Characterization of GdFFD, a D-amino acid-containing neuropeptide that functions as an extrinsic modulator of the Aplysia feeding circuit

Abstract

During eukaryotic translation, peptides/proteins are created using L-amino acids. However, a D-amino acid-containing peptide (DAACP) can be produced through post-translational modification via an isomerase enzyme. General approaches to identify novel DAACPs and investigate their function, particularly in specific neural circuits, are lacking. This is primarily due to the difficulty in characterizing this modification and due to the limited information on neural circuits in most species. We describe a multipronged approach to overcome these limitations using the sea slug Aplysia californica. Based on bioinformatics and homology to known DAACPs in the land snail Achatina fulica, we targeted two predicted peptides in Aplysia, GFFD, similar to achatin-I (GdFAD versus GFAD, where dF stands for D-phenylalanine), and YAEFLa, identical to fulyal (YdAEFLa versus YAEFLa), using stereoselective analytical methods, i.e. MALDI MS fragmentation analysis and LC-MS/MS. Although YAEFLa in Aplysia was detected only in an all L-form, we found that both GFFD and GdFFD were present in the Aplysia CNS. In situ hybridization and immunolabeling of GFFD/GdFFD-positive neurons and fibers suggested that GFFD/GdFFD might act as an extrinsic modulator of the feeding circuit. Consistent with this hypothesis, we found that GdFFD induced robust activity in the feeding circuit and elicited egestive motor patterns. In contrast, the peptide consisting of all L-amino acids, GFFD, was not bioactive. Our data indicate that the modification of an L-amino acid-containing neuropeptide to a DAACP is essential for peptide bioactivity in a motor circuit, and thus it provides a functional significance to this modification.

Keywords: Analytical Chemistry; Aplysia; D-Amino Acid-containing peptides; Epimer; Invertebrates; MALDI-TOF/TOF MS; Mass Spectrometry (MS); Peptides; Post-translational Modification; Stereoselective.

FIGURE 1.

Sequence and localization of expression of apALNP and apFLNP, and peptide profiles from apALNP-positive cells. A and B, precursor protein sequences of apALNP and apFLNP (35). Predicted peptides are underlined, and peptides that have been verified chemically in this report are shown in boldface. A, apALNP includes the predicted peptide GFFD; B, apFLNP includes the predicted peptide YAEFLa. All peptides from apFLNP are amidated. C and D, in situ hybridization showed that apALNP (GFFD) mRNA was expressed in a cluster of neurons near the root of pedal nerve 7 (P7) and pedal nerve 9 (P9) on the dorsal surface of the pedal ganglion. C, left pleural-pedal ganglia; D, right pleural-pedal ganglia. In both panels, the pedal commissure (PC) is facing upward, and the pleural ganglion (Pleural g.) is facing to either the left or right. Calibration, 200 μm. E and F, peptide profile detected by MALDI-TOF MS from the cells stained positive for cDNA corresponding to apALNP (GFFD/GdFFD) in in situ hybridization. E, several of the larger molecular weight peptide products from apALNP are detected from these cells by accurate mass match. Achatin-like (86–106)- and achatin-like (24–51)-NH₂ were detected at 44 and 12 ppm accuracy, respectively. F, detection of peptide products from apALNP using MALDI-TOF MS settings optimized for detection of smaller molecular weight species. Several products, including GFFD/GdFFD and YYGS, are detected from the cells by accurate mass matching. GFFD/GdFFD and YYGS were detected at 206 and 818 ppm accuracy, respectively.

FIGURE 2.

Localization of expression of apALNP and apFLNP. A, in situ hybridization shows that apFLNP (YAEFLa) mRNA is expressed on the ventral surfaces of the cerebral ganglion. A single neuron is located in the E cluster near the root of the cerebral-buccal connective (CBC and white arrow, only present at the right. The left CBC was accidentally cut.), and a cluster of small neurons is located between the roots of the cerebral-pedal connective (CPe) and cerebral-pleural connective (CPl). White arrows point to the labeled cells at the left side. AT, anterior tentacular nerve; UL, upper labial nerve. Calibration, 500 μm. B and C, immunocytochemistry with GdFAD antibody. B, dorsal surface of the pedal ganglion. The cluster of positive neurons matched in situ hybridization with apALNP, suggesting that GdFAD antibody can label apALNP-positive neurons. C, buccal ganglion. Consistent with in situ hybridization, there are no GdFAD-positive somata in the buccal ganglion, but there are GdFAD-positive fibers and varicosities, e.g. in the neuropile, commissure (C), and the radula nerve (RN). Presumably, the staining suggests that apALNP-positive fibers and varicosities are in the buccal ganglion. Calibration, 500 μm.

FIGURE 3.

GFFD and GdFFD can be distinguished by their different fragmentation patterns using MALDI-TOF/TOF MS. A, overlaid representative MS/MS spectra from GFFD standard (black), GdFFD standard (blue), and isolated pedal neurons (red), normalized to the y2 ion as % intensity. B, each epimer, i.e. GFFD or GdFFD, produces a unique ratio using two of the most discriminative fragment ions, y2 and b3. The ratio measured from the isolated neurons appears to be closer to the value for GdFFD, suggesting that the neurons contain both GFFD and GdFFD.

FIGURE 4.

