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• W2030309070 abstract "Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides. Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides. high performance liquid chromatography rapid amplification of cDNA ends compound action potential heptafluorobutyric acid γ-aminobutyric acid Conus is a genus of predatory marine gastropods that envenomate their prey. Prey capture is accomplished through a sophisticated arsenal of peptides that target specific ion channel and receptor subtypes. Each Conus venom appears to contain a unique set of 50–200 peptides. The structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels, ligand-gated ion channels, and G-protein-linked receptors. Conus peptides that target voltage-gated ion channels include those that delay the inactivation of sodium channels as well as blockers specific for sodium channels, calcium channels, and potassium channels. Peptides that target ligand-gated ion channels include antagonists of N-methyl-d-aspartate and serotonin receptors as well as competitive and non-competitive nicotinic receptor antagonists. Peptides that act on G protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented targeting selectivity of conotoxins derives from specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (see Ref. 1McIntosh J.M. Olivera B.M. Cruz L.J. Methods Enzymol. 1998; 294: 605-624Crossref Scopus (74) Google Scholar for review). Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders (2Jones R.M. Bulaj G. Curr. Pharmaceutical Des. 2000; 6: 1249-1255Crossref PubMed Scopus (106) Google Scholar). In this report we describe the isolation of a new peptide from the venom of the marble cone, Conus marmoreus. C. marmoreus is found in the Indo-Pacific, from India to the Marshall Islands and Fiji. It preys upon various gastropods including other cone snails (3Röckel D. Korn W. Kohn A.J. Manual of the Living Conidae I: Indo-Pacific Region. Verlag Christa Hemmen, Wiesbaden, Germany1995Google Scholar). We previously reported the isolation and characterization of a peptide from this venom that potently inhibits voltage-gated sodium channels (4McIntosh J.M. Hasson A. Spira M.E. Gray W.R. Li W. Marsh M. Hillyard D.R. Olivera B.M. J. Biol. Chem. 1995; 270: 16796-16802Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In this report, we describe the isolation of a novel peptide that appears antinociceptive and likely represents a defining member of a new family of Conus peptides. The venom of C. marmoreus was obtained from snails collected in the Philippines. The venom was lyophilized and stored at −70 °C until use. Crude venom was extracted using previously described methods (5McIntosh J.M. Olivera B.M. Cruz L.J. Gray W.R. J. Biol. Chem. 1984; 259: 14343-14346Abstract Full Text PDF PubMed Google Scholar). Reverse phase HPLC purification was accomplished with an analytical (4.6 mm internal diameter × 25 cm) Vydac C18 column. Column pore size was 300 Å. Additional conditions are described in Fig. 1. The peptide was reduced, and cysteines were carboxymethylated as described previously (6Gray W.R. Protein Sci. 1993; 2: 1732-1748Crossref PubMed Scopus (235) Google Scholar). The alkylated peptide was purified with a Vydac C18 analytical column using a linear gradient of 0.1% trifluoroacetic acid and 0.092% trifluoroacetic acid in 60% acetonitrile. Alkylated peptide was sequenced by Edman degradation at the Peptide Core Facility at the University of Utah. Electrospray ionization mass spectra were measured using a