Peptidomics Analysis of Neuropeptides Involved in Copulatory

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employ...
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Peptidomics Analysis of Neuropeptides Involved in Copulatory Behavior of the Mollusk Lymnaea stagnalis Z. El Filali,† J. Van Minnen,† W. K. Liu,‡ A. B. Smit,† and K. W. Li*,† Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, De Boelelaan 1085, Vrije Universiteit, 1081 HV, Amsterdam, The Netherlands, and Department of Anatomy, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong Received January 13, 2006

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens. Keywords: MALDI-TOF/TOF MS • peptidomics • cDNA cloning • snail

1. Introduction Neuropeptides comprise a class of structurally diverse primary messengers that regulate many aspects of animal physiology and behavior.1 The complexity in vertebrate neuronal systems, however, pose considerable difficulty for the detailed analysis of the functional contribution of the peptides. In past decades, several simple and experimentally accessible invertebrate model systems have been explored.2-7 The neuropeptidergic regulation of male copulation behavior in the freshwater snail, Lymnaea stagnalis, is an example of such an advantageous model system.8-10 Male copulation in Lymnaea is a flexible behavior consisting of a series of locomotory events that are synchronized with eversion of the penis from the body cavity, intromission, transfer of semen, and finally retraction of the penial complex back into the body cavity.11 These consecutive events are thought to be regulated by central neurons located in the right parts of the cerebral anterior lobes and ventral lobes, the pedal Ib clus* To whom correspondence should be addressed. Tel: 31-20-5987107. Fax: 31-20-5989281. E-mail: [email protected]. † Center for Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences. ‡ Faculty of Medicine, Chinese University of Hong Kong. 10.1021/pr060014p CCC: $33.50

 2006 American Chemical Society

ter, and a number of scattered cells in the pleural and parietal ganglia. These neurons use different classes of peptide co-transmitters that are transported from the central neurons via the penis nerve to the relevant muscular systems for the controlled movement of the penial complex and the vas deferens. For example, there are five isoforms of myomodulin that are contained in a single precursor. Functional studies showed that the peptides have overlapping yet distinct modulatory effects on the contraction and relaxation rate of the penis retractor muscle.10 The structurally highly related peptides, FMRFamide/ FLRFamide and SDPFLRFamide/GDPFLRFamide, are contained in two distinct precursors that are products of alternatively spliced forms of a single gene. These peptides are expressed in a mutually exclusive manner in different penis motoneurons, and have different effects on the penis retractor muscle.9 Although a number of peptides have been described in the penial system, direct mass spectrometric analysis of the penis nerve revealed the presence of additional molecular ion species in the mass range of 600-5000 Da,12 suggesting that additional peptides may be involved in the regulation of the activity of the penial complex. Liquid chromatography-tandem mass spectrometry approaches have been used successfully for the characterization Journal of Proteome Research 2006, 5, 1611-1617

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research articles of peptides from tissue extracts13,14 and even single neurons15 of vertebrate and invertebrate species such as crustaceans, insects and nematodes.16 In the present study, we employed a similar approach to characterize peptides contained in the male copulatory system of the snail Lymnaea stagnalis. In addition to the previously reported neuropeptides, several novel peptides that share the carboxyl termini of -FVRIamide have been detected. cDNA cloning revealed the organization of the precursor, which contains 20 neuropeptide domains with the carboxyl termini of -F(X)RIamide that are flanked by the conventional KR sites and the less commonly used (G)K or (G)R proteolytic sites. Furthermore, several putative monobasic R and dibasic RR and KK sites may also be used. As peptidomics did not characterize all the -F(X)RIamide peptides and since the generation of mature peptides cannot be predicted unequivocally based on the precursor structure, it is necessary to use an alternative approach to confirm the presence of all the mature peptides. We have separated the native peptides from the penis nerve extract using capillary C18 column, and collected the fractions off-line onto a MALDI metal plate. The 192 fractions on the metal plate were analyzed with MALDIMS. This MS survey scan detected the presence of all the molecular ion species that have the same masses as the predicted mature peptides. These molecular ion species were selected automatically and subjected to high energy CID consecutively. We have characterized all the mature -F(X)RIamide peptides, and revealed that only one of the three putative monobasic R sites is used for processing. Immunocytochemistry revealed that the -F(X)RIamide peptides were expressed in a number of neurons located in the right anterior cerebral ganglion, the pedal Ib cluster and in the penis nerve. Within the penial complex, immunoreactivities were present in structures resembling synapses that made close contact with muscle cells. Finally, application of synthetic -F(X)RIamide peptides inhibited the spontaneous contraction/relaxation cycle of the vas deferens, a part of the male copulatory system.

