vasopressin-related peptides in the cuttlefish Sepia officinalis

Peptides 46 (2013) 159–166

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Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis Joël Henry a,b,c , Valerie Cornet a,b , Benoit Bernay c , Céline Zatylny-Gaudin a,b,∗ a b c

Université de Caen Basse-Normandie, FRE3484 BioMEA, F-14032 Caen, France CNRS INEE, FRE3484 BioMEA, F-14032 Caen, France Post Genomic Platform PROTEOGEN, Université de Caen Basse-Normandie, SF ICORE 4206, Caen, France

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 10 May 2013 Accepted 13 May 2013 Available online 11 June 2013 Keywords: Sepiatocin Oxytocin/vasopressin Neuropeptide Neurohormone Sepia officinalis Cephalopod

a b s t r a c t Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called prosepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Oxytocin and vasopressin (OT/VP) were discovered in the early XXth century from the mammalian posterior pituitary [1,13]. These regulatory peptides have a wide range of partially overlapping biological activities. OT is mainly known to stimulate uterus contractions during delivery and milk ejection [17], whereas VP regulates the osmotic balance and the contractions of smooth muscle cells in arteries [10]. Vasopressin and oxytocin are cyclic nonapeptides that contain two cysteines (positions 1 and 6) linked by a disulfide bond (Table 1). The main structural difference between vasopressin and oxytocin is the occurrence of a basic amino acid residue (mainly Arg) at position 8 in vasopressin as opposed to a neutral residue in oxytocin (Table 1). In the early 1980s, vasopressin and oxytocin preprohormones were cloned in rats [19,28]. Both precursor proteins contained, in addition to the OT/VP sequences, a larger polypeptide sequence named neurophysin, initially thought to be a vasopressin- or oxytocin-carrier protein but now believed to play a crucial role in the proper folding and sorting of vasopressin and oxytocin prohormones [11].

∗ Corresponding author at: Université de Caen Basse-Normandie, FRE3484 BioMEA, F-14032 Caen, France. Tel.: +33 2 31 56 55 96; fax: +33 2 31 56 53 46. E-mail address: [email protected] (C. Zatylny-Gaudin). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.05.004

In cephalopods, the common octopus, Octopus vulgaris, possesses two peptides belonging to the oxytocin/vasopressin superfamily: cephalotocin (CT) and octopressin (OP) [24,30]. Takuwa-Kuroda and collaborators suggest that octopressin is a multifunctional neuropeptide that contributes to reproduction, cardiac circulation and feeding, and that cephalotocin plays important roles in metabolism as a neurohormone. In the cuttlefish Sepia officinalis, immunoreactivity was observed in several structures of the nervous system [4]. The present paper describes the identification of two novel members of the oxytocin/vasopressin family in S. officinalis through the cloning of the preprohormone using sequence homologies of the neurophysin domain. 2. Materials and methods 2.1. Animals and biological sample collection All the cuttlefish were trapped in the Bay of Seine between January and June. They were maintained in 1000-L outflow tanks at 16 ◦ C at the Marine Station of Luc sur Mer (University of Caen, France) under a natural photoperiod. Mature cuttlefish were anesthetized using ethanol 3% [25]. Then hemolymph samples were taken, and several organs were dissected, i.e. the optic gland, the optic lobe, the sub-esophageal mass,

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Table 1 Structures of vasopressin, oxytocin, and some selected oxytocin-and vasopressin like peptides identified in vertebrates and invertebrates. Name

Peptide structure

Source

OT family Oxytocin Pro-oxytocin Isotocin

CYIQNCPLGa CYIQNCPPGa CYISNCPIGa

Mammals [13] Callithrix jacchus [20] Fish [3]

VP family Vasopressin Lys-vasopressin Phenypressin Vasotocin

CYFQNCPRGa CYFQNCPKGa CFFQNCPRGa CYIQNCPRGa

Mammals [1,14] Pig, some marsupials[6] Some marsupials [7] Non-mammalian vertebrates [2]

among the OT/VP superfamily. NR and NR2 primers were synthesized for 5 -RACE from the neurophysin domain of the octopressin gene (GenBank accession no. AB108429). Spia1 and Spia2 primers, designed from OT/VP-like precursors, were used for 3 -RACE. For RT-PCR mapping, SepOT, SepOTTPP and SepOT2Rev were used (Fig. 1). Primers designed from Actin and Elongation factor ␥ were used to amplify reference genes. 2.4. 3 -RACE (rapid amplification of cDNA ends) and 5 -RACE

