The hypertrehalosaemic neuropeptide conformational twins of cicadas consist of only l-amino acids: are they cis–trans isomers?

It is known for almost 25 years that the corpora cardiaca (neurosecretory glands) of cicadas synthesize two isobaric peptides with hypertrehalosaemic activity denominated Placa-HrTH-I and II. Both decapeptides have the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn amide) and mass, but differ in their chromatographic retention time. The slightly more hydrophobic peptide, Placa-HrTH-II, co-elutes with the synthetic peptide of the same sequence and is less active in biological assays than Placa-HrTH-I. Ion mobility separation in conjunction with high-resolution mass spectrometry detected the differing structural feature between both peptides in the region Pro6-Ser7-Trp8. Here, it was shown that Placa-HrTH-I co-eluted with a synthetic peptide containing d-Pro in position 6, while dextrorotatory amino acid residues in positions 7 and 8 could be excluded in this way. Amino acid hydrolysis followed by chiral analysis using a relative of Marfey’s reagent was then used to validate the presence of d-Pro in Placa-HrTH-I. Interestingly, this experiment unambiguously proved both the absence of d-Pro and the presence of l-Pro in Placa-HrTH-I. Racemization as a reason for the structural differences of the twin adipokinetic hormones was hence ruled out and cis–trans isomerism as the likely alternative came into focus. It remains to be investigated if Pro6 in cis-conformation is indeed present and responsible for the increased bioactivity of Placa-HrTH-I.