Tyrosine-rich Conopeptides Affect Voltage-gated K+ Channels

Abstract

Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12–18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom. Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12–18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom. The venom of marine gastropods in the genus Conus has yielded numerous structurally and functionally diverse peptidic components (1Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (814) Google Scholar). The increasing variety of bioactive peptides identified in cone snail venoms has provided insight into the seemingly endless variety of directions taken by Conus species in evolving neuroactive molecules to suit their diverse biological purposes. The bioactive peptides in Conus (“conopeptides”) are classified into two broad groups: the non-disulfide-rich and the disulfide-rich (1Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (814) Google Scholar). The latter are conventionally called conotoxins. The non-disulfide-rich class includes conopeptides with no cysteines (contulakins (2Craig A.G. Norberg T. Griffin D. Hoeger C. Akhtar M. Schmidt K. Low W. Dykert J. Richelson E. Navarro V. Macella J. Watkins M. Hillyard D. Imperial J. Cruz L.J. Olivera B.M. J. Biol. Chem. 1999; 274: 13752-13759Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), conantokins (3Olivera B.M. McIntosh J.M. Clark C. Middlemas D. Gray W.R. Cruz L.J. Toxicon. 1985; 23: 277-282Crossref PubMed Scopus (43) Google Scholar), and conorfamides (4Maillo M. Aguilar M.B. Lopez-Vera E. Craig A.G. Bulaj G. Olivera B.M. Heimer de la Cortera E.P. Toxicon. 2002; 40: 401-407Crossref PubMed Scopus (51) Google Scholar)) and conopeptides with two cysteines forming a single disulfide bond (conopressins (5Cruz L.J. de Santos V. Zafaralla G.C. Ramilo C.A. Zeikus R. Gray W.R. Olivera B.M. J. Biol. Chem. 1987; 262: 15821-15824Abstract Full Text PDF PubMed Google Scholar) and contryphans (6Jimenez E.C. Olivera B.M. Gray W.R. Cruz L.J. J. Biol. Chem. 1996; 281: 28002-28005Abstract Full Text Full Text PDF Scopus (134) Google Scholar)). The conopeptides that compose the disulfide-rich class have two or more disulfide bonds (1Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (814) Google Scholar); among the major classes of molecular targets identified for these structurally diverse conopeptides are members of the voltage-gated and ligand-gated ion channel superfamilies. In this work, we present the discovery and characterization of a novel class of peptides, which we designate the conopeptide Y (CPY) 2The abbreviations used are:CPYconopeptide YHPLChigh pressure liquid chromatographyTFEtrifluoroethanolRACErapid amplification of cDNA ends. family, from the marine snails Conus planorbis and Conus ferrugineus (see Fig. 1). Both of these species belong to a distinct clade of Conus (7Espiritu D.J.D. Watkins M. Dia-Monje V. Cartier G.E. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 1899-1916Crossref PubMed Scopus (146) Google Scholar, 8Duda Jr., T.F. Kohn A.J. Palumbi S.R. Biol. J. Linn. Soc. 2001; 73: 391-409Crossref Google Scholar). The first two peptides (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle Scholar) belonging to the CPY family have a high frequency of tyrosine residues, in addition to basic and hydrophobic residues. The peptides are shown to be biologically active when injected into mouse and the nematode Caenorhabditis elegans. Activity on the Kv1 channel subfamily is demonstrated, and the subtype selectively within the subfamily is defined. conopeptide Y high pressure liquid chromatography trifluoroethanol rapid amplification of cDNA ends. Extraction and Fractionation of C. planorbis Venom—Snails were collected in Cebu and Marinduque Islands, Philippines, and dissected. Venom was pressed out of freshly dissected venom ducts that were kept on ice. Venom pooled from several snails was lyophilized and stored at –70 °C. A 500-mg portion was resuspended in 35 ml of 30% acetonitrile and 0.2% trifluoroacetic acid using a Vortex mixer for 2× 1 min with an interval of 5 min on ice. The mixture was sonicated using a Branson LS-75 probe for 3× 0.5 min on ice with 1-min rest periods, and the sediment was pelleted in a Beckman Avanti centrifuge with an F650 rotor. Centrifugation was done for 30 min at 37,500 × g. The supernatant was diluted with 0.1% trifluoroacetic acid, centrifuged again to remove all residual particles, and applied to a preparative Vydac C18 high pressure liquid chromatography (HPLC) column (2.5 × 25 cm). Venom peptides were eluted from the column with a linear gradient of 4.5–90% acetonitrile and 0.1% trifluoroacetic acid at 0.9% acetonitrile/min. The flow rate was 20 ml/min, and the absorbance of the eluate was monitored at 220 nm. An analytical Vydac C18 HPLC column (4.6 × 250 mm) with a linear gradient at 0.45% acetonitrile/min in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min was used for