Invertebrate OT/VP-like peptides CLITNCPRGa Inotocin Arg-conopressin CIIRNCPRGa Lys-conopressin CFIRNCPKGa Crustacean OT/VP-like peptide Vasotocin Nematocin Annetocin Echinotocin

CFITNCPPGa CFVRNCPPGa CFLNSCPYRRYa CFVRNCPTGa CFISNCPKGa

Cephalotocin Octopressin Sepiatocin Pro-sepiatocin

CYFRNCPIGa CFWTSCPIGa CFWTTCPIGa CFFRNCPPGa

Various insects [18,23,29] Conus geographicus [9] Leech various mollusks [9,26,32] Daphnia pulex [29] Platynereis dumerilii [31] Caenorhabditis elegans [5] Various annelids [22] Strongylocentrotus purpuratus [15] Octopus vulgaris [24] Octopus vulgaris [30] Sepia officinallis (this paper) Sepia officinalis (this paper)

the supra-esophageal mass, the vena cava, the oviduct, the male genital tract, the penis. These organs were frozen in liquid nitrogen and stored at −80 ◦ C until RNA isolation or peptide extraction. Rectum, penis, oviduct, gill and vena cava were dissected for myotropic bioassays. These organs were maintained in sterile seawater supplemented with glucose. No specific permits were required for the described field studies. The common cuttlefish is not an endangered or protected species. 2.2. Total RNA and mRNA preparation Total RNA was isolated from the nervous system or genital organs of mature cuttlefish using Tri-Reagent (Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. mRNAs were isolated using oligodT coupled to magnetic beads as described by the manufacturer (Dynal, Invitrogen, Carlsbad, CA, USA). 2.3. Oligonucleotide primers Oligonucleotide primers (Table 2) were purchased from Eurobio (France) and Genecust (Luxembourg). RACE Primers were designed based on sequences of neurophysin domains, which are conserved Table 2 Oligonucleotide sequences. Name

Sequence

NR NR2 Spia Spia2 SepOT sepOTPP sepOT2Rev Actin ActinRev EF␥ EF␥Rev

5 -GCAAACACCGTCGGCAACACATCGTCCTTGT-3 5 -GCGAGTGTGCCTATGATGCAACCGTCTTT-3 5 -GGGGTTCCTGGGCTCTTTTGCTGCTTAT-3 5 -CGGGCCGATTCCTCTTTTCATCCACCAAG-3 5 -GCGAAGTGCTTCAGAATTCAGAG-3 5 -GAGAAGCGAAATGGGGTCGGG-3 5 -CTTTTTGTCTTGATGTTAGGGAACG-3 5 -TCCATCATGAAGTGCGATGT-3 5 -TGGACCGGACTCGTCATATT-3 5 -TACAGCGGGGCAAACGTGACTG-3 5 -GGGTGATACGTTCACCCACCAGA-3

