PROCESSING OF THE FMRFAMIDE PRECURSOR PROTEIN IN THE SNAIL LYMNAEA-STAGNALIS - CHARACTERIZATION AND NEURONAL LOCALIZATION OF A NOVEL PEPTIDE, SEEPLY

In the pulmonate snail Lymnaea stagnalis, FMRFamide-like neuropeptides are encoded by a mufti-exon genomic locus which is subject to regulat ion at the level of mRNA splicing. We aim to understand the post-trans lational processing of one resulting protein precursor encoding the te trapeptide FMRFamide and a number of other putative peptides, and dete rmine the distribution of the final peptide products in the central ne rvous system (CNS) and periphery of Lymnaea. We focused on two previou sly unknown peptide sequences predicted by molecular cloning to be enc oded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and s tructural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDVVLQSEEPLY. The novel peptide is col ocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ h ybridization and immunocytochemistry. Its detection in heart excitator y motoneurons and in nerve fibres of the heart indicated that the nove l peptide may play a role, together with FMRFamide, in heart regulatio n in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 ami no acids), was at very low abundance in the CNS and was only occasiona lly detected. Our current findings, suggestive of a distinct pattern o f post-translational processing, allowed the reassessment of a previou sly proposed hypothesis that the two equivalent sequences in the Aplys ia FMRFamide gene constitute a molluscan homologue of vertebrate corti cotrophin releasing factor-like peptides.