2. Materials and Methods 2.1. Animals. Mature L. stagnalis (22-30 mm) were used. They were bred under standard laboratory conditions in tanks with a continuous water supply, at 20 °C and a photoperiod of 12-h light/12-h dark. The animals were fed lettuce ad libitum. 2.2. Tissue Extraction and Partial Purification. 2.2.1. Sample for ESI-MS Analysis. Extraction of peptides from the penial complex was carried out as described.8-10 In brief, fifty penial complexes were dissected and stirred in 30 mL 100% acetone overnight, and then centrifuged for 30 min at 10 000 × g at 4 °C. The supernatant was diluted 25 times in 7.5 mM trifluoroacetic acid (TFA), and loaded into a 3 mL C18 solid-phase extraction column (Supelclean LC-18; from Supelco). The bound peptides were eluted with 2 mL of 60% acetonitrile in 7.5 mM TFA. The eluate was concentrated to 600 µL in a speedvac, centrifuged, and the supernatant was subjected to high performance gel permeation chromatography using an I-125 Protein Pak column (Waters Associates). The solvent was 30% acetonitrile in 7.5 mM TFA; the flow rate was 1 mL/min and 1 min fractions were collected. Fractions 11-13 were pooled and the peptides were partially fractionated by reversedphase HPLC using a Nucleosil C18 column (5 µm; 2.1 × 250 mm, Macherey-Nagel). The running solvent was 0.2% acetic acid, and the peptides were eluted using a linear gradient of acetonitrile from 0% to 36% in 60 min and from 36% to 60% in 1612

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20 min. The flow rate was 250 µL/min; one min fractions were collected and the peptides analyzed by ESI-mass spectrometry. 2.2.2. Sample for MALDI-MS Analysis. Fifty penis nerves were dissected and extracted in acetone:HCl:water (40:1:6 v/v). After centrifugation, the supernatant was diluted 25× in water and loaded into a 1 mL C8 solid-phase extraction column (Supelclean from Supelco). The bound peptides were eluted, dried in speedvac, and redissolved in 25 µL 7.0 mM TFA. A 10µL portion was injected into a C18 column (3 µm; 0.1 × 150 mm). The running solvent was 0.1% TFA, with an increasing linear gradient of acetonitrile to 50% in 30 min. The flow rate was 500 nL/min. Each 15 s fractions were mixed with 250 nl R-cynao-4-hydroxycinnaminic acid (5 mg/mL in 50% acetonitrile and 0.1% TFA) in a robotic device (Probot from Dionex), and then deposited sequentially on the 192 spotting position of the metal target plate from Applied Biosystems. After the fractions were dried the target plate was inserted into the mass spectrometer for analysis. 2.3. Functional Study. Synthetic peptides were synthesized by The Netherlands Cancer Institute (Amsterdam, The Netherlands) on a Millipore system. The posterior vas deferens was dissected under a microscope, suspended in a continuous perfusion bath (1 mL), and hooked to a length transducer using thin threads.8-10 Prior to testing substances, the preparation was superfused in snail Hepes ringer for at least 1 h by using a peristaltic pump at a flow rate of 1 mL/min. 2.4. Mass Spectrometry. For the characterization of peptides isolated from the penial complex an ESI-tandem mass spectrometer (Qtof from Micromass) was used as described.17 For each HPLC fraction, 6 µL was loaded into an ESI capillary, which was pulled from borosilicate glass capillary GC 100F-10 with a microcapillary puller. An internal wire electrode inserted inside the capillary was used for the measurement. For mass spectrometry analysis performed in MS1 mode, the quadrupole was operated in the rf-only mode and mass analysis was performed using the TOF analyzer. It was scanned between 250 and 1200 mass/charge. For tandem mass spectrometric experiments, precursor ion was selected using the quadrupole, fragmented in the collision chamber using energy around 20 eV and argon as the collision gas, and the daughter ions detected by the TOF analyzer. For primary sequence determination from penis nerve extract the MALDI-TOF/TOF-MS (Proteomics Analyzer 4700 from Applied Biosystems) was used.18 In brief, a MALDI target plate with the format of 192 sample spot positions was used to collect fractions that were eluted from the C18 HPLC capillary column. The mass spectrometer performed a survey scan to generate a peptide profile across the 192 fractions. All of the molecular ion species with masses corresponding to the predicted masses of -F(X)RIamide peptides were automatically selected and subjected to high-energy collision induced dissociation, using a collision energy of 1 keV and ambient air for collision. The fragment ions were detected by the second TOF detector. 2.5. Cloning and Sequencing of -FVRIamide cDNA. A degenerate oligonucleotide [RIa1: 5′-AAGGATCCGCNTT(T/C)GTNAGNAT(A/T/C)GG-3′] was synthesized based on the amino acid sequence of peptide GSAFVRIamide, and a λ-primer (5′CGCCAGGGTTTTCCCAGTCACGAC-3′) was synthesized based on a sequence in the cloning vector λZAP II. Using these, a partial -RIamide-encoding cDNA was amplified on cDNA of a λZAP II cDNA library of the central nervous system (CNS) of

