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

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.