Micromass Quattro II Triple Quadrupole Mass Spectrometer with Micromass MassLynx operating system. The samples (∼100 pmol) were resuspended in 0.1 ml of 50% acetonitrile with 0.05% trifluoroacetic acid and automatically infused with a flow rate of 0.05 ml/min in the same solvent system. The instrument was scanned over them/z range 50–2,000 with a capillary voltage of 2.95 kV and a cone voltage of 64 V. The resulting data were analyzed using MassLynx software. Peptides were synthesized, 0.45 mmol/g, on a RINK amide resin (Fmoc-Cys(Trityl)-Wang, Novabiochem (04-12-2050)) using Fmoc (N-(9-fluorenyl)methoxycarboxyl) chemistry and standard side chain protection except on cysteine residues. Cys residues were protected in pairs with either S-trityl orS-acetamidomethyl groups. Amino acid derivatives were from Advanced Chemtech (Louisville, KY). All three possible disulfide forms of the peptide were synthesized. The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (7Walker C. Steel D. Jacobsen R.B. Lirazan M.B. Cruz L.J. Hooper D. Shetty R. DelaCruz R.C. Nielsen J.S. Zhou L. Bandyopadhyay P. Craig A. Olivera B.M. J. Biol. Chem. 1999; 274: 30664-30671Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The sequence of the mr10a peptide was used to design degenerate oligonucleotide primers for use in 5′ and 3′ RACE amplification of the mr10a precursor cDNA. A 3′ RACE forward primer (mr10a-F, CAGGATCC AA(T/C) GGI GT(C/T/G) TG(T/C) TG(T/C) GG) was based on the peptide sequence NGVCCG. A 5′ RACE reverse primer (mr10a-R, CTGGATCC GG (A/G)TG (A/G)CA (C/A/G)A(G/A) (T/C)TT (G/A)TA ICC) was based on the peptide sequence GYKLCHP. C. marmoreus venom duct RNA was prepared, and cDNA appended with 3′ and 5′ adapter sequences was synthesized by standard methods (8Frohman M.A. Innis M.A. PCR Protocols. Academic Press, Inc., San Diego, CA1990: 28-45Google Scholar). To facilitate cloning of the RACE amplification products, the mr10a primers incorporated BamHI sites, and the 3′ and 5′ adapter primers contained XhoI sites. RACE amplifications were performed using a “touchdown” cycling protocol consisting of an initial denaturation of 95 °C for 30 s followed by 30 cycles of 95 °C for 10 s , 65 °C for 15 s, decreasing 0.5 °C each cycle, 72 °C for 10 s, then 15 cycles of 95 °C for 10 s, 50 °C for 10 s, and 72 °C for 10 s. Polymerase chain reaction amplifications were performed using Taq polymerase (PE Applied Biosystems, Foster City, CA), and amplification reactions were analyzed by electrophoresis on 2% agarose gels. RACE amplification products were isolated from the gel (Qiaex II DNA purification resin; Qiagen, Inc., Santa Clarita, CA), digested with BamHI and XhoI, and cloned into the plasmid vector pBluescript II SK−(Stratagene, La Jolla, CA). Plasmids containing inserts of the appropriate size were selected and sequenced on an ABI Prism 373 Fluorescent DNA Sequencer. Adult male CF-1 mice (25–35 g) were used for all experiments. Mice were housed five per cage, maintained on a 12-h light/dark cycle, and allowed free access to food and water. Analgesic activity was assessed by placing mice in a plexiglass cylinder (10.2-cm diameter × 30.5 cm high) on a hot plate (Mirak model HP72935, Barnstead/Thermolyne, Dubuque, IA) maintained at 55 °C. Thirty minutes before the hot plate test, animals were treated with a dose of mr10a or vehicle (0.9% saline) by free-hand intrathecal injection (9Hylden J. Wilcox G. Eur. J. Pharmacol. 