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Lymnaea. PCR was performed in a 100 µL solution containing 10 mM Tris/HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.1 mg/ mL gelatin, 200 µM of each of the four dNTPs, 200 pmol FV1, 25 pmol λ-primer, and 0.5 U Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany), using 40 cycles: 94 °C for 20 s, 54 °C for 45 s, and 72 °C for 2 min. Amplified cDNA was digested with Bam H1, and a ∼250 nt fragment was cloned in pBluescript and sequence verified. Approximately 40 000 clones of a Lymnaea CNS-specific λZAP II cDNA library were plated at a density of 20 000 pfu/ 400 cm2, absorbed to charged Nylon membranes (Boehringer Mannheim), and hybridized to the Bam HI-digested PCR product randomly labeled with 10 µCi [R-32P]dATP (specific activity: >109 dpm/µg). Prehybridization and hybridization were performed in 6× SSC (1× SSC: 150 mM NaCl/15 mM Na-Citrate, pH 7.4), 5× Denhardt’s (according to Maniatis et al., 1982), 0.1% SDS, 20 µg/mL yeast tRNA at 65 °C for 3 and 20 h, respectively. Filters were washed in 0.5× SSC/0.1% SDS at 65 °C for 40 min and autoradiographed. Positive clones were re-screened at lower plaque density. RIa cDNA-containing pBluescript was generated from λZAP II by in vivo excision and sequenced in both orientations from universal primer sites and by primer walking using internal primers. 2.6. Antisera and Immunocytochemistry. Polyclonal antibodies raised against ASHFVRIamide were produced in mouse. Brains were fixed in 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 24 h. After dehydration and embedding in paraffin, serial 7 µm sections were adhered to double-coated (0.5% gelatin, 0.5% chrome alum in distilled water) microscope slides. Sections were de-paraffinized and rehydrated, washed in TBS (50 mM Tris-Hcl; pH 7.6, 150 mM NaCl) containing 0.25% gelatine and 0.5% Triton X-100, and incubated for 1618 h at 4 °C in primary antiserum (1:500 in the washing buffer). After rinsing with TBS the slides were incubated in Alexa 568conjugated secondary antibodies (Molecular Probes), 1:400 in washing buffer for 1 h at room temperature. The immunostained slides were examined using a Nikon Eclipse fluorescent microscope.