The transcriptional start site was determined by oligo-capping RACE methods using a GeneRacer kit (Invitrogen). First strand cDNA was synthesized from mRNA with the GeneRacer Oligo dT Primer supplied in the GeneRacer kit (Invitrogen) according to the manufacturer’s instructions. The first PCR was performed using the GeneRacer 5 primer and primer NR with Platinum TaqDNA Polymerase High fidelity (Invitrogen), under the following conditions: 2 min at 94 ◦ C, 5 cycles of 30 s at 94 ◦ C and 2 min at 72 ◦ C, 5 cycles of 30 s at 94 ◦ C and 2 min at 68 ◦ C, and 25 cycles of 30 s at 94 ◦ C, 30 s at 62 ◦ C, 2 min at 72 ◦ C (7 min for the last cycle). The second PCR was performed using the GeneRacer 3 nested primer and NR2 under the following conditions: 94 ◦ C for 5 min, 35 cycles of 30 s at 94 ◦ C, 30 s at 62 ◦ C, 2 min at 72 ◦ C (7 min for the last cycle). The second PCR products were subcloned and sequenced by genomexpress (Grenoble, France). The 3 -ends of the cDNAs were determined as follows: the first template was amplified using the GeneRacer 3 primer and Spia or Spia2. Each of the first PCR products was re-amplified using the GeneRacer 3 nested primer and Spia or Spia2. Both first and second PCR reactions were performed for 5 min at 94 ◦ C, and then 35 cycles of 30 s at 94 ◦ C, 30 s at 62 ◦ C, and 2 min at 72 ◦ C (7 min for the last cycle). The second PCR products were subcloned and sequenced by genomexpress (Grenoble, France). 2.5. Patterns of tissue-specific expression We examined the patterns of tissue-specific expression for the sepiatocin transcripts. The variations in expression were assayed via RT-PCR from 5 tissues of the two sexes: the female genital tract (oviduct), the male genital tract, the sub-esophageal mass, the supra-esophageal mass, the optic lobes and the optic glands. After RNA quantification (Nanodrop, Thermoscientific), genomic DNA was digested for 30 min at 37 ◦ C with 1U of RQ1 DNAse (Promega). Then, 1 ␮g of total RNA was reverse-transcribed 1 h at 37 ◦ C, followed by 10 min at 70 ◦ C with 200 U of MMLV-RT (Promega), 24 U of RNAsin and 250 ng of Random Primers. The resulting cDNAs were used to assay sepiatocin expression in different organs and actin gene expression was taken as a reference. Amplification reactions were performed in a Thermocycler (Biorad), at 95 ◦ C for 3 min, and then 30 cycles of 95 ◦ C for 30 s, 55 ◦ C (or 60 ◦ C with EF␥ primers) for 30 s, 72 ◦ C for 45 s, and final elongation for 10 min at 72 ◦ C. The reaction mixes were composed of 1 ␮L of cDNA, 1 U of Go Taq (Promega) with the following primers: SepOTPP, SepOT and SepOT2Rev or Actin and ActinRev or EF␥ and EF␥Rev. For each set of primers, an equal amount of PCR amplicon (15 ␮l) from each template was electrophoresed at 100 V for 30 min in a 1.8% agarose gel, stained with ethidium bromide and visualized under UV light (alpha imager EP, alpha Innotech). The targetspecific amplified products had expected sizes of 513 and 360 bp for Pro-Sep and Sep, respectively, 261 bp for actin and 406 bp for elongation factor ␥. 2.6. Recovery of material from tissues Tissues were dissected from mature cuttlefish anesthetized using ethanol 3% [24], frozen in liquid nitrogen and stored at −80 ◦ C until peptide extraction.

J. Henry et al. / Peptides 46 (2013) 159–166

161

Fig. 1. Nucleotide sequence alignment of sepiatocin and pro-sepiatocin cDNAs. Oligonucleotide primers for RT-PCR are highlighted in gray; oligonoucleotide primers for 3 RACE are underlined.

Two protocols were used for peptide extraction: 1 Three animal-equivalents of each organ studied (sub-esophageal mass and supra-esophageal mass) were crushed in liquid nitrogen and extracted under stirring at +4 ◦ C for 1 h in 0.1% TFA containing 50 mM DTT (1 g of tissue for 10 mL of extraction medium), centrifuged for 30 min at 35,000 × g at +4 ◦ C, and then the supernatants were frozen at −20 ◦ C. 2 Three animal-equivalents of each organ studied (sub-esophageal mass, supra-esophageal mass and hemolymph were crushed in liquid nitrogen and extracted under stirring at +4 ◦ C for 1 h in a mix made of methanol, water and acetic acid (90/9/1) adjusted to 50 mM DTT (1 g of tissue for 10 mL of extraction medium), centrifuged for 30 min at 35,000 × g at +4 ◦ C. The supernatants were evaporated and the dry pellets were resuspended in 0.1% TFA before being pooled with the TFA extracts obtained from the first extraction protocol. The extracts were finally concentrated and desalted on Chromafix C18 solid phase extraction cartridges (Macherey-Nagel), evaporated and kept at +4 ◦ C until use. Hemolymph extraction was performed as described above with 1 mL of frozen hemolymph for 10 mL of extraction medium. 2.7. Reduction and alkylation The dry pellets were resuspended in 60 ␮L of water and centrifuged for 5 min at 15,000 × g. One hundred microliter of 8 M urea

