Oxidation products of tryptophan and proline in adipokinetic hormones – artefacts or posttranslational modifications?

(1) Background: Adipokinetic hormones (AKHs) regulate important physiological processes in insects. AKHs are peptides of 8-10 amino acids with blocked termini and Trp in position 8. In more than half of the known AKHs, Pro occupies position 6. Posttranslational modifications have been found for AKHs including hydroxyproline ([Hyp6]) and kynurenine ([Kyn8]). Our re-cent data from studies with cockroaches, flies and beetles suggest that the Hyp- and Kyn-containing AKHs occur more often than originally thought. Here, we investigate, if this is the case and if such modified AKHs are natural or artefactual. (2) Methods: From crude extracts of the corpora cardiaca (CC) of various insect species, AKHs were analyzed using liquid chro-matography coupled to high-resolution mass spectrometry (LC-MS). A few [Hyp6]-AKHs were synthesized and tested in an in vivo metabolic assay in cockroaches. Freshly dissected Periplaneta americana and Blaberus atropos CCs (with precautions taken against oxidation) were immediately subjected to peptide extraction and LC-MS analysis. B. atropos CC were also placed into a depo-larizing saline (high potassium concentration) and the AKHs released into the saline were ob-served via LC-MS. (3) Results: Hyp was detected in several decapeptides from cockroaches that structurally resemble the hypertrehalosemic peptide of Blaberus discoidalis (i.e. Bladi-HrTH) and that bear the amino acid recognition sequence for the enzyme prolyl 4-hydroxylase, viz. SPG. The modified form accompanies the "parent" AKH at concentrations from <1% up to 7% and is thus not always well detected depending on sample availability and AKH concentration. In the CC extract of the American cockroach P. americana, we found some indication for Hyp of the endog-enous octapeptides Peram-CAH-I and -II at low spectral quality, which may be associated with a weak enzyme-substrate recognition sequence in these peptides (SPN, TPN). The [Hyp6]-AKHs of B. atropos were clearly present also in fresh CC preparations that were kept under conditions that exclude oxidation processes, and were shown to be releasable from the CC ex vivo. The available synthetic [Hyp6]-containing peptides tested positively in a hypertrehalosemic bioassay. Moreo-ver, from the CC extract of the termite Kalotermes flavicollis, hydroxyprolination was detected for the octapeptide Manto-CC and in the grasshopper, Tetrix subulata, [Hyp6]-Tetsu-AKH was seen. Finally, the singly and doubly oxidized Trp- as well as the Kyn-containing form of Nicve-AKH were found at similarly low concentrations in CC extracts from various species of the burying beetle genus Nicrophorus, but not in the extracts from the examined cockroaches, termite and grasshopper. (4) Conclusions: Both the formation of Hyp and Kyn could be the result of endog-enous processes, but in, particular, Trp oxidation is known to occur easily during sample han-dling. From the MS data on cockroach CCs protected from oxidative processes, we are confident that the detected Hyp forms of the decapeptides code-named Bladi- and Blaat-HrTH are endog-enous and present in the CCs. In general, artefactually generated peptide modifications need to be carefully distinguished from biological forms.