1980; 67: 313-316Crossref PubMed Scopus (1725) Google Scholar) in a volume of 5 μl. The time from being placed on the plate until each mouse either licked its hind paws or jumped was recorded with a stopwatch by a trained observer unaware of the treatments. An arbitrary cut-off time of 60 s was adopted to minimize tissue injury. Hot plate test data were analyzed by analysis of variance followed by Dunnett's test for multiple comparison, with p < 0.05 considered significant. Statistical analysis was performed with GraphPad Prism software (San Diego, CA). Shortly after the hot plate test, mice were placed on a 3-cm-diameter rotarod turning at 6 rpm (model 7650, Ugo Basile, Comerio, Italy). Mice were considered impaired if they fell three times in 1 min. Rana pipiens, 2.5 to 3 inches long, were used. The dissected preparation consisted of the 9th and 10th spinal nerves and their continuation down the sciatic nerve to the posterior crural nerve, freed from the skin that it innervates (see e.g. Ref.10Ecker, A. (1889) The Anatomy of the Frog, (Haslam, G. H., translator) Oxford at the Clarendon Press, Oxford.Google Scholar). Each spinal nerve was severed from the spine several millimeters proximal to its sympathetic ramus such that it could be electrically stimulated without activating sympathetic axons (see e.g.Ref. 11Dodd J. Horn J.P. J. Physiol. 1983; 334: 255-268Crossref PubMed Scopus (83) Google Scholar). For extracellular stimulation and recording, the preparation was placed in a chamber fabricated from the silicone elastomer Sylgard (Dow Corning). The chamber was essentially a series of wells, each separated from its closest neighbor by about 1 mm. The proximal ends of the 9th and 10th nerves were placed in separate wells; the sciatic portion lay in a third well, whereas the attached lateral crural nerve spanned two additional wells, each 4 mm in diameter. All wells were filled with frog Ringer's solution (111 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 10 mm Na-HEPES (pH 7.2)). All portions of the nerve that draped over the partitions between wells and otherwise exposed to air were covered with Vaseline. One of a pair of stainless steel wire stimulating electrodes was placed in the well on either side of the Vaseline gap covering a spinal nerve. One of a pair of stainless steel wire recording electrodes was placed in the well on either side of the Vaseline gap covering the lateral crural nerve. Thus for both stimulation and recording, one electrode was in the same well that containing a cut end of the nerve, whereas its counterpart was in the well containing the portion of the nerve just distal (stimulation) or proximal (recording) to the cut end. The stimulating electrodes led to a Grass SIU5 stimulus isolation unit connected to a Grass S-88 stimulator. The recording electrode in the most distal well, containing the cut end of the posterior crural nerve, led to the positive input of a Grass P-55 differential preamplifier, whereas its counterpart, led to the negative input. Recordings were made with a preamplifier gain of 1000, RC-filtered (1 Hz alternating current and 1 kHz low pass) and digitized at a sampling rate of 4 kHz with a National Instruments Lab-NB board in a Macintosh Quadra 840 computer. Homemade virtual instruments (National Instruments LabVIEW) were used for data acquisition and analysis. Supramaximal stimuli (1 ms, ∼30 V) were applied to a spinal nerve once a minute while recording propagated compound action potentials (CAPs) reaching the other end of the preparation. Conopeptide was introduced into the well just proximal to that containing the distal cut end of the posterior crural nerve. Xenopus oocytes were injected with cRNA encoding human nAChR subunits as described previously (12Luo S. Kulak J.M. Cartier G.E. Jacobsen R.B. Yoshikami D. Olivera B.M. McIntosh J.M. J. Neurosci. 