3. Results 3.1. Isolation and Characterization of Peptides from the Penial Complex. The acetone extracts of 50 penial complexes were size fractionated by high performance gel permeation chromatography. Mass spectrometric analysis revealed that fractions 11-13 contained molecules in the mass range of 5001500 Da. These three fractions were pooled and loaded into a C18 HPLC column for further separation. Major UV absorbance peaks clustered around 46-80 min (Figure 1) and were used for further ESI-mass spectrometric analyses. A large number of singly charged molecular ion species were detected by mass spectrometric analysis of the HPLC fractions. Collision-induced fragmentation of a number of them showed that they were not peptidergic in nature (data not shown). The identities of these molecular ion species are currently unclear. It is known that bioactive peptides are often carboxyl terminal amidated and/or contain a basic amino acid in the carboxyl terminus, and will be detected as multiple charged species by ESI-mass spectrometry. We therefore focused on doubly charged ion species, and successfully characterized 25 peptides (Table 1) belonging to the previously described myomodulins, small cardioactive peptides, FMRFamide, conopressin, pedal peptide, -FVamide, SPTR, -LFRFamide as well as a number of novel peptides. Several single peptides were present in

Figure 1. Reversed-phase HPLC separation of molecules contained in the size exclusion chromatography fractions 11-13 of the penial complex extract. The chromatogram reveals that most UV absorbing molecules eluted around 46-80 min. Table 1. Peptidomics Analysis of the Penial Complex and Penis Nervea ESI-MS/MS analysis of peptides from penial complex extract

ASSFVRIamide (45) ASNFVRIamide (47) LSSFVRIamide (47, 48) NPSSFVRIamide (45, 46) SPSSFVRIamide (45, 46) GLQMLRLamide (70) GLQM*LRLamide (64) PMSMLRLamide (67) PM*SM*LRLamide (67, 68, 69, 70) SLSMLRLamide (69, 70) SMSMLRLamide (65) SMSM*LRLamide (65) SGYLAFPRMamide (59) SGYLAFPRM*amide (59) QNYLAFPRMamide (70) GTLLRFamide (65) TDPLFMamide (59) FMRFamide (59) SDPFLRFamide (59) GDPFLRFamide (60) PFDSISGSHGLSGFA (65) CFIRNCPKGamide (59) SPTRTDEVLQEASGLALD (59) EI/LGSGI/LDDNI/Lamide (59) ENGSGI/LSDNI/Lamide (59)

MALDI data-dependent MS/MS analysis of -F(X) RIamide peptide from penis nerve extract

ASSFVRIamide ASNFVRIamide LSSFVRIamide NPSSFVRIamide SPSSFVRIamide SSREGQSSFVRIamide MNKFLRIamide PSSFVRIamide GSAFVRIamide ASHFVRIamide PNSFLRIamide IPSSAFVRIamide YPMNRFIRIamide

a The Ile and Leu, and Gln and Lys, repectively, could not be distinguished by the ESI-MS/MS analysis. Their identities are assigned based on previous studies and/or their predicted structures deduced from the cDNA studies. For novel peptides ambiguities of amino acid residues often exist. As their structural identities are not yet supported by independent analysis their (partial) sequences should be considered as tentative. The fraction numbers of the identified peptides eluted from HPLC are enclosed within brackets. The oxidized methionine residues are indicated with an asterisk.

multiple fractions, which were caused primarily by oxidation of the methionine residues. Novel peptides were also detected. Of notice is a class of peptides that share the same carboxyl termini of -FVRIamide. Although it is not possible to distinguish Leu from Ile by ESImass spectrometry because they have the same molecular mass, a subsequent cDNA study (see below, Figure 2) indicated that the carboxyl terminal residue must be Ile-amide. Peptides with similar amino acid sequence have previously been isolated from other species19,20 and have shown to have modulatory effects on muscle contraction. As structure and function of Journal of Proteome Research • Vol. 5, No. 7, 2006 1613

