Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

Cg-TGF-beta, a TGF-beta/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection.

Transforming growth factor-beta (TGF-beta) members represent a widespread protein superfamily in the animal kingdom, but few members have been characterised in lophotrochozoans, a major clade of invertebrates. Here, we report the identification of Crassostrea gigas-TGF-beta (Cg-TGF-beta), a homologue of vertebrate TGF-beta and activin, from the bivalve mollusc C. gigas. Phylogenetic analysis suggests an early ancestral origin of this subgroup of TGF-beta superfamily member. Investigation of the spatio-temporal expression of Cg-TGF-beta gene by real-time quantitative RT-PCR showed a ubiquitous pattern in all adult tissues. These findings imply that Cg-TGF-beta has multiple functions as described for its vertebrate counterparts. Moreover, Cg-TGF-beta was upregulated in haemocytes during infection by a Gram-negative bacterium, suggesting that it could act as a cytokine involved in immunity in molluscs.

Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria.

The transforming growth factor beta (TGFbeta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s.). These ligands have been shown to play a key role in numerous biological processes including early embryonic development and immune regulation. They transduce their signal through a hetromeric complex of type I and type II receptors. Such receptors have been identified in ecdysozoans but none have been found as yet in the other major protostomal clade, the lophotrochozoans. Here, we report the identification of the first lophotrochozoan TGFbetas.s. type I receptor (Cg-TGFbetaRI) from the mollusk Crassostrea gigas. The phylogenetic and structural analyses as well as the expression pattern during early development suggest Cg-TGFbetaRI to belong to the TGFbetas.s./activin type I receptor clade and functional studies corroborate these deductions. The use of the zebrafish embryo as a reporter organism reveals that either Cg-TGFbetaRI or its dominant negative acting truncated form, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning due to a strong modulation of ventrolateral mesoderm patterning. Finally, a Cg-TGFbetaRI cytokine activity during immune regulation in C. gigas has been investigated by real-time PCR in haemocytes and mantle edge during an in vivo bacterial LPS challenge. One piece of evidence from this study suggests that the molecular mechanisms controlling mesodermal patterning and some immune regulations across all bilateria could be conserved through a functional TGF-beta s.s. pathway in lophotrochozoans.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas.

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.

Identification of new bone morphogenetic protein-related members in invertebrates.

Although believed to be widely distributed among the animal kingdom, transforming growth factor-beta (TGF-beta) superfamily members have mainly been characterized in vertebrate and in invertebrate model organisms such as Drosophila and C. elegans. To characterize such new factors in invertebrates, a PCR screen was performed on genomic DNA from different animal phyla, using degenerate primers deduced from consensus sequences of known members of the TGF-beta superfamily. Four new members were identified from a cnidaria, an echinoderm and from two classes of molluscs. These new proteins exhibit a high degree of identity with human bone morphogenetic proteins (BMP2/4). Sequence comparisons suggest an early origin and an evolutionary conservation of the molecular conformation. However, their possible involvement in distinct regulatory pathways is discussed.

Structure and expression of mGDF, a new member of the transforming growth factor-beta superfamily in the bivalve mollusc Crassostrea gigas.

To gain insight into the evolution of the structure and functions of transforming growth factor (TGF)-beta superfamily members, a cDNA encoding a new member from the bivalve mollusc Crassostrea gigas named mGDF (molluscan growth and differentiation factor) was identified by PCR using degenerate primers. The mGDF precursor exhibits characteristic features of the TGF-beta superfamily and shows highest homology with human BMP2 and Drosophila DPP. Conversely, the mgdf gene displays a distinct pattern of expression during development. Indeed mgdf transcripts were not detected early in development but increased markedly before metamorphosis. These findings raise the possibility that mGDF could play a central role in the biological processes that allow larvae to become competent to metamorphose.

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.

Stimulation of alpha-amylase release in the scallop Pecten maximus by the myosuppressins. Structure-activity relationships.

The insect myosuppressin LMS (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2) elicits potent stimulation of the release of the digestive enzyme alpha-amylase from cell suspensions of the stomach-digestive gland complex of the scallop Pecten maximus. The myosuppressins are members of the FMRFamide-like peptide superfamily, which immunocytochemical data confirm is present in the scallop. Structure-activity studies indicated that the two most critical residues for bioactivity are Arg and Phe. Bioactivity of the peptide can be maintained if the basic, aromatic residue His is replaced by another basic residue (Lys) and another aromatic residue (Trp), but not the aromatic Tyr, indicating a sensitivity to the introduction of a phenolic OH group. A restricted-conformation analogue containing a cyclopropyl-Ala residue in position 8 (Cpa-MS) demonstrates an ability to antagonize the amylase secretion activity of LMS at microM concentrations. This result provides evidence that the myosuppressins adopt a tight turn in the C-terminal tetrapeptide active core region while binding to the scallop digestive gland receptor. Cpa-MS may provide a useful tool to neuroendocrinologists studying in vitro and in vivo digestive processes in mollusks and other invertebrates.

Structure of the cDNA encoding the precursor for the neuropeptide FMRFamide in the bivalve mollusc Mytilus edulis.