1998; 18: 8571-8579Crossref PubMed Google Scholar, 13McIntosh J.M. Gardner S. Luo S. Garrett J.E. Yoshikami D. Eur. J. Pharmacol. 2000; 393: 205-208Crossref PubMed Scopus (43) Google Scholar). Oocytes injected with α4β2 subunits were placed in a recording chamber (∼30 μl), voltage-clamped, and assessed for expression of receptor using a 1-s pulse of 300 μmacetylcholine (see Lu et al. (14Lu Y. Grady S. Marks M.J. Picciotto M. Changeux J.P. Collins A.C. J. Pharmacol. Exp. Ther. 1998; 287: 648-657PubMed Google Scholar) for details). Peptide was then added to the bath to achieve a final concentration of 10 μm. ω-Conotoxin GVIA was iodinated by previously described methods (15Cruz L.J. Olivera B.M. J. Biol. Chem. 1986; 261: 6230-6233Abstract Full Text PDF PubMed Google Scholar). The binding protocol was a modification of that described in Hillyard et al.(16Hillyard D.R. Monje V.D. Mintz I.M. Bean B.P. Nadasdi L. Ramachandran J. Miljanich G. Azimi-Zoonooz A. McIntosh J.M. Cruz L.J. Imperial J.S. Olivera B.M. Neuron. 1992; 9: 69-77Abstract Full Text PDF PubMed Scopus (437) Google Scholar). Crude brain membranes from Harlan Sprague-Dawley rats were prepared as described by Catterall (17Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1980; 20: 15-43Crossref PubMed Scopus (1006) Google Scholar), with modifications in buffer components as described in Cruz and Olivera (15Cruz L.J. Olivera B.M. J. Biol. Chem. 1986; 261: 6230-6233Abstract Full Text PDF PubMed Google Scholar). The binding of125I-labeled ω-conotoxin GVIA to rat brain membrane was measured in a 200-μl assay mix that contained approximately 10 μg of membrane protein, 100,000 cpm of carrier-free ω-[125I]conotoxin GVIA (approximately 150 pm), 0.2 mg/ml lysozyme, 0.32 m sucrose, 100 mmNaCl, and 5 mm HEPES/Tris (pH 7.4). Nonspecific binding was measured by preincubating the membrane preparation with 1 μm unlabeled ω-conotoxin GVIA for 30 min on ice before the addition of ω-[125I]conotoxin GVIA. mr10a was assessed for activity by preincubating the toxin for 30 min on ice. The final assay mix was then incubated at room temperature for 30 min and diluted with 1.5 ml of wash buffer containing 160 mm NaCl, 1.5 mm CaCl2, 2 mg/ml bovine serum albumin, 5 mm HEPES/Tris (pH 7.4). Membranes were collected on glass fiber filters (Whatman GF/C soaked in 0.1% polyethyleneamine) using a Brandell apparatus model M-24 and washed with 1.5 ml of wash buffer four times. The amount of radioactivity in the filters was then measured. The procedure of Pabreza et al. (18Pabreza L.A. Dhawan S. Kellar K.J. Mol. Pharmacol. 1991; 39: 9-12PubMed Google Scholar) was used. [3H]Cytisine (15–40 Ci/mmol) was from PerkinElmer Life Sciences. Rat forebrain membrane was incubated for 75 min at 4 °C in 50 mm Tris-HCl (pH 7.0 at room temperature) containing 120 mm NaCl, 5 mm KCl, 1 mm MgCl2, and 2.5 mmCaCl2. Nonspecific binding was defined with 10 μm nicotine. The remaining assays were carried out under contract with Novascreen (Hanover, Maryland). The essentials of the procedures used are summarized below. Rat forebrain membranes were incubated with 0.3 nm [3H]prazosin (70–87 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.7) at 25 °C for 60 min. Prazosin (1.0 μm) was used to define nonspecific binding (19Timmermans P. Ali F.K. Kwa H.Y. Schoop A.M.C. Slothorst-Grisdijk F.P. van Zwieten P.A. Mol. Pharmacol. 