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Figure 2. Organization of -F(X)RIamide precursor as deduced by cDNA studies. There are several conventional KR processing site, and less commonly occurred processing sites (G)R, (G)K, and KK. At the carboxyl terminal region there are three monobasic R and a single RR sites (enclosed in dotted boxes) of which their processing cannot be predicted with certainty. The potential processing sites are enclosed in boxes. The peptides identified by MALDI-MS/MS as shown in Figure 3 are highlighted in bold.

neuropeptides are often conserved during evolution, we hypothesized that the -FVRIamide peptides regulate Lymnaea muscular systems including those in the penial complex, and so they may be involved in the copulation processes. We have therefore carried out in depth studies on this class of peptides. 3.2. cDNA Cloning of -FVRIamide Peptide Precursor. The -FVRIamide peptides share the same carboxyl termini suggesting that they may be derived from a single precursor. Although -FVRIamide peptides have been isolated from several species, the precursor structure of the peptides has not been reported. To elucidate the organization of the precursor, oligonucleotides were constructed based on the sequence of GSAFVRIamide (GSAFVRIG) and used for further cDNA cloning studies. Figure 2 shows the predicted sequence of the peptide precursor, which contains a signal peptide followed by 20 peptide domains and the acidic connecting peptide domains. In addition to the KR processing sites, many peptide domains are 1614

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flanked by (G)R, (G)K, or KK sites. Furthermore, at the carboxyl terminal region there are three monobasic R sites at amino acid residues 389, 393, and 429, and a single dibasic RR site at amino acid residue 425-426. These putative processing sites are encoded within two peptide domains; therefore some of these processing sites would not be used to generate peptides. The carboxyl termini of most of the predicted bioactive peptides are -FVRIamide, and a few with -FLRIamide or -FIRIamide. The common carboxyl terminus for the peptides is thus -F(X)RIamide. All together, seven ASSFVRIamide, two SPSSFVRIamide and single copies of eleven related -F(X)RIamide peptides can be processed from the precursor. There are a number of (acidic) peptide domains that connect the -F(X)RIamide domains together and may be involved in precursor processing. These peptides generally do not have a functional role. They degrade rapidly and are often not detected as mature peptides.21 3.3. Confirmation of the Presence of all the Predicted -F-

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Figure 3. MALDI-high energy-CID tandem Mass spectrum of ASSFVRIamide from penis nerve extract. Penis nerve peptides were separated by a capillary rpHPLC column and deposited directly onto a metal target in a total of 192 fractions. A mass spectrometric survey scan of the fractions detected those containing the molecular ion species corresponding to the calculated masses of -F(X)RIamide peptides. These peptides were automatically selected for high-energy collision induced dissociation analysis. As a representative example the fragment ion spectrum of one of the peptides is shown. Extensive b- and y- ions and some side chain cleavage unequivocally confirm the peptide identity as ASSFVRIamide. The fragment ion spectra of all other -F(X)RIamide peptides are shown in the supplementary Figure.

(X)RIamide Peptides in Penis Nerve. The cDNA cloning study indicated that 13 related -F(X)RIamide peptides can be formed from the -F(X)RIamide precursor. The off-line ESI-MS/MS analysis has identified only a subset of these peptides. To characterize all the -F(X)RIamide peptides that may be generated from the precursor protein we used a data dependent MALDI- TOF/TOF mass spectrometric technique to specifically detect and characterize these peptides. We took advantage of the predicted processing sites present in the precursor protein to calculate the expected molecular masses of the mature peptides, and used them to construct an inclusion list of parent ion selection for MS/MS experiment. A peptide extract from 50 penis nerves was separated by a 100 µm ID C18 HPLC column and the fractions deposited directly on the metal plate for MALDI TOF/TOF mass spectrometric analysis. Many small molecular ion species were detected around 600-3000 Da. A survey mass spectrometric scan of the fractions detected molecular ion species with masses corresponding to the calculated masses of -F(X)RIamide peptides. There are molecular ion species with similar masses but different hydrophobicity eluting at different fractions. As molecular mass alone and/or in combination with elution position from C18 column cannot identify the peptides with certainty, all of these molecular ion species were selected automatically and subjected sequentially to high energy collision dissociation for structural characterization. As an example Figure 3 shows the fragment ion mass spectrum of ASSFVRIamide. The mass spectra of all other F(X)RIamide peptides are shown in the Supporting Information Figure. All of these fragment ion spectra have extensive sequence coverage of the -F(X)RIamide peptides with strong b- and y- ion series, and some internal fragment ions and side chain cleavage fragments. This demonstrates the presence of all the -F(X)RIamide peptides in the penis nerve, and confirms the use of the (G)R, (G)K, KR, KK, and R (residue 393) and RR (residue 425-426) processing sites. 3.4. Immunocytochemistry Localization of the -F(X)RIamide Containing Neurons. Immunocytochemistry of Lymnaea brain sections revealed that the -FVRIamide immunoreactive neurons were located in the buccal ganglia, in and