in 0.2 M Tris and 10 ␮L of 450 mM DTT were added to 50 ␮L of sample supernatant. The reduction of disulfide bonds was carrying out for 45 min at 55 ◦ C. Alkylation was performed at room temperature for 20 min in the dark by adding 20 ␮L of 500 mM iodoacetamide. The samples were finally concentrated and desalted on Zip Tip C18 (Omix, Varian). 2.8. Mass spectrometry analyses The samples were desalted and concentrated on a ␮C18 Omix (Varian) before nano-LC–MS/MS analysis. The chromatography step was performed on a nano-LC system (Prominence, Shimadzu). Peptides were concentrated on a Zorbax 5 mm × 0.3 mm C18 precolumn (Agilent) and separated on a Zorbax 150 ␮m × 75 ␮m C18 column (Agilent). Mobile phases consisted of 0.1% trifluoroacetic acid, 99.9% water (v/v) (A) and 0.1% trifluoroacetic acid, 20% water in 79.9% ACN (v/v/v) (B). The nanoflow rate was set at 300 nL/min, and the gradient profile was as follows: constant 7% B for 5 min, from 7 to 70% B in 183 min, from 70 to 100% B in 5 min, and back to 7% B. The 300 nL/min volume of the peptide solution was mixed with 1.2 ␮L/min volumes of 5 mg/mL CHCA matrix solution prepared in a diluent solution containing 50% ACN and 0.1% TFA. Thirty-second fractions were spotted by an AccuSpot spotter (Shimadzu) on stainless steel Opti-TOFTM 384 targets. MS experiments were carried out on an AB Sciex 5800 proteomics analyzer equipped with TOF, TOF ion optics and OptiBeamTM on-axis laser irradiation with 1000 Hz repetition rate. The system was calibrated just before analysis with a mixture of

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des-Arg-Bradykinin, Angiotensin I, Glu1-Fibrinopeptide B, ACTH (18–39), ACTH (7–38) and mass precision was better than 50 ppm in reflectron mode. A laser intensity of 3200 was typically employed for ionizing. MS spectra were acquired in the positive reflector mode by summarizing 1000 single spectra (5 × 200) in the 700–1200 Da mass range. MS/MS spectra were acquired in the positive MS/MS reflector mode by summarizing a maximum of 2500 single spectra (10 × 250) with a laser intensity of 3900. For tandem MS experiments, the acceleration voltage was 1 kV and air was used as the collision gas. Gas pressure medium was selected as settings. 2.9. Peptide sequencing The fragmentation pattern was used to determine the peptide sequence. Database searching was performed using the Mascot 2.2.04 program (Matrix Science). A specific database of invertebrate peptides including sepiatocin precursors was used. The various modifications allowed were as follows: C-terminal amidation, N-terminal pyroglutamate, C-carbamidomethyl, Kacetylation, methionine oxidation, and dioxidation. “No enzyme” was selected. Mass accuracy was set to 100 ppm and 0.6 Da for the MS and MS/MS modes, respectively. 2.10. In vitro bioassay The myotropic bioassay was performed with several contractile organs, i.e. rectum, gill, penis, oviduct and vena cava dissected from mature cuttlefish (S. officinalis). Each organ was suspended from a displacement transducer (Phymep, Bionic Instruments) connected to a computer that controlled the recorder. The muscle chamber was perfused at a flow-rate of 0.5 mL/min with synthetic seawater (Instant Ocean) containing 1 mM glucose and maintained at 15 ◦ C. Several concentrations of synthetic sepiatocin and pro-sepiatocin (Genecust, Luxemburg) or oxytocin (Sigma) were injected into the perfusing flow using a three-way valve in order to avoid mechanical and thermal stress. Peptide concentrations were determined according the volume of muscle chamber. The flow of the fractions into the muscle chamber was traced by adding phenol red. 3. Results

