Post-translational processing of the alternative neuropeptide precursor encoded by the FMRFamide gene in the pulmonate snail Lymnaea stagnalis

The neuropeptide gene encoding FMRFamide-like peptides in the pulmonate mollusc Lymnaea is subject to alternative splicing that generates cell-specific expression of distinct sets of peptides in the CNS. In this paper, we analyse the post-translational processing of the alternative protein precursor encoded by the exon I, III-V transcript (type 2 transcript). We raised anti-peptide antisera specific to distinct segments of the precursor in order to address the pattern of endoproteolytic cleavages, specifically around the tetrabasic site RRKR. We first showed that not all peptides predicted by the precursor structure are generated as final steady-state products. We then identified a novel peptide by biochemical purification, amino acid sequencing and mass spectrometry-the 35 amino acid SDPFFRFGKQQVATDDSGELDDEILSRVSDDDKNI, which we termed the acidic peptide, previously not predicted on the basis of the precursor structure, This novel peptide, abundant in the snail brain (0.7 pmol per central nervous system), includes the N-terminal sequence SDPFFRF, which was previously considered to be a variant of the known heptapeptide SDPFLRFamide, also encoded within the same protein precursor. We showed by in situ hybridization and immunocytochemistry that the acidic peptide is produced in all cells that transcribe type 2 FMRFamide mRNA. We mapped the expression of this novel peptide in the CNS and localized it mainly in three identifiable neuronal clusters-the E, F and B groups of cells-and some additional neurons, all situated in three of the eleven central ganglia. Immunoreactive neurons included the single identifiable visceral white interneuron (VWI or VD4), a key cell of the cardiorespiratory network.