around the anterior lobe of the right cerebral ganglia, in the right parietal ganglion, and within the pedal Ib cluster. As expected, -FVRIamide immunoreactive axons were prominently present in the penis nerve (Figure 4A). In the vas deferens, -FVRIamide immunoreactive neuronal endings often converged onto the muscle fibers (Figure 4B). Similar distribution patterns of the -FVRIamide immunoreactive neurons were observed in the penis. In the penis retractor muscle the -FVRIamide immunoreactive neurons could not be detected (data not shown). 3.5. Effect of -F(X)RIamide on the Contraction of Vas Deferens. The presence of -FVRIamide immunoreactive neuronal endings around the muscle fibers of the vas deferens suggests that the peptides may exert their effects on this tissue. We have tested the effect of several peptides, namely SPSSFVRIamide, PSSFVRIamide, PNSFLRIamide, and SSREGQSSFVRIamide. Addition of the peptides to the bioassay chamber inhibited the spontaneous contraction/relaxation cycle of the vas deferens in a dose-dependent manner (Figure 5). This confirms the immunocytochemical data indicating that the peptides may regulate penis muscle activity. SPSSFVRIamide and PSSFVRIamide have the same primary sequence with the exception of a single extra N-terminal amino acid for SPSSFVRIamide. Accordingly, these two peptides have similar potency on vas deferens activity. SSREGQSSFVRIamide has an extended N-terminal region and showed a lower potency than PSSFVRIamide and SPSSFVRIamide. PNSFLRIamide also has a lower potency. This peptide differs from PSSFVRIamide with amino acid substitutions at two positions.

4. Discussion Liquid chromatography in conjunction with ESI-tandem mass spectrometry identified a number of neuropeptides from the penial complex extract of L. stagnalis. Similar approaches have been applied to detect and characterize tens of neuropeptides from other animal species.13-14,16 Some of the peptides existed in native and methionine- oxidized form. Oxidation of methionine is frequently observed and may be an artifact during sample preparation and/or peptide isolation. In addition to previous identified peptides, we have characterized several Journal of Proteome Research • Vol. 5, No. 7, 2006 1615

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Figure 4. Central -FVRIamide immunoreactive neurons innervate the penial complex. (A) The penis nerve contains bundles of -FVRIamide immunoreactive axons (arrows). (B) Immunofluorescent labeling of the -FVRIamide axons (arrows) in the vas deferens, many of which appear to appose to muscle fibers (arrows). Arrowheads point at immunoreactive fibers in a nerve located in the connective tissue of the vas deferens. The alternating contractions of CMV and LMV generate peristaltic movements that transport sperm to the penis. The release sites of -F(X)RIamide are close to the muscle fibers suggesting that the peptides may regulate vas deferens muscle activity. L, lumen of vas deferens; CMV, circular muscle of vas deferens; LMV, longitudinal muscle of vas deferens. The bar represents 10 µm in length.