Whereas the N-terminal sequences of the sepiatocin and prosepiatocin precursors are different, the remaining parts (sepiatocin: Cys45 -Asn147 ; pro-sepiatocin: Cys57 -Asn158 ) are identical. The predicted N-terminal sequences of the two precursors (sepiatocin: Met1 -Gly26 ; pro-sepiatocin: Met1 -Ala32 ) contain hydrophobic amino acids, a characteristic feature of signal peptides. Each nonapeptide displays an oxytocin consensus sequence (C-xx-x-x–C-P-P/I-G) and is connected to a neurophysin domain by the Gly-Lys-Arg (G-K-R) sequence, a signal for carboxylterminal amidation and a dibasic proteolytic processing site. Sequence comparison with neurophysins of the OT/VP superfamily showed that the sepiatocin–neurophysin (Ser39 -Asn147 ) and prosepiatocin–neurophysin (Ala44 -Asn158 ) domains conserved the positions and numbers of Cys residues (Fig. 2). Since the structural organization of these precursors is typical of other known members of the OT/VP superfamily, this confirms that sepiatocin and pro-sepiatocin are members of that superfamily. 3.2. Tissue-specific expression patterns The expression of the two sepiatocin precursors was restricted to nervous structures (Fig. 4). The sepiatocin precursor was expressed in the brain (supra esophagal and subesophagal masses) and also in the optic lobes, whereas pro-sepiatocin precursor expression was only detected in the brain. Moreover, according the stable expression of reference genes, the pro-sepiatocin transcripts seemed to be more expressed in the sub-esophageal mass than in the supra esophagal mass, and more expressed than sepiatocin transcripts in the subesophagal mass. No difference in the expression of the two transcripts was observed between the nervous structures of the two sexes. 3.3. Peptide detection Among all the samples analyzed by mass spectrometry, a single one allowed us to detect one of the two nonapeptides in the female hemolymph. Indeed, the analysis of the MS/MS spectrum of the m/z 1153.56 peptide confirmed the amino acid sequence of mature prosepiatocin with mass adducts of 57 Da for each alkylated cysteine (Fig. 5A and B).

3.1. Cloning of sepiatocin preprohormones 3.4. Activity of sepiatocin We cloned two cDNAs coding for cuttlefish preprohormones, each containing a oxytocin/vasopressin like peptide. The first peptide was called sepiatocin and the second one pro-sepiatocin. The two cDNAs displayed 80.34% similarity and 78.93% identity (Fig. 1). The 3 regions were 100% similar and 100% identical. The full-length cDNAs encoding the sepiatocin and prosepiatocin precursors and the deduced amino acid sequences are shown in Fig. 2. The sepiatocin precursor cDNA of 600pb predicts a protein of 147 amino acids with a calculated molecular mass of 15,661. The pro-sepiatocin precursor cDNA consisted of 627 bp encoding 158 amino acids with a molecular mass of 16,713. Nucleotide sequence data were deposited in the DDBJ/EMBL/GenBank databases under the accession numbers KC763356 and KC763357 for sepiatocin and pro-sepiatocin sequences respectively. In the Fig. 3, the cuttlefish sequences were compared with New World monkey oxytocin, Lymnaea stagnalis conopressin, O. vulgaris octopressin and cephalotocin preprohormones. Sequences alignment shows that these proteins have conserved their structural organization since Proto- and deuterostomia split, 700 million years ago [12]. In the two precursors, the oxytocin-like peptide sequence is located directly after the signal sequence, followed by dibasic cleavage sites and neurophysin-like sequences (Fig. 2).

Increasing concentrations of synthetic peptides (Genecust) were applied on contractile organs such as the oviduct and the accessory sex glands of females, and the penis, vena cava, gills and rectum of males. Only sepiatocin induced an increase in tonus and amplitude when applied on perfused vena cava or genital organ like penis or oviduct (Fig. 6) with a threshold ranging between 10−7 M and 10−8 M. No myotropic activity was observed with pro-sepiatocin. Mammalian oxytocin induced an thin increase in tonus and amplitude on cuttlefish oviduct when applied at 10−6 M (Fig. 6C). 4. Discussion Two novel members of the OT/VP superfamily were characterized from the cuttlefish S. officinalis. These two neuropeptides were called sepiatocin (CFWTTCPIGa) and pro-sepiatocin (CFFRNCPPGa). The nature of the 8th amino acid, isoleucin for sepiatocin and proline for pro-sepiatocin, indicates an oxytocin structure for the two new OT/VP-related peptides. Sepiatocin is very similar to the octopressin identified in O. vulgaris (CFWTSCPIGa) with only one different amino acid in position 5 [30]. Pro-sepiatocin differs from cephalotocin identified in O. vulgaris by two amino acids in positions 2 and