subfractionation. Bioassay of HPLC Fractions on Nereis virens—Specimens (usually within 10–16 cm long) of N. virens were maintained in seawater at 0–4 °C in a cold room. During injections, the worms were taken outside the cold room in individual beakers containing cold seawater. The temperature was maintained close to 4 °C by keeping the beakers immersed in iced water. Each worm was laid out on a block of Styrofoam right before injection and returned to cold seawater within a few seconds after injection. Lyophilized samples resuspended in 10–12 μl of normal saline solution were injected using an insulin syringe at the anterior end of the worm. A needle lid with the tip cut off was used to obtain a consistent depth of needle puncture of ∼1.5 mm. The initial effects of injections were evaluated by transferring the worms to seawater at room temperature for 5–15 s at ∼1 h after injection. These were observed side by side with saline-injected controls continuously for at least 4 h and checked every day for at least 2 weeks. Characterization of Peptides—Samples were completely reduced with 10 mm dithiothreitol at 65 °C for 15 min and completely alkylated using 0.7% 4-vinylpyridine for 20 min in the dark at room temperature. Alkylation reactions were diluted 10-fold with 0.1% trifluoroacetic acid and fractionated in an analytical C18 HPLC column. Elution was done with a linear gradient of 1.8% acetonitrile/min in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Peptide sequencing by Edman degradation chemistry was done on alkylated samples after HPLC purification. Uncertain readings of C-terminal residues were verified by sequencing of fragments obtained after endopeptidase digestion with Arg-C. The reaction conditions for enzyme digestion were as recommended by the supplier (Roche Diagnostics). Mass determinations on both native and synthetic peptides were done by matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry at the University of Utah Mass Spectrometry and Proteomic Core Facility, the Salk Institute Peptide Biology Lab, and The University of Queensland Institute for Molecular Bioscience. Chemical Synthesis and Purification of CPY-Pl1 and CPY-Fe1—CPY-Pl1 and CPY-Fe1 were synthesized on an ABI 433 peptide synthesizer (Applied Biosystems, Foster City, CA) following the FastMoc chemistry procedure from ABI. Standard protecting groups for the amino acid side chains were used. The cleaved peptide was further purified by HPLC on a semi-preparative C18 column with a linear gradient of 0.18% acetonitrile/min in 0.1% trifluoroacetic acid. The flow rate was 2.5 ml/min, and absorbance at 220 and 280 nm was monitored. NMR Spectroscopy—Samples for 1H NMR measurements contained ∼2 mm peptide in 90% H2O and 10% D2O (v/v) at ∼pH 3 or 20% TFE, 80% H2O, and 10% D2O. Spectra were recorded at 288–298 K on a Bruker ARX-600 spectrometer equipped with a shielded gradient unit. Two-dimensional NMR spectra were recorded in phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension as described previously for other disulfide-rich peptides (10Daly N.L. Ekberg J.A. Thomas L. Adams D.J. Lewis R.J. Craik D.J. J. Biol. Chem. 2004; 279: 25774-25782Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Spectra were processed on a Silicon Graphics Indigo Workstation using XWINNMR software (Bruker). The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Chemical shifts were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate. NMR spectral assignments for CPY-Pl1 were made using established techniques (12Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley-Interscience, New York1986Crossref Google Scholar). Bioassay of Synthetic Samples in Mice and C. elegans—Intracranial injections were administered to mice (Swiss Webster) that were 15–17 days old. Peptide samples in HPLC solutions were lyophilized, resuspended in 12 μl of normal saline solution, and administered intracranially to mice using an insulin syringe. The peptide-injected mice were observed side by side with saline-injected controls continuously for 2–4 h and checked the next day. Microinjection of peptide samples into the pseudocoelomic region of the nematode C. elegans was done as described (13Mello C.C. Kramer J.M. Stirchcomb D. Ambros V. EMBO J. 1991; 10: 2959-3970Crossref Scopus (2453) Google Scholar). Electrophysiology—The Xenopus oocyte expression system was used to study the effect of the CPY peptides on K+ and Na+ channels (14Zhang M. Green B.R. Caitlin P. Fiedler B. Azam L. Chadwick A. Terlau H. McArthur J.R. French R.J. Gulyas J. Rivier J.E. Smith B.J. Norton R.S. Olivera B.M. Yoshikami D. Bulaj G. J. Biol. Chem. 2007; 282: 30699-30706Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and the E.C. V. Watkins M. M. Bulaj G. Olivera B.M. