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 (d 2-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 d 2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d 2-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.
Prohormone-derived peptides, such as neuropeptides and peptide hormones, undergo a number of post-translational modifications (PTMs) that greatly impact their biological functions. One unusual PTM is the enzyme-catalyzed conversion of a residue from the l-stereoisomer to the d-stereoisomer to generate a d-amino acid–containing peptide (DAACP). DAACPs have been identified in animals across several phyla, acting as neuropeptides, hormones, and toxins. Despite considerable progress in developing methods to identify DAACPs, l- to d-residue isomerization remains difficult to detect de novo because this PTM does not change a peptide's mass or chemical composition. As a result, relatively little is known about the full biological consequences of isomerization on peptide function.
In most cases of l- to d-residue isomerization, the DAACP appears to be significantly more biologically active than its all-l-residue diastereomer (for a list of known cell–cell signaling DAACPs; Table S1). Consistent with this observation, the only identified receptors for cell–cell signaling DAACPs, the achatin-like neuropeptide receptors, are activated by their DAACP ligands and not by their all-l-residue analogs. In fact, 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. However, these bioactivity tests would fail to identify peptides in which both the DAACP and the all-l-residue isomer have similar activities, and this possibility is rarely explored. To our knowledge, outside of the achatin receptors found in several animals, no additional receptors for DAACPs have been identified and studied. As a result, it is unclear whether l- to d-residue isomerization is always a critical mediator of receptor activation, as is the case in the achatin-like neuropeptide system. In addition to altering receptor activity, d-residues can also decrease the susceptibility of peptides to protease action relative to their all-l-residue counterparts, although few studies have directly examined how l- to d-residue isomerization influences the stability of cell–cell signaling DAACPs to endogenous proteases.
Allatotropins are a family of prohormone-derived peptides that play roles in multiple biological processes. The first allatotropin peptide was identified from the insect Manduca sexta, where it was isolated based on its stimulation of juvenile hormone secretion. Since this initial discovery, allatotropins have been found to act in a variety of biological processes in neuropeptide, hormonal, and myoactive functions. Allatotropin-related peptides have been identified in many animals across phyla, including insects, mollusks, and annelids, although the roles of these peptides remain poorly understood. Allatotropin receptors have been characterized from insects and annelids and predicted in several additional phyla based on sequence similarity. Furthermore, phylogenetic analyses show that allatotropin prohormones and their receptors are evolutionarily related to orexin prohormones and receptors found in vertebrates. We recently reported an allatotropin-related peptide (hereafter referred to as ATRP) from the model organism Aplysia californica and identified physiological roles for the all-l-residue form of this peptide as a regulator of peripheral and central nervous system functions in the feeding network. A receptor for ATRP, or to our knowledge any other molluscan allatotropin-related peptide, has not been experimentally verified.
Predatory cone snails produce venom consisting of hundreds of compounds, including disulfide-constrained conotoxins and neuropeptide/hormone-like compounds, which act on a variety of molecular targets. Several peptides from cone snail venom have been identified as DAACPs, including the peptide Conomap-Vt (here called d 2-Conp; see Fig. 1) from Conus vitulinus, which shows high sequence similarity to allatotropin and ATRP (see Fig. 1). The similarity in sequences of the DAACP d 2-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. Here, we determined that ATRP exists in the Aplysia CNS as two diastereomers, l-ATRP and d 2-ATRP. We examined the signaling of l- and d 2-ATRP through their newly identified receptor and in physiological experiments and studied their relative susceptibilities to action by endogenous proteases. Overall, 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.
Primary sequences of peptides relevant to this study. dF, highlighted in bold type, indicates a d-Phe residue. A red G indicates a 13 C-Gly residue containing one 13 C atom at the carbonyl carbon, incorporated to distinguish synthetic peptides from endogenous peptides.
The high sequence similarity between ATRP and d 2-Conp led us to hypothesize that ATRP may exist as two diastereomers bearing either l- or d-Phe at position 2 (l- or d 2-ATRP; Fig. 1). To determine the chirality of ATRP in the Aplysia CNS, we examined the chromatographic properties, mass spectral profiles, and protease stabilities for peptides extracted from ganglia by LC–MS and LC–MS/MS. Consistent with the hypothesis that ATRP exists in multiple diastereomers, LC–MS analysis of cerebral ganglia peptide extracts identified two major peaks with different retention times that each matched the predicted m/z value for ATRP of 511.9 for z = +3 (Fig. 2, A and B). The MS/MS fragment spectra for the two peaks were very similar (Fig. 2C), and the majority of the peaks present in these MS/MS spectra matched fragments predicted for ATRP and matched those of synthetic 13 C-d 2-ATRP (Fig. S1). Consistent with previously reported expression revealed by Northern blotting, ATRP was detected in all major ganglia by LC–MS (Fig. S2) and showed similar behavior.
Analysis of endogenous ATRP by LC–MS from cerebral ganglia extracts. A, extracted ion chromatogram for the ATRP parent mass (m/z = 511.9 ± 0.1, z = +3). B, representative MS spectra from peaks 1 and 2 from A. C, representative MS/MS fragmentation spectra for the 511.9 precursor ion from peaks 1 and 2 from A. See Fig. S1 for assignments of MS/MS fragments to predicted ATPR fragments and comparison to synthetic 13 C-d 2-ATRP.
To relatively rapidly test whether one of the peaks identified as 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 all-l-residue peptides but degrades DAACPs bearing a d-residue near their N terminus at a reduced rate. In fact, we have previously used APM digestion as a screening tool to identify putative DAACPs in complex biological mixtures. When incubated with APM for 15 h, we found that the later-eluting ATRP peak was completely degraded (Fig. 3). In contrast, the earlier-eluting ATRP peak resisted degradation over this time, consistent with the hypothesis that this species bears a d-residue near its N terminus (Fig. 3 and Fig. S3).
Extracted ion chromatograms for the ATRP parent mass (m/z = 511.9) from cerebral ganglia extracts incubated in the absence (Extracts) or presence (Extracts + APM) of 0.37 unit/ml aminopeptidase M, for 15 h at 37 °C. Peaks labeled with red asterisks correspond to ATRP, as judged by MS and MS/MS spectra. The peak at ∼27 min was identified through bioinformatics analysis as SFDRITDSSFRGF-NH 2 (from XP_005098921.1), which is not known to exist as a DAACP.
To directly test our hypothesis that the two major peaks present in the chromatograms corresponded to the two predicted diastereomers of ATRP, we spiked into cerebral ganglia extracts 13 C-l-ATRP or 13 C-d 2-ATRP, ATRP analogs bearing two 13 C-glycine residues (Fig. 1
