Five Neuropeptide Ligands Meet One Receptor: How Does This Tally? A Structure-Activity Relationship Study Using Adipokinetic Bioassays With the Sphingid Moth, Hippotion eson

Abstract

Adipokinetic hormones (AKHs) play a major role in mobilizing stored energy metabolites during energetic demand in insects. We showed previously (i) the sphingid moth Hippotion eson synthesizes the highest number of AKHs ever recorded, viz. five, in its corpus cardiacum: two octa- (Hipes-AKH-I and II), two nona- (Hipes-AKH-III and Manse-AKH), and one decapeptide (Manse-AKH-II), which are all active in lipid mobilization ( 1 ). (ii) Lacol-AKH from a noctuid moth showed maximal AKH activity in H. eson despite sequence differences and analogs based on Lacol-AKH with modifications at positions 2, 3, 8, or at the termini, as well as C-terminally shortened analogs had reduced or no activity ( 2 ). Here we report on N-terminally shortened and modified analogs of the lead peptide, as well as single amino acid substitutions at positions 1, 4, 5, 6, and 7 by an alanine residue. Ala 1 and Glu 1 instead of pGlu are not tolerated well to bind to the H. eson AKH receptor, whereas Gln 1 has high activity, suggesting it is endogenously cyclized. Replacing residue 5 or 7 with Ala did not alter activity much, in contrast with changes at position 4 or 6. Similarly, eliminating pGlu 1 , Leu 2 , or Thr 3 from Lacol-AKH severely interfered with biological activity. This indicates that there is no core peptide sequence that can elicit the adipokinetic effect and that the overall conformation of the active peptide is required for a physiological response. AKHs achieve a biological action through binding to a receptor located on fat body cells. To date, one AKH receptor has been identified in any given insect species; we infer the same for H. eson . We aligned lepidopteran AKH receptor sequences and note that these are very similar. The results of our study is, therefore, also applicable to ligand-receptor interaction of other lepidopteran species. This information is important for the consideration of peptide mimetics to combat lepidopteran pest insects.

Insight into mosquito GnRH-related neuropeptide receptor specificity revealed through analysis of naturally occurring and synthetic analogs of this neuropeptide family

“…Similar observations have been made with AKH analogs tested using both in vivo and in vitro assays whereby complete removal of the N-terminal pyroglutamate abolishes activity of the AKH analog whereas the alternatively blocked N-terminus (i.e. N-Acetyl-Ala), or even simply glutamate or glutamine that can spontantenously undergo cyclization (Schilling et al, 2008), were shown to have improved activity, albeit lower than the native AKH (Caers et al, 2016; Gäde and Hayes, 1995; Marco and Gäde, 2019b). Lastly, although alanine substitution for valine in the second position led to a relatively minor effect on ACPR activation (~70-fold reduced activity), the internally truncated analog omitting valine within the N-terminal region elicited no activation of the ACPR, indicating the spacing between the more critical residues, namely pyroglutamate and polar threonine, is essential for ACP peptide activity on the ACPR.…”

“…In light of the high fidelity of the three GnRH-related systems (Hansen et al, 2010; Oryan et al, 2018; Wahedi and Paluzzi, 2018), the current study set out to examine for the first time the ligand structure-activity relationship for an insect ACP receptor. This is of utmost importance in order to gain insight on the specific structural features of the ACP system given that extensive studies have been carried out previously on AKH receptors in a variety of species using natural and synthetic analogs of this neuropeptide family (Caers et al, 2012; Fox and Reynolds, 1991; Gäde et al, 2000; Gäde, 1992; Gäde, 1993; Gäde et al, 2016; Keeley et al, 1991; Lee and Goldsworthy, 1996; Lee et al, 1996; Lee et al, 1997; Marco and Gäde, 2015; Marco and Gäde, 2019b; Poulos et al, 1994; Velentza et al, 2000; Ziegler et al, 1991; Ziegler et al, 1998). By examining the activity of a variety of ACP analogs on the activation of the A. aegytpi ACP receptor (ACPR), including single alanine substitutions along with truncated or extended analogs, a few observations can be highlighted.…”

“…Observations correlating with these findings have been made in various studies utilizing in vitro heterologous assays as well as in vivo bioassays monitoring lipid- and/or carbohydrate-mobilizing actions of AKH analogs. Specifically, removal or replacement of the critical aromatics that are archetypal features of AKH neuropeptides found in positions four and eight demonstrates the absolute requirement of these features for receptor activation in heterologous systems or in vivo biological activity (Gäde, 1992; Gäde, 1993; Marco and Gäde, 2015; Marco and Gäde, 2019b).…”

Structural diversity of adipokinetic hormones in the hyperdiverse coleopteran Cucujiformia

“…Harax‐AKH in the Coccinelloidea and Trica‐AKH in the Tenebrionoidea (see Table ). Interestingly, the latter 2 peptides share the C‐terminal amidated tetrapeptide completely (STGW amide) or mostly (STDW amide) with the ancestral Schgr‐AKH‐II; a substitution at position 7 has previously not shown much difference in AKH receptor activation in vitro (Caers et al, ; Marco, Verlinden, Vanden Broeck, & Gäde, ; Marchal et al, ) or in vivo (Gäde & Hayes, ; Marco & Gäde, ).…”

“…In vivo structure‐activity studies with P. americana to test the biological response to various naturally occurring AKHs (Gäde, ) and a series with single amino acid substitutions have been performed (Gäde & Hayes, ). These studies demonstrated that the AKH receptor of P. americana can bind a fair number of different AKHs, but this may be explained by the fact that more than one AKH peptide structure is produced and active in this species, while all evidence seems to indicate that insects have only one AKH receptor, regardless of the number of AKH structures produced (Marco & Gäde, ). In H. abietis on the other hand, in which Peram‐CAH‐I is the sole conspecific AKH, five other naturally occurring AKHs were tested in an in vitro system in which the receptor was expressed with a G protein, and none of the heterospecific ligands could attain 40% or higher activation of the receptor when applied in the nM range (Marchal et al, ).…”