Identification and tissue mapping of APGWamide-related peptides in Sepia officinalis using LC-ESI-MS/MS.

This paper demonstrates for the first time the occurrence of tetrapeptides related to APGWamide in the mollusk cephalopod Sepia officinalis. LC-ESI-MS/MS analysis allowed the identification of the APGWamide-related peptides predicted by the two genes cloned previously in Lymnaea stagnalis and in Mytilus edulis, as well as the dipeptide GWamide released from the processing of the tetrapeptides by a dipeptidyl aminopeptidase (DPAP). TPGWamide and GWamide appeared to be exclusively located in the CNS, and the APGWamide in both the CNS and the nerve endings. The RPGWamide and the KPGWamide were not detected by LC-ESI-MS/MS suggesting they could be totally processed into GWamide. The in vitro processing of the tetrapeptides into GWamide by optic lobe extract revealed a differential processing for each, with APGWamide (44.7%)>RPGWamide(24.3%)>KPGWamide(19.3%)>TPGWamide (11.7%). The tissue mapping results, together with the processing efficiency data suggest that the GWamide is mainly produced from the M. edulis gene products RPGWamide and KPGWamide.

Characterization of a novel Sepia officinalis neuropeptide using MALDI-TOF MS and post-source decay analysis.

A novel neuropeptide acting as a myosuppressor on esophagus, funnel and mantle muscular fibers has been isolated from the stellar ganglia of the mollusk cephalopod Sepia officinalis by means of HPLC analysis. Fractions were monitored using a myotropic bioassay. After three separation steps, MALDI-TOF spectrum revealed one main peak at m/z 756.6. The partial N-terminal and C-terminal digestions by exopeptidases followed by MALDI-TOF analysis allowed the determination of the nature of the two C-terminal and N-terminal amino acids. Post Source Decay fragmentation of the molecular ion accurately determined the following primary sequence: Val-Tyr-Ser-Ala-Pro-Tyr-Gly-OH. The mapping of this heptapeptide performed in ESI-MS revealed that its distribution is restricted to the stellar ganglia, the giant fibers III, and the nervous bundle containing the giant fibers II and the palleal nerve. The neuropeptide was not detected in the hemolymph suggesting a release by nerve endings next to the targets.

The SepOvotropin: a new ovarian peptide regulating oocyte transport in Sepia officinalis.

In the cuttlefish Sepia officinalis, the successive steps of egg laying are controlled by multiple neuropeptides. Recent experiments led us to suppose that there was possible involvement of a second regulation pathway by the release of ovarian regulatory peptides in the genital tract. Using HPLC fractionation and an in vitro biological test, a C-terminal amidated peptide modulating the motility of the Sepia officinalis oviduct was isolated from an extract of vitellogenic ovarian follicles. The mass of this peptide as determined by MALDI-TOF (1501.8 Da) and analysis by Edman degradation led to the following sequence: Pro-Lys-Asp-Ser-Met-Leu-Leu-Leu-Gln-Val-Pro-Val-Tyr-amide. The peptide mapping performed by LC/MS revealed a distribution restricted to the follicles, the full grown oocytes and the eggs. This new peptide, called SepOvotropin, modulated contractions of the whole genital tract in physiological conditions from a threshold concentration between 10(-20) and 10(-19) M, demonstrating for the first time the occurrence of a specific peptidergic control of egg-laying in cephalopods.

ILME: a waterborne pheromonal peptide released by the eggs of Sepia officinalis.

A novel tetrapeptide modulating the oviduct contractions was characterized from egg mass of Sepia officinalis. After two purification steps by rpHPLC, an apparent pure fraction containing the biological activity was submitted to MALDI-TOF analysis. The mass spectrum revealed 6 peaks of m/z 293, 505, 596, 613, 728, and 745. The tissue peptide mapping performed in LC-MS demonstrated the occurrence of the m/z 505 peptide in the follicles, the full-grown oocytes, and in the eggs. This peptide was also recovered in the seawater after the incubation of full grown oocytes or eggs, demonstrating a release in the genital tract and in the environment. Edman degradation gave the following sequence: Ileu-Leu-Met-Glu. The synthetic peptide applied to the whole genital tract triggered a cyclisation of the contractions at 10(-14) M. ILME appeared to be a chemical messenger released by the oocytes and the eggs, and was able to exert both paracrine and pheromonal activity.