J. Henry et al. / Peptides 46 (2013) 159–166

A

163

1

GTC GAG GTT AAA AAA GCA TTT TTT GCA ATA CCA ATC CAC TGA TAC AAG AGA AGC GAA

58

M G S G R F L F S S T K C Q V A C V L ATG GGG TCG GGC CGA TTC CTC TTT TCA TCC ACC AAG TGC CAA GTA GCC TGC GTT CTG

Signal peptide 19

SepiatocinPP

115

C F F R N C N F C V F L I C T T D A F TTC AAT TTC TGT GTC TTT CTC ATC TGT ACG ACA GAT GCT TGT TTC TTC AGA AAC TGT

38

Neurophysin

P

G

G

57

172

K R A V A M N D G V A H K Q C CCA CCG GGT GGA AAA CGG GCC GTC GCA ATG AAT GAT GGC GTT GCA CAT AAA CAG TGC M A C G P E G K G R C C A G P N I C Q ATG GCT TGC GGT CCG GAA GGT AAA GGC CGG TGT GCT GGT CCC AAT ATC TGT TGC CAA

76

229

286

B

P

E G C I I G D M A K E C M Q E D E G K AAA GAA GGT TGC ATT ATT GGT GAC ATG GCC AAG GAG TGT ATG CAG GAA GAC GAG GGT

95

T V C E V K G I P C G A E G Q G R C T ACA ACT GTA TGT GAA GTG AAA GGG ATA CCG TGC GGT GCA GAG GGA CAA GGA CGT TGT

114

343

D T S A C V A A G V C C S T N S H C G GTG GCT GCT GGA GTC TGC TGT GAT ACA TCT GCT TGC TCG ACG AAC AGC CAC TGT GGT

133

400

A L P R T S S R R Q E L F S L L K R S TCC GCT CTG CCC CGA ACT TCC AGC CGA CGA CAA GAA TTA TTC AGT TTG CTG AAA AGG

152

457

I N K V N * T F P N I K T K R K * K L CTA ATC AAC AAA GTG AAT TAA ACG TTC CCT AAC ATC AAG ACA AAA AGA AAA TGA AAA

171

514 571

TAC AAA ATA AAC GTT GTC CAA AAA GAA TGA CAT TTG GCA AAA TAA TGT AAA ACT CTG

1

CTA ATA ATT TCC TGG TGC TCT ACA ACA TAT TAT TGT CTC ATC CAT TTC ACA TTC TCA

58

M A S Y R W G S W A L L L L I V V ACC GAC ATG GCA AGC TAC CGT TGG GGT TCC TGG GCT CTT TTG CTG CTT ATT GTA GTT

Signal peptide 17

Sepiatocin 115

L P L V S L V E G C F W T T C P I G G CTA CCC CTG GTT TCT CTT GTG GAA GGA TGT TTC TGG ACC ACA TGT CCT ATT GGG GGC

172

K R S A S E F R E C M A C G P E G K G AAG CGA AGT GCT TCA GAA TTC AGA GAG TGC ATG GCT TGC GGT CCG GAA GGT AAA GGC

36

Neurophysin

229

A G P N I C C R C Q K E G C I I G D M CGG TGT GCT GGT CCC AAT ATC TGT TGC CAA AAA GAA GGT TGC ATT ATT GGT GAC ATG

55

74

K E C M Q E D E G T T V C E V K G I A GCC AAG GAG TGT ATG CAG GAA GAC GAG GGT ACA ACT GTA TGT GAA GTG AAA GGG ATA

93

286

C G A E G Q G R C V A A G V C C D T P CCG TGC GGT GCA GAG GGA CAA GGA CGT TGT GTG GCT GCT GGA GTC TGC TGT GAT ACA

112

343

A C S T N S H C G S A L P R T S S R S TCT GCT TGC TCG ACG AAC AGC CAC TGT GGT TCC GCT CTG CCC CGA ACT TCC AGC CGA

131

400

Q E L F S L L K R L I N K V N * R CGA CAA GAA TTA TTC AGT TTG CTG AAA AGG CTA ATC AAC AAA GTG AAT TAA ACG TTC

147

457 514 571

CCT AAC ATC AAG ACA AAA AGA AAA TGA AAA TAC AAA ATA AAC GTT GTC CAA AAA GAA TGA CAT TTG GCA AAA TAA TGT AAA ACT CTG

Fig. 2. Nucleotide sequences and deduced amino acid sequences of the sepiatocin precursors (A) pro sepiatocin and (B) sepiatocin. The numbers on the left indicate nucleotide positions and those on the right indicate amino acid positions. Sepiatocin or pro-sepiatocin sequences are in bold. The dibasic cleavage sites are boxed. The 14 conserved Cys residues are underlined.

