Isolation and Characterisation of Conomap-Vt, a D-Amino Acid Containing Excitatory Peptide from the Venom of a Vermivorous Cone Snail

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FEBS Letters 580 (2006) 3860–3866

Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail

Se´bastien Dutertre1, Natalie G. Lumsden, Paul F. Alewood, Richard J. Lewis* Institute for Molecular Bioscience, The University of Queensland, Brisbane, 4072 Qld, Australia

Received 28 March 2006; revised 22 May 2006; accepted 2 June 2006

Available online 16 June 2006

Edited by Hans Eklund

containing a D-amino acid from Conus vitulinus venom. This Abstract Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic novel peptide, named conomap-Vt (Conp-Vt), showed no transmission. Here, we report the discovery of conomap-Vt homology to previously discovered conopeptides, but had up (Conp-Vt), an unusual linear tetradecapeptide isolated from to 65% sequence identity with ‘‘myoactive tetradecapeptides’’ Conus vitulinus venom. The sequence displays no homology to (MATPs), found in non-venomous molluscs, annelids and in- known conopeptides, but displays significant homology to pep- sects [4]. MATPs are fourteen amino acid long peptides with tides of the MATP (myoactive tetradecapeptide) family, which excitatory actions in invertebrate smooth muscles. However, are important endogenous neuromodulators in molluscs, annelids their precise mode of action has not been determined. and insects. Conp-Vt showed potent excitatory activity in several Biochemical studies revealed the presence of a D-phenylala- snail isolated tissue preparations. Similar to ACh, repeated doses nine in Conp-Vt at position 2. A D-amino acid is a rare of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic post-translational modification, even amongst the highly antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt con- post-translationally modified Conus peptides [5]. Indeed, only tractions were non-cholinergic in origin. Finally, biochemical 4 conopeptides containing a D-amino acid have been isolated studies revealed that Conp-Vt is the first member of the MATP to date, with no obvious pattern in the position of the isomer- family with a D-amino acid. Interestingly, the isomerization of ised residue, suggesting the presence of several isomerases in L-Phe to D-Phe enhanced biological activity, suggesting that this cone snail venoms [6–9]. D-amino acid peptides have also been post-translational modified conopeptide may have evolved for found in other venoms, including those from spiders and frogs prey capture. and an isomerase has been characterised from the American 2006 Published by Elsevier B.V. on behalf of the Federation of funnel-web spider Agelenopsis aperta venom and from Bombi- European Biochemical Societies. nae frog skin secretions [10,11]. Recently, several D-amino acid containing peptides were also identified in the venom of a Keywords: Cone snail; Conotoxin; Venom peptide; Post- translational modification; Molusc selective bioactive mammal, Ornithorhynchus anatinus the Australian platypus, along with evidence for the presence of an isomerase [12–14]. Clearly, the isomerization of amino acids is more widely dis- tributed than once thought, but the role of such modification is not well understood. We propose that L to D isomerization in Conp-Vt contributes to enhanced activity of the venom. 1. Introduction