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In K+ channel were and channels were as described B. M. J. A. Olivera B.M. Terlau H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were recorded at room temperature using an or a were at 3 and at 10 or 1 and at 4 The solution was normal B.M. PubMed Google Scholar) containing mm 2.5 mm mm and 10 mm was to the solution in all the The for block of the Kv1.6 channel were from the at a of 20 or to × is the and is the from a single of C. planorbis and C. ferrugineus were obtained using the for of from Each for C. planorbis and for C. was used in cDNA Scholar). from CPY-Pl1 and CPY-Fe1 were in Scholar) and on C. planorbis from the signal identified for CPY-Pl1 were used in Scholar) on C. planorbis and C. ferrugineus were done in a using high The was used for all sequencing was done at the University of Utah Institute Core using samples following the of CPY Peptides from the Venom of C. planorbis (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle C. planorbis venom was fractionated by HPLC as described activity on the marine worm N. virens was used to active from the preparative HPLC column and to further of the and were lyophilized, and on N. virens as described a as described individual were The in Fig. when injected into N. that injected or injected with the solution saline in when to seawater at room temperature. In worms in or injected worms were to cold seawater after 5–15 s of in seawater at room worms that the initial or in and worms for The in Fig. were and one the activity on N. virens described The active in Fig. was completely reduced and alkylated as described The reduced and alkylated was fractionated by HPLC the by the is CPY-Pl1, and the is The peptides were using Edman The peptide obtained a an high frequency of tyrosine residues, in addition to basic and hydrophobic residues. The of the two peptides are shown in Fig. the of and that were obtained to the for CPY-Pl1 and of the 30 residues are in the two peptides, including tyrosine residues. The high content of tyrosine in CPY-Pl1 and CPY-Fe1 is of the Y family (see Fig. we to these peptides as the conopeptide Y or CPY family. to these CPY family peptides from Conus venoms using the following The individual peptides be by the first two of the species by a the of peptides that belong to this family from The two peptides from was to be C. planorbis venom are to as CPY-Fe1 for and CPY-Pl1 for from described that both C. ferrugineus and C. planorbis venoms were present in the C. planorbis that was used. of the close of of the two species the of one species for the other was the that were to the pooled venom that yielded the purified Chemical Synthesis of CPY of the CPY peptides was out as described The HPLC for the of synthetic CPY-Fe1 and CPY-Pl1 with the native samples are shown in Fig. 4 and (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle Scholar). The in the mass spectra of both synthetic peptides in the spectra of the native NMR shifts of CPY-Pl1 in aqueous solution and in 20% are shown in Fig. In of shifts of peptides a of the of CPY-Pl1 in aqueous solution, the shifts that no is present on the of of consistent or in the of TFE, a of from shifts residues 12 and the of helical in this is to helical in peptides an helical and this to be the for Bioassay of CPY-Fe1 Mice and C. were injected intracranially with Mice injected with of of mouse as more or An to more by the lid the a a in a mouse injected with ∼1.5 of of A effect was when one peptide-injected mouse was two was a within s after the was The were observed within min after the we out a to as after all mouse injections of the CPY mice were from a of ∼1.5 the at min after injection. This was every min to min after injection. The to the mouse was observed after the first were observed in normal saline controls or after to within the after injection. Both peptides with the The was for the CPY-Fe1 a of 5 of the mice with to 20 injected of the mice and in C. elegans were made at a of of which for was observed in of worms In of the from the to the the worm from the to the anterior An initial on the effect of CPY-Fe1 on the of J. Thomas L. Adams A. E. Craik D.J. Lewis R.J. 2001; PubMed Scopus Google Scholar) any effect on both and with 10 for activity using CPY-Fe1 on channels in Xenopus to and In these the used were of the the CPY-Fe1 was to be active at 1 on were made to with for high block of Kv1.6 was obtained for both were using these a after a of as the measurements were was to obtain measurements using The for both CPY-Fe1 and CPY-Pl1 that CPY-Pl1 is more on the Kv1.6 The data shown in 1 and Fig. were obtained using Xenopus the were and of to 1 the obtained from in to CPY-Pl1 is more active CPY-Fe1 on both and Kv1.6 In further using CPY-Pl1 of the and activity and of the of CPY peptides on mammalian Kv1 isoforms in Xenopus in a new Molecular of the CPY cDNA made from C. planorbis venom ducts was used to the precursors of the CPY The including signal sequence, and peptide is shown in Fig. The the signal peptide, and the was the C. planorbis cDNA and from both peptides yielded for CPY-Pl1 no for Clones obtained in for both CPY