8 (CYFRNCPIGa) [24]. Pro-sepiatocin is the first oxytocin-like peptide with a proline in position 8 identified in mollusks and the second one among lophotrochozoa after the vasotocin identified in Platynereis dumerilii (CFVRNCPPGa) [31]. An oxytocin with proline in position 8 was recently identified in New World monkeys (CYIQNCPPGa) [20], demonstrating that proline in position 8 is not restricted to protostomes. In vertebrates, the coexistence of oxytocin and vasopressin is established, but in protostome invertebrates, only one of them (either oxytocin or vasopressin-like) is expressed: conopressin in gastropods [21], inotocin in insects [18,29] or annetocin in various annelids [17]. In cephalopods, two OT/VP-like peptides are

expressed, as shown in O. vulgaris [24,30] and in S. officinalis (our data). The occurrence of a proline in position 8 of pro-sepiatocin reveals a structural homology with a novel mammalian oxytocin identified in New World monkeys. The amino acid sequence homology between pro-oxytocin and pro-sepiatocin suggests that pro-sepiatocin is an OT-related peptide like sepiatocin with a isoleucine in position 8 characteristic of non mammalian vertebrate oxytocin [3]. Using a molecular approach, the present study reveals the expression of two new oxytocin-like peptides in the cephalopod S. officinalis, but it does not confirm the occurrence of a

164

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OT/VP related peptides conopressin oxytocin_callithrix pro-Sepiatocin sepiatocin octopressin cephalotocin

-------MMSSLCGMPLTYLLTAAVLSLSLTDACFIRNCPKGGKR--SLDTGMVTSRECM --------------MAGPSLACCLLGLLALTSACYIQNCPPGGKR--AALD--LDVRKCL -MGSGRFLFSSTKCQVACVLFNFCVFLICTTDACFFRNCPPGGKR-AVAMNDGVAHKQCM -------MASYRWGSWALLLLIVVLPLVSLVEGCFWTTCPIGGKR-SAS-----EFRECM ---------MSSIKSSVFAILIVV-VLLPLVKGCFWTSCPIGGKRSNIPATEP---RQCM -----MSQNCFAIVQLLFVLFTVCSLFIATTDGCYFRNCPIGGKR-ATPMSEQGSNQKCM : : ...*: .** **** ::*:

51 42 58 47 47 54

conopressin oxytocin_callithrix pro-Sepiatocin sepiatocin octopressin cephalotocin

KCGPGGTGQCVGPSICCGQDFGCHVGTAEAAVCQQENDSSTPCLVKGEACGSRDAGNCVA PCGPGGKGRCFGPNICCVEELGCFVGTAEALRCQEESYLPSPCQSGHKACGS--GGHCAA ACGPEGKGRCAGPNICCQKE-GCIIG-DMAKECMQEDEGTTVCEVKGIPCGAEGQGRCVA ACGPEGKGRCAGPNICCQKE-GCIIG-DMAKECMQEDEGTTVCEVKGIPCGAEGQGRCVA SCGPNGEGQCVGSNICCHKD-GCIIG-TLAKECNEENESTTACSVKGVPCGTDGQGRCVA SCGPNGEGQCVGSNICCHKD-GCIIG-TLAKECNEENESTTACSVKGVPCGTDGQGRCVA *.*.* *** * *:* *..*** :: ** :* * * :*. .: * .**: DGICCDSESCAVNDRCRDLDGNAQANRGDLIQLIHKLLKVRDYD 155 FGLCCSPDGCHADPAC-DVEATLSQH------------------ 125 AGVCCDTSACSTNSHCGSALPRTSSRRQELFSLLKRLINKVN-- 158 AGVCCDTSACSTNSHCGSALPRTSSRRQELFSLLKRLINKVN-- 147 DGVCCDESSCFTTDRCDRENHR-SMAMQKLLEIRDGIYYKK--- 145 DGVCCDESSCFTTDRCDRENHR-SMAMQKLLEIRDGIYYKK--- 152 * . *:**. ..* .