HPLC and electrospray ionization mass spectrometry as tools for the identification of APGWamide-related peptides in gastropod and bivalve mollusks: comparative activities on Mytilus muscles.

The APGWamide-related neuropeptides, predicted by the cDNA of the APGWamide precursor of Mytilus edulis, have been sought by means of HPLC and electrospray mass ionization. The three predicted peptides KPGWamide, RPGWamide and TPGWamide were detected in the three main muscles and surprisingly an ion at m/z 429 corresponding to the gastropod peptide APGWamide was also demonstrated. Similar investigations performed in Lymnaea stagnalis central nervous system (CNS) revealed the occurrence of mussel APGWamide-related peptides (APGWamide-RPs) demonstrating for the first time the presence and the expression of the two precursors in both gastropod and bivalve mollusks. The absence of homologous domain in the Mytilus precursor [P. Favrel, M. Mathieu, Molecular cloning of a cDNA encoding the precursor of Ala-Pro-Gly-Trp-amide related neuropeptides from the bivalve Mytilus edulis. Neurosci. Lett. 1996;205:210-214] and the Lymnaea precursor [A.B. Smit, C.R. Jiménez, R.W. Dirks, R.P. Croll, W.P.M. Geraerts, Characterization of cDNA clone encoding multiple copies of the neuropeptide APGWamide in the molluscs Lymnaea stagnalis. J. Neurosci. 1992;12:1709-1715] eliminates the hypothesis of an alternative splicing of a single gene and suggests the likelihood of two genes probably resulting from duplication of an ancestral gene before the divergence between gastropods and bivalves. The similar potency observed on contraction assay and the differential distribution of the various peptides suggest that they may exert distinct activities on multiple targets.

Evidence of 5-hydroxytryptamine synthesis in the follicles of Sepia officinalis and direct involvement in the control of egg-laying.

At the beginning of egg-laying, in the cuttlefish Sepia officinalis, the oocytes accumulated in the proximal oviduct are released into the mantle cavity by the contractions of the oviduct before being encapsulated and fertilised. A bioassay based on the recording of the contractile activity of the distal oviduct was performed to characterise the molecule(s) inhibiting the oviducal motility and then responsible for the storage of the oocytes before mating. From 200 full-grown oocytes, a factor lowering the oviducal contractions was purified and isolated by means of HPLC. ESI-MS as well as electrochemical detection following HPLC fractionation allowed identification of the 5-hydroxytryptamine in the pure fraction. The inhibition of the oviducal contractions by 5-HT was dose dependent with a threshold near 10(-7) M. An immunoenzymatic assay showed that 5-HT appeared in the follicles at the beginning of vitellogenesis and reached a maximum level in the full-grown oocytes. In vitro experiments revealed that 5-HT is synthesised by the follicular cells and the full-grown oocytes, before being released to target proximal oviduct. Thus 5-HT could be one of the molecules involved in the accumulation of oocytes in the oviduct before mating. Mol. Reprod. Dev. 55:182-188, 2000.

Peptidergic control of egg-laying in the cephalopod Sepia officinalis: involvement of FMRFamide and FMRFamide-related peptides.

The peptidergic control of egg-laying was investigated in Sepia officinalis by using a myotropic bioassay. Three myotropic high-performance liquid chromatography fractions were obtained from optic lobe extracts. In the first fraction, FMRFamide (FMRFa) and FLRFa were isolated and sequenced. FMRFa-related peptides then were sought by dotting immunobinding of optic lobes extracts. The four immunoreactive fractions detected revealed the occurrence of FMRFa, FLRFa, FIRFa, and ALSGDAFLRFa predicted by the precursor already cloned from the optic lobes of S. officinalis (J Exp Biol 200:1483-9;1997). These peptides clearly appeared to be involved in the regulation of oocyte transport through the oviduct: the tetrapeptides FMRFa and FLRFa stimulated the contractions, whereas FIRFa and ALSGDAFLRFa lowered the tonus, the frequency, and the amplitude of the contractions. The occurrence of FaRPs in the nervous endings of the accessory sex glands suggested that this peptide family is involved in the regulation of secretory processes of the egg capsule. Indeed, FMRFa modulates the contractions of the main nidamental glands in vitro and, thus, should induce mechanical release of the secretion in vivo during ovulation. These results show that the FaRPs could play an important role in the synchronization of ovulation and egg capsule coating.