Cone snails are predatory marine gastropods that rapidly immobilise their prey by injecting potent venom peptides 2. Materials and methods through a harpoon-like radula tooth [1]. Most conopeptides are antagonists, blocking receptors of key physiological role 2.1. Materials with high affinity and specificity. The few agonist conotoxins Boc protected amino acids and reagents used during chain assembly and HPLC purification (dimethylformamide, dichloromethane, aceto- identified typically show homology to endogenous neurotrans- nitrile (ACN) and trifluoroacetic acid (TFA)) were peptide synthesis mitters. While only a relatively small number of conotoxins grade and purchased from Auspep (Melbourne, Australia) and Nova- have been fully characterised, several are now widely used as biochem (San Diego, CA). MBHA-NH2 resin was obtained from Ap- research tools and a few are being developed as therapeutics plied Biosystems (Foster City, CA). Drugs were from Sigma (St. Louis, [2]. Conus venom peptides are usually highly constrained struc- MO, USA). D-phenylalanine was a gift from Peter Cassidy (Institute for Molecular Bioscience, Australia). tures, typically containing 1–3 disulfide bonds [3]. Here we report the isolation of a small linear excitatory peptide 2.2. Animals Male Wistar rats (150–250 g) sourced from the Central Animal *Corresponding author. Fax: +61 7 3346 2101. Breeding House, The University of Queensland (St. Lucia, Brisbane, E-mail address: [email protected] (R.J. Lewis). Australia) were killed by 100% CO2 followed by cervical dislocation before dissection of tissues. Pomacea bridgesi snails (5–15 g) purchased 1 Present address: Department of Neurochemistry, Max-Planck-Insti- from a local pet shop (Westside Pets & Aquarium, Brisbane, Australia) tute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt, were maintained for at least a week before use in an aerated aquaria Germany. containing dechlorinated tap water (neutral pH; ambient temperature; 12:12 dark:light cycle). All experiments involving the use of animals Abbreviations: ACN, acetonitrile; Conp-Vt, conomap-Vt; MATP, were conducted with the approval of the University of Queensland myoactive tetradecapeptide; TFA, trifluoroacetic acid Animal Experimentation Ethics Committee.

0014-5793/$32.00 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2006.06.011 S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866 3861

2.3. Isolation and sequencing 2.4. Mass spectrometry Specimens of C. vitulinus of the C. planorbis/vitulinus complex Molecular mass analyses of the purified active venom peptide, syn- (Fig. 1A inset shows the C. vitulinus form of this presently not well- thetic isomers, and LC–MS analysis on crude venom and digested pep- defined complex) were collected from the Great Barrier Reef, Queens- tides were performed on a PESciex API QSTAR Pulsar instrument land, Australia. The venom ducts were dissected and venom extract over m/z 400–2000. LC conditions were: Phenomenex 5 lmC18 prepared as previously described [15]. A total of 80 · 1 min fractions 2.1 · 150 mm column eluted with 0–60% B in 60 min, (A: 0.1% formic were collected and stored at 4 C until further use. Conp-Vt was puri- acid; B: 90% ACN, aqueous 0.09% formic acid) at 300 lL/min. Data fied from fraction 27 on analytical RP-HPLC (C18 Phenomenex col- were analysed using Analyst software. umn) with a linear gradient of solution B (B: 90% ACN, 0.45% TFA; A: 0.5% TFA). The flow rate was 1 mL/min and absorbance 2.5. Synthesis and purification of peptides monitored at 214 nm. Pure peptide (20 pmol) was submitted to the Biomolecular Research Facility (Newcastle, NSW, Australia) for All peptides were assembled in high yield using Boc chemistry with N-terminal sequencing. in situ neutralisation protocols on a 0.5 mmol scale [16]. Semiprepara- tive chromatographic separation of crude peptides from HF cleavage was achieved on a C18 Phenomenex RP-HPLC column using a linear gradient from 0% to 60% B (A: 0.5% TFA; B: 0.45% TFA, 90% ACN) in 60 min. The UV absorbance of the eluant was monitored at 220 nm, and collected fractions were analysed by electrospray ioni- sation mass spectrometry (ES-MS). Pure fractions as determined by analytical RP-HPLC containing the correct mass for each peptide were pooled and lyophilised. Peptide aliquots were prepared in MilliQ water and stored at 4 C.

2.6. Trypsin digestion Pure native and synthetic peptides were digested with trypsin enzyme (Trypsin TPCK treated from bovine pancreas, Sigma). Peptides and trypsin were dissolved in ammonium bicarbonate buffer (pH 8.5) to a concentration of 1 mg/mL. Trypsin was added to a 300 lL sample of peptide (ratio 1:50) and placed in an oven at 37 C. After 2 h and 24 h, 50 lL aliquots of the digest were injected in a Phenomenex C18 analytical RP-HPLC column. The peptides were eluted using a linear gradient of solution B (90% ACN; 0.45% TFA). The flow rate was 1 mL/min and absorbance monitored at 214 nm. Digest products were identified through LC–MS.