FMRFamide immunoreactivity is widespread in the tissues of bivalve molluscs, but some of this immunoreactivity may represent distinct related peptides (FaRPs) rather than the exact tetrapeptide FMRFamide. We have cloned the first full-length cDNA encoding the precursor protein for FMRFamide from this class of molluscs to investigate the possibility that additional peptides may be produced. The precursor contains one copy each of NFLRFamide, FLRFamide, ALAGDHFFRFamide and 16 copies of FMRFamide. This precursor is expressed in all three ganglia of the central nervous system. Since the gene encoding the FMRFamide precursor in pulmonate molluscs is alternatively spliced to give two distinct messages, we searched for evidence that the FMRFamide gene of Mytilus is also alternatively spliced. No evidence of alternative splicing was found.

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study.

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of 3H-leucine and 14C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated 3H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs.

Isolation and identification of a novel Ala-Pro-Gly-Trp-amide-related peptide inhibiting the motility of the mature oviduct in the cuttlefish, Sepia officinalis.

A novel myotropic neuropeptide was isolated from 110 optic lobes (OL) of mature females of the cuttlefish Sepia officinalis L. by mean of high performance liquid chromatography (HPLC). The peptide inhibits the motility of the oviduct by decreasing the tonus, the frequency and the amplitude of the contractions. The primary structure of the peptide was determined as Gly-Trp-NH2. This new dipeptide is closely related to the Ala-Pro-Gly-Trp-NH2 family first identified in gastropod molluscs. On the perfused oviduct, GWa appeared to be 3000 times more potent than APGW-amide. The processing of synthetic APGWa into GWa by diaminopeptidyl activity has been clearly observed in OL extract. Nevertheless, the analysis in MALDI-MS of HPLC OL fractions did not reveal any APGWa related peptides of the known: APGWa, KPGWa, RPGWa and TPGWa. GWa could be processed from a not yet identified APGWa related peptide.

Molecular cloning of a cDNA encoding the precursor of Ala-Pro-Gly-Trp amide-related neuropeptides from the bivalve mollusc Mytilus edulis.

We isolated and characterised a cDNA clone encoding the precursor of neuropeptides related to the molluscan neuropeptide Ala-Pro-Gly-Trp amide from the marine mussel Mytilus edulis. The preproprotein is posttranslationally processed to generate a 20 amino acid signal peptide together with five sequences of the neuropeptide Arg-Pro-Gly-Trp amide (RPGWamide), one Lys-Pro-Gly-Trp amide (KPGWamide), one Thr-Pro-Gly-Trp amide (TPGW amide) as well as a putative 31 amino acid long C-terminal peptide. In situ hybridisation showed that the gene encoding this precursor is expressed in discrete neurons within the three ganglia of the central nervous system.

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc, Haliotis tuberculata.

A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [3H]-leucine and [3H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [3H]-leucine or for [3H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.

A new biological activity for the neuropeptide FMRFamide: experimental evidence for a secretagogue effect on amylase secretion in the scallop Pecten maximus.

FMRFamide immunoreactivity in the digestive tract of the bivalve mollusc Pecten maximus was investigated by immunocytochemistry. Positive FMRFamide-like immunoreactivity was detected in nerve fibres in close contact with exocrine alpha amylase secreting cells. Physiological studies on enzymatically dissociated cells of the stomach-digestive gland complex demonstrated the involvement of FMRFamide and analogues in the control of alpha amylase release from the cells. The FMRFamide-induced secretion was shown to be time- and dose-dependent. In contrast to most naturally occurring vertebrate secretagogues which are hormones, FMRFamide appears to work in our in vitro system as a paracrine factor.

Structure and biological activity of crustacean gastrointestinal peptides identified with antibodies to gastrin/cholecystokinin.

Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.

Immunochemical and biochemical characterization of gastrin/cholecystokinin-like peptides in Palaemon serratus (Crustacea Decapoda): intermolt variations.

Gastrin/cholecystokinin (G/CCK)-like peptides cross-reacting with an antiserum specific for the carboxyamide terminal pentapeptide of gastrin and CCK have been detected in the eyestalks and in the stomach of the prawn Palaemon serratus using immunocytochemical methods. In the eyestalks, immunoreactivity is present in the neuroendocrine cells, the X organ-sinus gland tractus and the neurohemal organ itself. This suggests, for the first time, the existence of a neuroendocrine secretion of G/CCK-like peptides. Hemolymph G/CCK level is about 18 pM. In the stomach, G/CCK-like material has been observed in epithelial cells in the cuticle and in the lumen. Molecular sieving of crude extracts of the medulla terminalis from the eyestalks, the stomach, and the hemolymph samples on a Sephadex G-50 filtration column exhibited a molecular heterogeneity of the G/CCK immunoreactive material. Large components were observed principally in the medulla terminalis and in the hemolymph, and smaller forms in the stomach. A fraction common for the three tissues had an apparent molecular weight of 2500 Da. That fraction was characterized further by HPLC and shown to be more hydrophobic than human G17 I. By radioimmunoassay relatively low levels were detected in all the aforementioned organs. Although the concentration of the G/CCK-like components varies during the intermolt cycle, this was the case mainly in the hemolymph and in the stomach. These observations suggest a possible role of G/CCK-like peptides in molting processes.