De Novo Molecular Modeling and Biophysical Characterization of Manduca sexta Eclosion Hormone

Abstract

Eclosion hormone (EH) is an integral component in the cascade regulating the behaviors culminating in insect emergence from the old exoskeleton. Little is known regarding the EH solution structure, consequently, we utilized a computational approach to generate a hypothetical structure for Manduca sexta EH. The de novo algorithm exploited the restricted conformational space of disulfide bonds (Cys14-Cys38, Cys18-Cys34, and Cys21-Cys49) and predicted secondary structure elements to generate a thermodynamically stable structure characterized by 55% helical content, an unstructured N-terminus, a helical C-terminus, and a solvent exposed loop containing Trp28 and Phe29. Both the strain and pseudo energies of the predicted peptide compare favorably with those of known structures. The 62 amino acid peptide was synthesized, folded, assayed for activity, and structurally characterized to confirm the validity of the model. The helical content is supported by circular dichroism and hydrogen-deuterium exchange mass spectrometry. Fluorescence emission spectra and acrylamide quenching are consistent with the solvent exposure predicted for Trp28, which is shielded by Phe29. Furthermore, thermodynamically stable conformations that deviated only slightly from the predicted Manduca EH structure were generated in silico for the Bombyx mori and Drosophila melanogaster EHs indicating that the conformation is not species dependent. In addition, the biological activity of known mutants and deletion peptides were rationalized with the predicted Manduca EH structure and we found that, based on sequence conservation, functionally important residues map to two conserved hydrophobic clusters incorporating the C-terminus and the first loop.

In silico Neuropeptidome of Female Macrobrachium rosenbergii Based on Transcriptome and Peptide Mining of Eyestalk, Central Nervous System and Ovary

“…In our study, the Mro-EH transcript was discovered in both the eyestalk and CNS transcriptomes. Based on multiple sequence alignment, the Mro-EH mature peptide displays six conserved cysteine residues that may be responsible for the formation of three-disulfide bridges [ 81 , 82 ]. RT-PCR showed that the Mro-EH gene was expressed in the brain and thoracic ganglia, but not in the other tissues, including the eyestalk, probably due to low or no expression.…”

Genomics and Peptidomics of Neuropeptides and Protein Hormones Present in the Parasitic Wasp Nasonia vitripennis

“…Interestingly, an alignment of EH sequences from various insects shows that the Nasonia EH contains only two of the three disulfide bridges usually present in EHs ( Figure 6). 42,43 ETH. In insects, ecdysis triggering hormone (ETH) is expressed in Inka cells and initiates molting.…”

Ecdysis Triggering Hormone, Eclosion Hormone, and Crustacean Cardioactive Peptide Play Essential but Different Roles in the Molting Process of Mud Crab, Scylla paramamosain

“…Even the initiation of the molting process has started, there is no guarantee that the animal will molt successfully as the fulfillment of the subsequent events must be straightly followed. In insects, neuropeptides that induce molting behavior include ecdysis-triggering hormone (ETH) (Shi et al, 2019a;Minh Nhut et al, 2020;Shen et al, 2021), eclosion hormone (EH) (Hull et al, 2009;Zhou et al, 2017;Scott et al, 2020), crustacean cardioactive peptide (CCAP) (Veelaert et al, 1997;Arakane et al, 2008;Jackson et al, 2009), etc.…”

“…Eclosion hormones (EH) is a neuropeptide that influences several aspects of pupal-adult ecdysis as well as larval-larval ecdysis (Hull et al, 2009;Scott et al, 2020). It was first characterized in the moth Bombyx mori as a brain neuropeptide (Truman and Riddiford, 1970).…”

“…In this study, we have monitored the expression of several key molting-related genes ETH, EH and CCAP that induce the molting process the crab (Fort et al, 2007;Hull et al, 2009;Shen et al, 2021). Also, we have developed in vivo assays in juvenile crabs during the premolt stage of the molting crab.…”