2.7. Bioassays Conp-Vt was tested for biological activity on molluscan and mammalian isolated tissue preparations. In vitro preparations were mounted in 5 mL isolated organ baths containing a physiological salt solution (specified below), which was bubbled with carbogen (95% O2 and 5% CO2). Preparations were equilibrated in the organ bath for at least 30 min before addition of drugs. Responses were measured isometrically via a Grass force displacement trans- ducer (FTO3) and recorded on a MacLab system (ADInstruments, Castle Hill, NSW, Australia). When required, electrical field-stimu- lation was generated with a Grass S44 stimulator (Quincy, MA, USA). The left and right rat atria were isolated, bisected and mounted onto electrodes or tissues holders, respectively, under 0.75 g resting tension in organ baths containing Tyrodes solution (composition in mM: 137 NaCl, 2.7 KCl, 1.4 CaCl2, 0.5 MgCl2, 12 NaHCO3, 0.4 NaH2PO4 and 5.6 D-glucose) maintained at 37 C. The left atrium was electrically stimulated (7–12 V, 5 ms duration, 2 Hz). The isolated rat ileum was dissected into segments (2 cm) and mounted onto tissue holders in organ baths containing Tyrodes solution under 0.4 g resting tension at 34 C. Ileal contractions induced by 10 lM ACh was used to define the maximal tissue response and any changes to this response at the end of each experiment. Tissues were exposed to 1 lM of each peptide. A recovery period of 3–10 min after peptide or drug addition as well as after washes was allowed between experiments. Adult snails (P. bridgesii) were dissected on a sylgard petri dish in snail ringer (composition in mM: 80 NaCl, 4 KCl, 10 CaCl2, 5 MgCl2, 10 TRIS, 5 glucose; pH 7.4). The whole buccal mass was removed with cerebral ganglion and mounted onto a tissue holder at 0.6 g resting tension in organ baths containing the snail ringer. Other snail tissues, including 1 cm section of crop, penis retractor and foot muscles, were mounted as described for the buccal mass. The contraction in- Fig. 1. Analytical RP-HPLC chromatograms. (A) C. vitulinus crude duced by 100 lM ACh was used as a positive control (100% maximal venom fraction 27. Inset shows a C. vitulinus shell. (B) Purified native response) and applied at the beginning and end of each experiment. Conp-Vt. (C) Purified synthetic L-Phe-Conp-Vt. This synthetic version Responses to the peptide and its analogues were expressed as a per- of Conp-Vt eluted 5 min earlier than native Conp-Vt on the same C18 centage of the response induced by 100 lM ACh. Tachyphylaxis of tis- column under the same conditions, indicating the presence of a post- sues to Conp-Vt or ACh was determined by repeat application to the translational modification in native Conp-Vt. Monoisotopic masses tissue (without washing between additions) until no further response are indicated. was observed. 3862 S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866