peptides on C. planorbis cDNA using from both peptides the CPY-Pl1 with the from which has a region to the from A on C. planorbis cDNA from the signal CPY-Pl1 to the A for CPY peptides in C. ferrugineus cDNA using one of the from the signal and the Scholar) for The signal and are completely shared by the two peptides to the of the 30 residues of the peptide are shared to the is in the of CPY-Fe1 and CPY-Pl1 The unique signal revealed from precursors of these peptides a new gene family of conopeptides, which we to as the conopeptide Y or CPY family. The peptides characterized in this study are in their high content of an amino acid. Conus venom peptides are by disulfide a the Conus venom peptides that disulfide cross-links to be in the disulfide-rich conotoxins. The CPY peptides that are characterized are the conopeptides shown to be biologically In addition to the high tyrosine these peptides are in and hydrophobic residues and in CPY-Pl1, these amino acids for of the Both CPY-Fe1 and CPY-Pl1 were (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle Scholar) from a of venom that to have from of C. The however, that one of the CPY peptides that was from the pooled be from cDNA from a C. ferrugineus venom CPY-Pl1 were from C. planorbis shown in Fig. are of C. ferrugineus or C. planorbis that be as one or the the of the of species to C. planorbis Each cDNA was obtained from a single venom of the more obtained by from venom pooled from or have demonstrated that these peptides are of voltage-gated K+ channels belonging to the Kv1 Both of the peptides characterized selectivity for Kv1.6 among the Kv1 subfamily channels with CPY-Pl1 from venoms of Conus species as C. to for mammalian the conopeptides from venoms of cone the data are with the native of this peptide the Kv1.6 in the worm of C. is that an K+ channel be in mammalian as as in The data in 1 that these peptides have a for Kv1.6 with the that the native is a of as an Kv1.6 and selectivity for a Kv1 that has more one of is a that to be The of in in addition to the K+ channel the CPY peptides be these are hydrophobic peptides, the that into to or the oocyte in other the in the that the data shown in Fig. and 1 be A peptide that voltage-gated K+ channels was previously from Conus the subfamily selectivity was S. G. P. K. B. 2004; PubMed Scopus Google Scholar). The CPY peptide that has CPY-Pl1, is for the Kv1.6 is that CPY-Pl1 is more on Kv1.6 a disulfide-rich peptide that was previously from the venom (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle Scholar, Daly N.L. Craik D.J. A. Terlau H. Lopez-Vera E. Olivera B.M. PubMed Scopus Google Scholar). Conus species have a structurally diverse of peptides to the Kv1 the first peptide characterized was K. M. Terlau H. W. R. C. M. Watkins M. Hillyard Gray W.R. Olivera B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), from the venom of the cone snail Conus this peptide has disulfide bonds and to the when Kv1 peptides from two other of were peptides structurally to were A. D. M. Gulyas J. Rivier J.E. Olivera B.M. T. Terlau H. 2004; PubMed Scopus Google Scholar) from Conus has disulfide bonds to the and N. Olivera B.M. A. M. Terlau H. Soc. 2007; Scholar, M. V. M. R. J. Imperial J. J.E. Olivera B.M. Terlau H. M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) is a K+ channel from Conus The of C. planorbis venom revealed K+ peptides to the classes two to other that both a peptide with two disulfide bonds and previously called (9Imperial, J. S. (2007) Novel Peptides from Conus planorbis, Terebra subulata, and Hastula hectica. Ph.D. thesis, The University of QueenslandGoogle Scholar, Daly N.L. Craik D.J. A. Terlau H. Lopez-Vera E. Olivera B.M. PubMed Scopus Google Scholar), and CPY-Pl1, the peptide characterized in this is unprecedented for venom from one species to peptides that the ion channel has previously shown that peptides the subtype in C. C. M. C. K. Cruz L.J. Gray W.R. Dykert J. Rivier J. Yoshikami D. Olivera B.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. R. Cartier G.E. C. Gray W.R. Watkins M. Hillyard Rivier J. J. Yoshikami D. Olivera B.M. PubMed Scopus (83) Google Scholar) and Conus M. McIntosh J.M. Olivera B.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) In both the peptides were structurally and were shown to on the This to be of the that cone snails for that the of C. planorbis has a K+ channel that be an molecular in the for disulfide cross-links Conus peptides are injected into the of the the CPY peptides are unstructured in aqueous solution, to be to rapid a of Conus peptide right at the of in the of cone K+ channel are of the of that a of in the of the injection which in effect the to that the CPY peptides in C. planorbis and C. ferrugineus venoms an in this were the the peptides right at the injection to their more and to by J. S. for peptide for peptide and Lewis for in using for the for the of CPY-Pl1 on and for on P. C. Yoshikami for on oocyte