Figure 5. -F(X)RIamide peptides induced dose dependent inhibition of the vas deferens spontaneous contraction/relaxation cycle. The four -F(X)RIamide peptides all show inhibitory effect on the contraction frequency of vas deferens, but differ slightly in their potency. The maximum effect of the -F(X)RIamide, i.e., the inhibitory effect of SPSSFVRIamide at 10-4 M on vas deferens contraction/relaxation cycle, was set at 100%. Dark blue line, SPSSFVRIamide; light blue line, SSREGQSSFVRIamide; pink line, PSSFVRIamide; yellow line, PNSFLRIamide.

novel -FVRIamide peptides. The de novo sequencing of peptides is not straightforward, because several isobaric amino acids such as Lys/Gln and Leu/Ile cannot be differentiated by low-energy collision fragmentation. Furthermore, in most instances only partial fragment ion series were observed. As the genome of Lymnaea has not been sequenced, the sequence tag from incomplete fragment ion series does not allow us to characterize the peptides. For this reason, the cDNA cloning study has been carried out to reveal the structural organization of the peptide domains in the precursor. The -F(X)RIamide peptides are encoded in a single precursor, i.e., 7 copies of ASSFVRIamide, 2 copies of SPSSFVRIa and single copies of LSSFVRIamide, ASHFVRIamide, etc. The peptide domains are flanked by the KR, (G)R, (G)K, KK, and putative R and RR processing sites. The (G)K and (G)R sites occur less frequently than the KR site. Nevertheless, as glycine serves for translational amidation during precursor processing it is very likely that (G)R and (G)K are used as processing sites. Indeed, our recent mass spectrometric single cell analysis on the processing of another peptide precursor, the S/GDPFLRFamide precursor, revealed that the (G)K and (G)R are the functional processing sites.22 In line with the previous studies, here we have structurally characterized all of the -F(X)RIamide peptides by data dependent MALDI-tandem mass spectromet1616

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ric analysis and confirmed the use of the KR, (G)K, (G)R, RR, KK, and monobasic R processing sites. The structural diversity of the -F(X)RIamide peptides appears predominantly at the amino termini, and this is in line with the general finding for many small peptide messengers contained in single precursors.23 Previously, it has been suggested that the structural determinant of the biological activity of the peptides resides in the carboxyl termini, which are evolutionary conserved. The amino termini are less conserved and may be responsible for the differences that exist among the isoforms.23 Indeed, SPSSFVRIamide and PSSFVRIamide have similar potency in inhibiting contraction/relaxation cycle of the vas deferens, whereas the N-terminal extended form SSREGQSSFVRIamide has a lower potency. The lower potency of PNSFLRIamide may be caused by the amino acid substitution at the N-terminal and/or the substitution of Leu for Val within the consensus sequence of -F(X)RIamide. Recently, studies of the structure-activity of diverse -F(X)RIamide peptides on the electric activity of identified central neurons of Helix aspersa reported a similar finding.24 Several -F(X)RIamide peptides have been identified in molluscan species (Helix pomatia, Achatina fulica, Fusinus ferrugineus, and Andonta cygnea) and other invertebrates (Echiura, and Annelida).19 The LSSFVRIamide isolated from the ganglia of F. ferrugineus was shown to exhibit bi-phasic effects on the twitch contraction of the buccal radula retactor muscle.18 In Lymnaea, the -FVRIamide immunoreactive neurons were also detected in the buccal ganglia and shown to innervate the buccal muscles. Therefore, the -F(X)RIamide peptides in Lymnaea may regulate the buccal system involved in feeding behavior, in addition to their effects on the penial complex. Over the years, we have functionally characterized many neuropeptides from the penial complex. A future challenge will be to clarify how neurotransmission of different peptides is coordinated to produce the series of copulation events during mating.

Acknowledgment. We thank The Netherlands research organization (ALW, NWO) for the financial supports of the project to K.W.L., and the Centre for Medical Systems Biology from Netherlands Genomics Initiatives for the support of mass spectrometry to K.W.L. and A.B.S.; Miss Nederlof, M. and Miss Wang, H.M. for their technical assistance. Supporting Information Available: The mass spectra of all other F(X)RIamide peptides (Supporting Information

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Figure). This material is available free of charge via the Internet at http://pubs.acs.org.

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