2.8. Data analysis tus of cone snails is thought to have evolved from digestive or- Data analysis was performed with a one-way analysis of variance gans [20], which may explain the origin of Conp-Vt in the (ANOVA) followed by a Bonferroni-corrected multiple t test using venom. GraphPad Prism 4.0. Values of P < 0.05 were considered significant. Data were expressed as means ± S.E.M. 3.2. Conp-Vt contains a D-amino acid Conp-Vt and MATP-Ls peptides were synthesised in high 3. Results and discussion yield using Boc chemistry. MATP-Ls peptide was chosen for comparative studies because of its high sequence identity 3.1. Isolation and sequencing of Conp-Vt (65%) with Conp-Vt and its established biological activity Conp-Vt was isolated from the crude venom of C. vitulinus in mollusc tissues. Unexpectedly, analytical RP-HPLC chro- as a major peak at fraction 27 of a semipreparative C18 frac- matograms revealed that synthetic Conp-Vt did not co-elute tionation. The peptide was purified to homogeneity on RP- with the native peptide (Fig. 1C), suggesting that a post-trans- HPLC using a linear gradient of ACN (Fig. 1A and B). Mass lational modification may be present in Conp-Vt. Since both spectrometry established a monoisotopique mass of synthetic and native peptides exhibit the same mass, the pres- 1488.86 Da that was unchanged after TCEP reduction. This ence of a D-amino acid in native Conp-Vt was suspected. To result suggesting that the peptide contained no disulfide bonds further explore the possibility that a D-amino acid was present was subsequently confirmed by the Edman sequence, in Conp-Vt and to start to determine its position in the se- AFVKGSAQRVAHGY. The calculated mass (1489.77 Da) quence, both native and synthetic peptides were enzymatically was consistent with an amidated C-terminal, a common digested and the products analysed using RP-HPLC and LC– modification in Conus peptides [5]. MS. Conp-Vt possesses one arginine and one lysine allowing This novel sequence had no homology to known conopep- trypsin digestion to produce 5 different peptide fragments tides. However, a BLAST search using the full-length sequence (Table 2). In theory, peptide fragments of the native Conp- revealed high similarity with a family of MATP from molluscs Vt that contain a D-amino acid will display a different elution and annelids, and significant homology with a family of peptide time compared to the synthetic peptide fragments, while those hormones from insects (Table 1). Given its similarity to without a D-amino acid should have identical retention times. MATPs, we named this C. vitulinus venom peptide conomap- After 2 h of trypsin digestion, the mixture of digested native Vt (Conp-Vt). All peptides of the MATP family induce contrac- and synthetic peptides revealed 4 peaks when injected into ana- tions in mollusc, annelid or insect isolated muscle preparations lytical RP-HPLC (Fig. 2A and B). LC–MS identified fragment [17]. Conp-Vt had 65% sequence identity with a Lymnaea 1, 3, 4 and 5, while fragment 2 was not found, indicating that peptide MATP-Ls [18]. Interestingly, while this endogenous trypsin was more efficient to cleave after the arginine than after peptide from Lymnaea can modulate the activity of various the lysine. Only fragment 1 and 5 had a different elution time in peripheral muscles, it is found in the nervous system in high native and synthetic peptide digests. Therefore, a D-amino acid quantity, suggesting that MATP-Ls is a neuropeptide that was predicted to occur in the first 4 residues of the native might be produced in the CNS [17]. In addition, tissue localisa- peptide. tion of a related peptide (ETP) from the worm Eisenia foetida Other linear molluscan peptides containing a D-amino acid revealed a high immunoreactivity in the CNS and digestive have a contrasting pattern of epimerisation compared to what tract including oesophagus and intestine, while other tissues has been found for the Conus peptides (see Section 2.4.). In- including the body wall muscles, sexual organs and nephridia deed, all D-amino acid containing peptides isolated from mol- contained low ETP immunoreactivity [19]. The venom appara- luscs other than Conus have incorporated a modified amino acid at position 2, regardless of the nature of the amino acid [4]. Conp-Vt is a small linear peptide that resembles the molluscan peptides shown in Table 3 more than disulfide contain- Table 1 MATP related peptides ing conopeptides possessing D-amino acids. As a consequence, Conp-Vt was hypothesised to contain a D-amino acid at Phylum-species Sequence Name position 2. Mollusca It is believed that in the precursor gene of D-amino acid con- Conus vitulinus AFVKGSAQRVAHGY* Conomap-Vt taining peptides the D-amino acid is encoded as a L-amino acid (Conp-Vt) Lymnaea stagnalis GFRANSASRVAHGY* Lymnaea MATP that later undergoes enzyme isomerization [9]. Therefore, it is (MATP-Ls) * Achatina fulica GFRQDAASRVAHGY Achatina MATP1 * Achatina fulica GFRGDAASRVAHGF Achatina MATP2 Table 2 Fusinus ferrugineus GFRMNSSNRVAHGF* Fusinus MATP Predicted Conp-Vt fragments after trypsin digestion Peptide fragments (Fragment Average Monoisotopic Annelida number) mass mass Eisenia foetida GFKDGAADRISHGF* ETP Pheretima vittate GFRDGSADRISHGF* PTP AFVKGSAQRVAHGY* (Parent) 1489.70 1488.79