111 100 116 105 105 112

conopressin oxytocin_callithrix pro-Sepiatocin sepiatocin octopressin cephalotocin

Fig. 3. Comparison of the amino acid sequences of the OT/VP superfamily precursors. The underlined sepiatocin and pro-sepiatocin precursors are aligned by CLUSTAL W (2) with the precursors of conopressin (GenBank accession no. AAA29289), octopressin (GenBank accession no. BAC84977.1), cephalotocin (GenBank accession no. BAC84978), and Callithrix jacchus oxytocin preproprotein (GenBank accession no. AEM43540.1). Identical amino acid residues in all OT/VP-like peptides are highlighted in gray. Cys residues are in black boxes. Vertical arrows indicate amino acid residues essential for binding neurophysin to peptides.

vasopressin-like peptide described by Bardou et al. [4] using an immuno-staining approach with antibodies raised against mammalian oxytocin and vasopressin. Moreover, on the one hand the functional classification based on the residue at position 8 is not relevant in invertebrates where only one OT/VP like-peptide was identified, and on the other hand residues 2–5 are important for high-affinity binding to the receptor and biological efficacy [27,32,33]. In S. officinalis, we demonstrated that the occurrence of two members of OT/VP family with a structure near than oxytocin peptide. The C-terminal domain of the cuttlefish peptides is involved in the biological activity. Indeed, sepiatocin, like mammalian oxytocin with less affinity, modulates the contractile activity of the cuttlefish oviduct whereas pro-sepiatocin has no effect on that muscle. The homolog C-terminal sequence P-I/L-Gamide shared by sepiatocin and mammalian oxytocin appears to be directly involved in the biological activity observed on oviduct. The high level of conservation of cysteines in the neurophysin domain of OT/VP precursors across prostostomes and deuterostomes is probably correlated to the involvement of the C-terminal domain in the folding and sorting of OT/VP peptides in the secretory pathway. Indeed, the Glu78 and Arg39 amino acid residues of bovine neurophysins are essential for the binding of VP and OT [8]. These significant residues are also conserved in sepiatocin neurophysins (Glu81 and Arg44 ) and pro-sepiatocin neurophysins

(Glu104 and Lys55 ), suggesting that each Sepia neurophysin could bind sepiatocin or pro-sepiatocin and perhaps mammalian OT/VP. Although the mature sepiatocin neuropeptide was not detected by mass spectrometry (maybe ionization was inefficient), its expression was clearly demonstrated in several parts of the central nervous system. Moreover, its ability to modulate the contractions of the vena cava, the penis and the oviduct revealed an OT/VP-like activity similar to that described in vertebrates. The sepiatocin, like is described for OT/VP related peptides [16], is clearly involved in reproductive physiology modulating contractile activity of organ responsible of gamete emission of cuttlefish. The detection of the mature pro-sepiatocin neuropeptide in the hemolymph supports the hypothesis of a C-terminal amidation and is consistent with the high expression level measured in the subesophageal mass. Indeed, that part of the CNS is associated to the single neurohemal area described in S. officinalis, which releases neurosecretions into the vena cava. As observed for cephalotocin in a previous paper [24], the function of pro-sepiatocin is not elucidated in this study, future research will have to address the potential role in metabolism and homeostasis as has also been suggested for cephalotocin. Nevertheless, the occurrence of pro-sepiatocin in hemolymph of cuttlefish during egg laying suggest an involvement of this neurohormone in reproductive function.

Fig. 4. Tissue-specific expression patterns for Sepiatocin precursors via reverse transcription PCR. Expression patterns for sepiatocin, pro-sepiatocin elongation factor EF␥ and actin transcripts in SupM, supra esophageal mass, SubM, sub-esophageal mass, OL, optic lobes, OG, optic glands, GT, genital tract (penis or oviduct). Ctrl−, negative control (no template added to the PCR mix).

J. Henry et al. / Peptides 46 (2013) 159–166

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Fig. 5. MALDI-TOF analyses (A) MS spectrum of hemolymph extract. (B) MS/MS of ion 1153.56. (C*) Cysteine with iodoacetamide adduct.

Fig. 6. Activity of sepiatocin and mammalian oxytocin on the vena cava (A) oviduct (B and C) and penis (D) of cuttlefish. The peptide was applied at the times indicated by the arrows (scale bar: 5 min).

Acknowledgments We thank Grégory Chitel, the captain of the professional fishing boat “La Virgule” and his crew, and Stéphane André, the captain of the professional fishing boat “Fabrice-Daniel” for help in providing cuttlefish. Mass spectrometry analyses were performed by the technical platform PROTEOGEN of the University of Caen and the cuttlefish were bred in the marine station of Luc-sur-Mer (CREC).

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