Statistical analysis demonstrates that peptides from isolated neurons are more similar to GdFFD, such that GdFFD is likely the major contributor to m/z 484. 9 in MALDI-TOF MS. A, PCA demonstrates the clustering of GdFFD standard (inverted triangle), GFFD standard (upright triangle), and biological samples (circles) based substantially on two principal components (PCs), with the biological samples clustering more closely with the GdFFD standard based primarily on PC1. Each symbol corresponds to the fragmentation profile of one sample. The average peak area from three acquisitions of each sample, normalized to the sum of the peak areas of all ions in the respective MS/MS spectrum, is used for PCA analysis. B, seven fragment ions are the variables used in the PCA analysis. Their impact regarding to the two principal components are shown in the figure. There are two distinctive groups regarding PC1. Fragment ions such as b3 and the Fi ion are in one group showing similar impact in group forming, and fragment ion y2 is another. The differences in these two groups of variables have contributed to assigning the two clusters. C, table of Eigenvalues shows that only PC1 and PC2 have an Eigenvalue higher than 1, and these two are the components that explain the majority of the variations. Cum, cumulative.

FIGURE 5.

GFFD and GdFFD are detected from neurons isolated from the pedal ganglia from A. californica using LC-MS. A, LC separation of GFFD and GdFFD. B, MS/MS fragmentation spectrum of endogenous GFFD (21.4 min). C, MS/MS fragmentation spectrum of endogenous GdFFD (24.3 min). D, retention times on the LC and fragmentation patterns (E and F) of both peaks match well with those of synthetic peptides, which confirm the identification. E, MS/MS fragmentation spectrum of synthetic GFFD (21.4 min). F, MS/MS fragmentation spectrum of synthetic GdFFD (24.3 min).

FIGURE 6.

GdFFD is detected in whole buccal ganglia extracts from A. californica using LC-MS. A, LC separation of buccal ganglia peptides. Arrow points to a peak at the retention time matching that of the GdFFD standard. B, MS/MS of GdFFD (31.4 min). The fragmentation patterns and retention time of GdFFD matches the synthetic peptide in C, which confirms the identification.

FIGURE 7.

YAEFLa, but not YdAEFLa, is detected using LC-MS to analyze A. californica cerebral ganglia peptide extracts. A, solid arrow points to the retention time for YAEFLa, and dotted arrow points to the retention time for YdAEFLa. Although the separation in A used a C18 column, the retention times were too close for confident assignment, and a hypercarb column was used as the stationary phase for the separations in B, where the solid arrow points to a peak matching the retention time of the YAEFLa standard (37.7 min) and the dotted arrow points to a retention time expected for YdAEFLa (41.0 min), although no m/z 641.3 is observed at the mass spectrum at this retention time. C, MS/MS fragmentation spectrum of YAEFLa. D, MS/MS fragmentation spectrum of synthetic YAEFLa (37.7 min). E, MS/MS fragmentation spectrum of synthetic YdAEFLa (41.0 min). The MS/MS fragmentation pattern of the endogenous YAEFLa matches the expected fragmentation pattern of Y(l/d)AEFLa at a retention time matching the YAEFLa standard, confirming that this peptide is predominantly in the all l-form.

FIGURE 8.

GdFFD induces cyclic activity in the buccal central pattern generator. Each cycle of programs consists of a protraction (open bar, marked by I2 nerve activity) − retraction (closed bar, marked by hyperpolarization of B65 interneuron) sequence. A, control: at rest, there was little activity in the buccal CPG protraction interneurons B40 and B65, radula closure motoneuron B8, or the I2 nerve. B, during the perfusion of 10⁻⁵ m GdFFD, the feeding network became cyclically active. The motor programs induced were mostly egestive because B8 activity occurred during the protraction phase (represented by B65 bursting). Consistently, B40, a neuron important for ingestive programs, was not active, although it received cyclic synaptic inputs. C, after washout of the peptide, the preparation gradually returned to the inactive state, beginning with the trace, 4.2 min after the start of the washout. D, summary data (n = 4) showing the effects of 10⁻⁶ and 10⁻⁵ m GdFFD on the activity of the buccal CPG. The frequency of the motor programs is the number of cycles per min measured over a period of 7 min or more in each experimental condition. Both 10⁻⁶ and 10⁻⁵ m GdFFD increased the frequency of spontaneously occurring motor programs. Bonferroni post hoc test (with selected comparisons between peptide applications versus the control (Ctrl) or the wash) is as follows: *, p < 0.05; **, p < 0.01. Error bars represent S.E. E and F, effects of GFFD and GdFFD on motor programs elicited by the command-like interneuron CBI-2. B34 is a buccal protraction interneuron active during protraction (open bar, marked by I2 nerve activity); CBI-2 is an interneuron that triggers programs by activating protraction interneurons in the buccal ganglion, which, in turn, activate the retraction neurons. In addition, CBI-2 receives inhibitory feedback during retraction (65), and it is not or only weakly active during retraction. Thus, single cycle motor programs were elicited by stimulation of CBI-2 at 9 Hz throughout the protraction phase with inter-trial intervals of 1 min. E1–3, representative examples. E1, control: CBI-2 stimulation induced an ingestive motor program, in which B8 was predominantly active during retraction (filled bar, marked by hyperpolarization of B34 interneuron). E2, perfusion of 10⁻⁵ m GFFD had no obvious effect on the CBI-2-elicited program. E3, washout of the peptide. F1–3, representative examples. F1 and F3 are the same as E1 and E3, except with the perfusion of (F2) 10⁻⁶ m GdFFD, which increased B8 firing during protraction and reduced B8 firing during retraction. It also decreased the duration of protraction. F4–7, group data (n = 4) showing the effects of GdFFD on (F4) protraction duration, (F5) retraction duration, (F6) B8 activity during protraction, and (F7) B8 activity during retraction. Bonferroni post hoc test is as follows: **, p < 0.01; ***, p < 0.001.