Arthropoda AFVK- (1) 463.57 463.27 Aedes aegypti APFR-NSEMMTARGF* -GSAQRVAHGY* (2) 1044.13 1043.52 Manduca sexta GFK-NVEMMTARGF* Allatotropin -GSAQR- (3) 517.54 517.26 Locusta migratoria GFK-NVALSTARGF* Lom-AG-MT I -VAHGY* (4) 544.61 544.27 * indicates an amidated C-terminal. Underlined residue indicates a AFVKGSAQR- (5) 963.10 962.52 D-amino acid. Sequences of the peptides are from [17] and references * indicates an amidated C-terminal. Arrows indicate the trypsin therein. cleavage sites. S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866 3863

Table 3 Small linear molluscan peptides containing a D-amino acid residue Mollusc Sequence Name Achatina GFAD Achatin-I Octopus GFGD Achatin-RP-I Octopus GSWD Achatin-RP-II Achatina FNEFV* Fulicin Achatina YAEFL* Fulyal Mytilus ALAGDHFFRF* FFRFamide Helix LFCNGYGGCGNL* CCAP-RP-III Aplysia NWF* NdWFamide * indicates an amidated C-terminal. Underlined residues indicate a D-amino acid. Sequences of peptides are from [4] and references therein.

3.3. Conp-Vt has a D-phenylalanine in position 2 A synthetic Conp-Vt containing a D-phenylalanine in position 2 was synthesised as described for the all L peptides. Syn- thetic D-Phe-Conp-Vt was subjected to the same trypsin digestion protocol described for the L-Phe-Conp-Vt and native peptide. After 2 h of digestion, the mixture was injected into analytical RP-HPLC. The chromatogram revealed an identical pattern of digestion compared to the native peptide (Fig. 2C). In addition, synthetic and native Conp-Vt also coeluted. Therefore, the correct sequence for Conp-Vt is AFVKG- SAQRVAHGY-NH2, where F is D-phenylalanine. The all L analogue will be referred to as L-Phe-Conp-Vt. The L to D isomerization appears to be a rare post-translational modification, even amongst the highly post-translationally modified Conus peptides. The first conopeptides shown to contain a D-amino acid were the contryphans, discovered in the venom of Conus radiatus and C. purpurascens [7,8]. The modified amino acid was found in position 4 in both peptides. More recently, 2 novel families of conopeptides containing a D-amino acid have been identified [6,9]. First, a large excitatory peptide (46 a.a.) isolated from C. radiatus venom and belonging to the I superfamily was found to have a D-Phe in position 44 (the second to the last amino acid of the polypep- tidic chain). Second, two small peptides (8 a.a.) isolated from the venom of Conus gladiator and Conus mus were found to have a D-hydroxyvaline in position 6. Therefore, it appears that the epimerisation in conopeptides occurs at various posi- Fig. 2. Analytical RP-HPLC chromatograms of peptide trypsin tions in the sequence, suggesting the presence of several highly digests. (A) Native Conp-Vt, (B) L-Phe-Conp-Vt analogue and (C) synthetic Conp-Vt trypsin digests (2 h). After 2 h of trypsin digestion, specific isomerases in the venom. the mixture was injected into analytical RP-HPLC. For each peptide, 4 peaks were obtained, corresponding to fragments 1, 3, 4 and 5 3.4. Conp-Vt is a potent and mollusc specific excitatory peptide (Table 2). Fragments 1 and 5 had elution times that differed between Peptides belonging to the MATP family are known to exert native and L-Phe-Conp-Vt, suggesting that a D-amino acid was present at position 1, 2, 3 and/or 4 in the peptide. In contrast, synthetic Conp- a concentration-dependent excitatory effect on a variety of Vt containing a D-phenylalanine in position 2 exhibited the same invertebrate tissues [18,19]. Therefore, the biological activity elution profile as native Conp-Vt. Monoisotopic masses of each peak of Conp-Vt was tested on different molluscan muscle prepara- are indicated. tions including the isolated buccal mass, crop, penis retractor muscle and foot. All tissues responded to the application of Conp-Vt (Fig. 3). Except for the foot muscle, the initial con- expected that both L and D isoforms of Conp-Vt could be centration of Conp-Vt (10 nM) produced a contraction or en- found in the crude venom of C. vitulinus. Accordingly, an hanced existing spontaneous contractions indicating that ion extraction over the range 745.2–745.6 Da was performed Conp-Vt had a potent excitatory effect, particularly on the on an LC–MS spectrum of C. vitulinus crude venom and 2 buccal mass. Conp-Vt was also tested on the rat isolated left peaks separated by 5 min corresponding to the mass of and right atrium, and rat isolated ileum preparations. No Conp-Vt (MH2+ = 745.4 Da) were identified (data not shown). activity was detected at concentrations up to 1 lM (data not However, the L form (assumed to be the early eluting peak) shown). Hence, the snail isolated buccal mass preparation represented only 0.6% of the D form, indicating that the was chosen for further pharmacological characterisation. enzyme responsible for the epimerisation of Conp-Vt is an 1 lM Conp-Vt produced a response similar in amplitude to efficient converting enzyme. 100 lM ACh but this contracture was overlayed with spikes of 3864 S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866

