet and development

“…Methods for LC-MS/MS detection of ecdysteroids have been developed (Blais et al, 2010;Hikiba et al, 2013;Li et al, 2006;Miyashita et al, 2011). However, crude larval extracts are difficult to analyze because of the abundance of other lipids.…”

cDNA cloning of <i>ecdysone receptor</i> (<i>EcR</i>) and <i>ultraspiracle</i> (<i>USP</i>) from <i>Harmonia axyridis</i> and <i>Epilachna vigintioctopunctata</i> and the evaluation of the binding affinity of ecdysone agonists to the <i>in vitro</i> translated EcR/USP heterodimers

“…Tebufenozide did not bind to the scorpion EcR. 4) In HaEcR and EvEcR, valines at 384 and 416 were also changed to methionine (M384) and threonine (T416), respectively. These differences may be the reason for the reduction of the binding affinity of DAHs to HaEcR and EvEcR.…”

“…3) Mostly in arthropods, 20E is used as a molting hormone, even in scorpions. 4) However, other ecdysteroids such as ecdysone, 5) makisterone A 6) and ponasterone A (PonA; Fig. 1) 7) are used as molting hormones in a few species.…”