Aplysia allatotropin-related peptide and its newly identified d-amino acid–containing epimer both activate a receptor and a neuronal target

James W Checco,Guo Zhang,Wang-Ding Yuan,Zi-Wei Le,Jian Jing,Jonathan V Sweedler

doi:10.1074/jbc.ra118.004367

Abstract

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

Highlights

L- to D-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals
To relatively rapidly test whether one of the peaks identified as allatotropin-related peptide (ATRP) may contain a D-residue, we incubated Aplysia cerebral ganglia peptide extracts with aminopeptidase M (APM), a relatively nonspecific peptidase that rapidly degrades most allL-residue peptides but degrades D-amino acid– containing peptide (DAACP) bearing a D-residue near their N terminus at a reduced rate
We have identified a novel DAACP, D2-ATRP, from the CNS of Aplysia, and have identified a GPCR, named apATRPR, which is activated by both L- and D2-ATRP

Summary

To whom correspondence may be addressed

Dept. of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801. E-mail: jsweedle@ illinois.edu. Most DAACPs have been identified based on studies in which the synthetic all-L-residue peptide fails to recapitulate the biological activity of the isolated endogenous compound. In addition to altering receptor activity, D-residues can decrease the susceptibility of peptides to protease action relative to their all-L-residue counterparts [3, 6, 16, 17], few studies have directly examined how L- to D-residue isomerization influences the stability of cell– cell signaling DAACPs to endogenous proteases [15]. The similarity in sequences of the DAACP D2-Conp in the cone snail venom and ATRP in the Aplysia CNS led us to hypothesize that allatotropin-related peptides in some species may undergo L- to D-residue isomerization. Our results show that that L- to D-residue isomerization in cell– cell signaling DAACPs is not always critical for receptor activation, demonstrating that even “well characterized” peptides may exist as multiple diastereomers and that L- to D-residue isomerization could be more widespread than previously thought

Results

Discussion

Experimental procedures