Fig. 4. Non-cumulative additions of ACh and Conp-Vt in the snail buccal mass. (A) ACh (1 = 10 lM; 2 = 100 lM; 3 = 200 lM; W = wash) and Conp-Vt (4 = 10 nM; 5 = 100 nM; 6 = 1 lM; W = wash). Application of drug is indicated by the arrowheads. Vertical bar represents 0.5 g and horizontal bar 10 min. (B) Concen- tration–response curve for Conp-Vt in the snail buccal mass (EC50 = 8 ± 0.25 nM; n = 5). The amplitude of contraction induced by diﬀerent Conp-Vt doses was normalised to the initial contractile response to 100 lM ACh (0.92 ± 0.14 g, n = 30).

defined as the acute loss of response upon repeated stimula- tions with agonists [21]. Similar to the activity produced by ACh, Conp-Vt induced tachyphylaxis after a few repeat appli- cations (Fig. 5A and B). However, while ACh tolerant tissue failed to generate a Conp-Vt response, Conp-Vt tolerant tissue Fig. 3. Cumulative additions of Conp-Vt in the snail (A) buccal mass, still responded normally to ACh (Fig. 5A and B). This result (B) crop, (C) foot muscle and (D) penis retractor muscle. Application suggests that Conp-Vt develops tachyphylaxis independent of of peptide is indicated by the arrowheads, with 1 = 10 nM; cholinergic receptors. To start to dissect the direct and indirect l 2 = 100 nM; 3 = 1 M; W = wash. Conp-Vt initiated a contraction in components of the Conp-Vt contractile response, ACh antag- all tissues tested, with the greatest response observed in the buccal mass. Vertical bar represents 0.5 g and horizontal bar represents onists a-bungarotoxin, d-tubocurarine and atropine were 10 min. tested for their ability to block the Conp-Vt induced contraction. Surprisingly given the lack of response in ACh tolerant tissue, responses to 100 nM and 1 lM of Conp-Vt were not spontaneous contractions (Fig. 4A). A complete concentra- modified (Fig. 5C) following pre-treatment with a high concen- tion–response curve for Conp-Vt confirmed the high potency tration of a mix of all three cholinergic antagonists. However, of this peptide (EC50 = 8 ± 0.25 nM; Fig. 4B). responses to ACh were also unmodified in the presence of these antagonists (data not shown). Limitations in characterising 3.5. Conp-Vt interacts with non-cholinergic receptors cholinergic activity in molluscan isolated preparations are also To further study the mechanism of action of Conp-Vt it was highlighted in other studies [22–24], which found inconsisten- tested for its ability to induce tachyphylaxis. Tachyphylaxis is cies in the ability of atropine and d-tubocurarine to antagonise S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866 3865

