Post‐translational Processing of the Alternative Neuropeptide Precursor Encoded by the FMRFamide Gene in the Pulmonate Snail Lymnaea stagnalis

Abstract

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, Ill-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 spectromettythe 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. lmmunoreactive neurons included the single identifiable visceral white interneuron (VWI or VD4), a key cell of the cardiorespiratory network.

Insights into early molluscan neuronal development through studies of transmitter phenotypes in embryonic pond snails

“…The posterior and peripheral locations of the first somata along with their extensive and anteriorly directed fibres not only challenge current thoughts about probable inductive cues and mechanisms of axonal navigation important in the development of the molluscan nervous system, but also raise numerous new questions that must be addressed in future studies. Importantly, these basic findings have now been confirmed by Voronezhskaya and Elekes (1997) using both antibodies raised against SEEPLY, another peptide sequence encoded by the FMRFamide gene, and two FMRFamide-related peptides known as the acidic peptide ACP (Santama et al, 1996) and the pentapeptide EFLRIamide (Santama et al, 1995b). Recently, Dickinson and Croll (2000a) also reported that the c-EFAP cell, but not the r-or l-EFAP cells, could be labelled using antibodies raised against neurocalcin, a member of the calmodulin superfamily of proteins (Dyer et al, 1996).…”

Expression of FMRFamide gene encoded peptides by identified neurons in embryos and juveniles of the pulmonate snail Lymnaea stagnalis

“…Antisera specifically recognizing different members of the FMRFa family have been successfully raised. Those antibodies allow specific labeling of the neurons containing pentapeptide EFLRIamide (EFLRIa, Santama et al 1995b), the non-FMRFamide-like 22-amino-acid peptide SEQPDVDDYLRDVVLQSEELY ("SEEPLY," Santama et al 1993), and the 35-amino-acid SDPFFRFGKQQVATDDSGELDDEILSRVSDDDKNI ("acidic peptide," Santama et al 1996). The presence and distribution of these peptides in the CNS of adult Lymnaea has been described in detail.…”

“…The presence and distribution of these peptides in the CNS of adult Lymnaea has been described in detail. EFLRIa and SEEPLY are expressed in the same cells where exon II is transcribed (type 1 mRNA) and acidic peptide is produced in all cells that transcribe type 2 mRNA Santama et al 1993Santama et al , 1995aSantama et al , 1995bSantama et al , 1996.…”

Detailed Review Paper (DRP) on Molluscs Life-cycle Toxicity Testing

“…Most work has been restricted to relatively few species of opisthobranch and pulmonate gastropods, particularly Aplysia and Lymnaea while there is some information on the bivalve Mytilus (de Lange and van Minnen, 1998;Lagadic et al, 2007;Nassel, 1996;Norekian and Satterlie, 1997;Santama et al, 1996). Lagadic et al (2007), who are the prime source for this paragraph, describe the considerable amount of information about the neuroendocrine control of reproduction in the hermaphrodite Lymnaea stagnalis.…”

“…The FMRFamide family is now the best known, characterised and most widespread of all invertebrate neuroendocrine hormones. Its activities in molluscs are manifest in a range of physiological processes of which the regulation of heartbeat is the best characterised (Greenberg and Price, 1983;Favrel et al, 1998;Nassel, 1996;Santama et al, 1996;Suzuki et al, 1997;Tensen et al, 1998).…”