Fig. 5. Preliminary pharmacological characterisation of Conp-Vt. (A) Tachyphylaxis to (A) ACh and (B) Conp-Vt was achieved by repeated administration of 100 lM ACh or 1 lM Conp-Vt, respectively (n = 4 each). (C) The effect of cholinergic antagonists on Conp-Vt contractions in the snail isolated buccal mass. a-Bungarotoxin (1 lM), d-tubocurarine (10 lM) and atropine (10 lM) were added at least 10 min prior to the addition of Conp-Vt (n = 4). (D) The contractile activity produced by 1 lM of conp-Vt and related peptides. A = MATP-Ls, B = D-Phe-MATP-Ls, C = L-Phe- Conp-Vt and D = Conp-Vt (n = 4 for each peptide). * P < 0.05 indicating the response was significantly different to the response produced by A, BorD.

ACh responses. Indeed, the excitatory effect of two other pose that Conp-Vt has evolved from the cone snail’s own MATPs (ETP and PTP) was not affected by atropine [19]. Ta- endogenous MATP peptide for specialised venom use. ken together, an action of Conp-Vt at non-cholinergic receptors to cause the release of ACh and contraction is Acknowledgements: We thank Christina Schroeder for performing the consistent with the antagonist and tachyphylaxis experiments. HF cleavage of the synthetic peptides, Natalie Steen for help with the rat atria preparations, Marion Loughnan for the HPLC fractionation of the C. vitulinus crude venom preparation and Alun Jones for assis- 3.6. Role of D-phenylalanine in Conp-Vt tance with mass spectrometry. This work was supported by grants The biological activity of Conp-Vt, L-Phe-Conp-Vt and from the Australian Research Council and the National Health and MATP-Ls were compared in the snail isolated buccal mass Medical Research Council (to R.J.L. and P.F.A.), and a postgraduate scholarship from the University of Queensland (to S.D.). preparation (Fig. 5D). While 1 lM of Conp-Vt and MATP- Ls produced responses of similar amplitude, 1 lMofL-Phe- Conp-Vt was less potent. Remarkably, this phenylalanine residue at position 2 is one of only two absolutely conserved References amino acids in all MATP peptides (Table 1). As Conp-Vt is the first known member to contain a D-amino acid, it was of [1] Olivera, B.M., Gray, W.R., Zeikus, R., McIntosh, J.M., Varga, J., Rivier, J., de Santos, V. and Cruz, L.J. (1985) Peptide neurotoxins interest to determine if a D-phenylalanine could enhance the from fish-hunting cone snails. Science 230, 1338–1343. activity of other MATP. Synthetic D-Phe-MATP-Ls was [2] Lewis, R.J. and Garcia, M.L. (2003) Therapeutic potential of found to be as active as the MATP-Ls peptide (Fig. 5D). Since venom peptides. Nat. Rev. Drug Discov. 2, 790–802. a synthetic MATP-Ls peptide was not compared with the [3] Olivera, B.M., Rivier, J., Clark, C., Ramilo, C.A., Corpuz, G.P., native compound, we cannot rule out the possibility that a Abogadie, F.C., Mena, E.E., Woodward, S.R., Hillyard, D.R. and Cruz, L.J. (1990) Diversity of Conus neuropeptides. Science D-Phe-MATP-Ls also occurs in Lymnaea [18]. Conp-Vt is a 249, 257–263. major component of the venom of C. vitulinus, suggesting that [4] Muneoka, Y., Morishita, F., Furukawa, Y., Matsushima, O., this peptide may play an important role in prey capture. In- Kobayashi, M., Ohtani, M., Takahashi, T., Iwakoshi, E., deed, 85–90% of all cone snail species including C. vitulinus Fujisawa, Y. and Minakata, H. (2000) Comparative aspects of invertebrate neuropeptides. Acta Biol. Hung. 51, 111–132. prey on worms and molluscs, and a peptide that can disrupt [5] Loughnan, M.L. and Alewood, P.F. (2004) Physico-chemical vital functions such as muscle contraction would constitute characterization and synthesis of neuronally active a-conotoxins. an efficient weapon in prey subjugation and capture. We pro- Eur. J. Biochem. 271, 2294–22304. 3866 S. Dutertre et al. / FEBS Letters 580 (2006) 3860–3866

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