1981; 20: 295-301PubMed Google Scholar, 20Reader T.A. Briere R. Grondin L. J. Neural Transm. 1987; 68: 79-95Crossref PubMed Scopus (26) Google Scholar). Rat cortical membranes were incubated with 1.0 nm [3H]RX821002 (40–67 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) at 25 °C for 75 min. RX821002 (0.1 μm) was used to define nonspecific binding (20Reader T.A. Briere R. Grondin L. J. Neural Transm. 1987; 68: 79-95Crossref PubMed Scopus (26) Google Scholar, 21O'Rourke M.F. Blaxall H.S. Iversen L.J. Bylund D.B. J. Pharmacol. Exp. Ther. 1993; 263: 1362-1367Google Scholar). Rat cortical membranes were incubated with 0.2 nm (−)[125I]iodopindolol (2200 Ci/mmol) and 120 nm ICI-118,551 (to block adrenergic β2 receptors). Reactions were carried out in 50 mmTris-HCl (pH 7.5) containing 150 mm NaCl, 2.5 mm MgCl2, and 0.5 mm ascorbate at 37 °C for 60 min. Alprenolol HCl (10 μm) was used to define nonspecific binding (22Minneman K.P. Hegstrand L.R. Molinoff P.B. Mol. Pharmacol. 1979; 16: 34-46PubMed Google Scholar, 23Kalaria R.N. Andorn A.C. Tabaton M. Whitehouse P.J. Harik S.I. Unnerstall J.R. J. Neurochem. 1989; 53: 1772-1781Crossref PubMed Scopus (142) Google Scholar). Bovine cerebellar membranes were incubated with 5.0 nm[3H]GABA (70–90 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) at 0–4 °C for 60 min. GABA (1.0 μm) was used to define nonspecific binding (24Enna S.J. Collins J.F. Snyder S.H. Brain Res. 1977; 124: 185-190Crossref PubMed Scopus (131) Google Scholar, 25Falch E. Hedegaard A. Nielsen L. Jensen B.R. Hjeds H. Krogsgaard-Larsen P. J. Neurochem. 1986; 47: 898-903Crossref PubMed Scopus (78) Google Scholar). d-aspartate Agonist Site Binding Assay—Rat forebrain membranes were incubated with 2.0 nm [3H[CGP 39653 (25–60 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) at 0–4 °C for 60 min. N-Methyl-d-aspartate (1.0 mm) was used to define nonspecific binding (26Murphy D.E. Schneider J. Boehm C. Lehmann J. Williams M. J. Pharmacol. Exp. Ther. 1987; 240: 778-784PubMed Google Scholar, 27Lehmann J. Hutchinson A.J. McPherson S.E. Mondadori C. Schmutz M. Sinton C.M. Tsai C. Murphy D.E. Steel D.J. Williams M. J. Pharmacol. Exp. Ther. 1988; 246: 65-75PubMed Google Scholar). Rat spinal cord membranes were incubated with 16.0 nm[3H]strychnine (15–40 Ci/mmol). Reactions were carried out in 50 mm Na2HPO4 and 50 mm KH2PO4 (pH 7.1) containing 200 mm NaCl at 0–4 °C for 60 min. Strychnine nitrate (1.0 mm) was used to define nonspecific binding (28Young A.B. Snyder S.H. Mol. Pharmacol. 1974; 10: 790-809Google Scholar, 29Ruiz-Gomez A. Garcia-Calvo M. Vazquez J. Marvizon J.C. Valdivieso F. Mayor F.J. J. Neurochem. 1988; 52: 1775-1780Crossref Scopus (44) Google Scholar). Bovine cerebellar membranes were incubated with 2.0 nm[3H]pyrilamine (15–25 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.5) at 25 °C for 60 min. Triprolidine (10 μm) was used to define nonspecific binding (30Chang R.S. Tran V.T. Snyder S.H. J. Neurochem. 1979; 32: 1653-1663Crossref PubMed Scopus (195) Google Scholar, 31Haaksma E.E.J. Leurs R. Timmerman H. Pharmacol. Ther. 1990; 47: 73-104Crossref PubMed Scopus (65) Google Scholar, 32Martinez-Mir M.I. Pollard H. Moreau J. Arrang J.M. Ruat M. Traiffort E. Schwartz J.C. Palacios J.M. Brain Res. 1990; 526: 322-327Crossref PubMed Scopus (230) Google Scholar). Rat cortical membranes were incubated with 0.15 nm[3H]quinuclidinylbenzilate (30–60 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) at 25 °C for 75 min. Atropine (0.1 μm) was used to define nonspecific binding (33Yamamura H.I. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 1725-1729Crossref PubMed Scopus (1241) Google Scholar, 34Luthin G.R. Wolfe B.B. J. Pharmacol. Exp. Ther. 1984; 228: 648-655PubMed Google Scholar, 35Luthin G.R. Wolfe B.B. Mol. Pharmacol. 1984; 26: 164-169PubMed Google Scholar). Rat forebrain membranes were incubated with 2.0 nm [3H]neurotensin (70–120 Ci/mmol). Reactions were carried out in 50 mmTris-HCl (pH 7.4) containing 0.04% bacitracin, 0.1% bovine serum albumin, and 1 mm Na2EDTA at 25 °C for 60 min. Neurotensin (1.0 μm) was used to define nonspecific binding (36Goedert M. Pittaway K. Williams B.J. Emson P.C. Brain Res. 1984; 304: 71-81Crossref PubMed Scopus (94) Google Scholar, 37Gully D. Canton M. Boigegrain R. Jeanjean F. Molimard J.C. Poncelet M. Gueudet C. Heaulme M. Leyris R. Brouard A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 65-69Crossref PubMed Scopus (396) Google Scholar). Rat forebrain membranes were incubated with 1.0 nm [3H]deltorphin II (30–60 Ci/mmol). Reactions were carried out in 50 mmTris-HCl (pH 7.4) at 25 °C for 90 min. [d-Pen2,d-Pen5]-enkephalin (1.0 μm) was used to define nonspecific binding (38Akiyama K. Gee K.W. Mosberg K.W. Yamamura H.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2543-2547Crossref PubMed Scopus (117) Google Scholar, 39Sofuoglu M. Protoghese P.S. Takemori A.E. Eur. J. Pharmacol. 1992; 216: 273-277Crossref PubMed Scopus (39) Google Scholar). Guinea pig cerebellar membranes were incubated with 0.75 nm [3H]U-69593 (40–60 Ci/mmol). Reactions were carried out in 50 mm HEPES (pH 7.4) at 30 °C for 120 min. U-69593 (1.0 μm) was used to define nonspecific binding (40Lahti R.A. Mickelson M.M. McCall J.M. Von Voigtlander P.F. Eur. J. Pharmacol. 1985; 109: 281-284Crossref PubMed Scopus (448) Google Scholar, 41Kinouchi K. Pasternak G.W. Eur. J. Pharmacol. 1991; 207: 135-141Crossref PubMed Scopus (23) Google Scholar, 42Rothman R.B. Bykov V. Xue B.G. Xu H. De Costa B.R. Jacobson A.E. Rice K.C. Kleinman J.E. Brady L.S. Peptides ( Orlando ). 1992; 13: 977-987Crossref PubMed Scopus (47) Google Scholar). Rat forebrain membranes were incubated with 1.0 nm[3H]Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO) (30–60 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) at 25 °C for 90 min. Naloxone (1.0 μm) was used to define nonspecific binding (43Gillan M.G.C. Kosterlitz H.W. Br. J. Pharmacol. 1982; 77: 461-469Crossref PubMed Scopus (279) Google Scholar, 44Goldstein A. Naidu A. Mol. Pharmacol. 1989; 36: 265-272PubMed Google Scholar). Rat forebrain membranes were incubated with 1.0 nm[3H]nisoxetine (60–85 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) containing 300 mm NaCl and 5 mm KCl at 0–4 °C for 4 h. Desipramine (1.0 μm) was used to define nonspecific binding (45Langer S.Z. Raisman R. Briley M. Eur. J. Pharmacol. 1981; 72: 423-424Crossref PubMed Scopus (72) Google Scholar, 46Raisman R. Sette M. Pimoule C. Briley M. Langer S.Z. Eur. J. Pharmacol. 1982; 78: 345-351Crossref PubMed Scopus (104) Google Scholar). N1E-115 cells were incubated with 0.35 nm [3H]GR65630 (30–70 Ci/mmol). Reactions were carried out in 20 mm HEPES (pH 7.4) containing 150 mm NaCl at 25 °C for 60 min. MDL-72222 (1.0 μm) was used to define nonspecific binding (47Hoyer D. Neijt H.C. Mol. Pharmacol. 1988; 33: 303-309PubMed Google Scholar, 48Lummis S.C.R. Kilpatrick G.J. Eur. J. Pharmacol. 1990; 189: 223-227Crossref PubMed Scopus (44) Google Scholar, 49Tyers M.B. Therapie ( Paris ). 1991; 46: 431-435PubMed Google Scholar). Rat forebrain membranes were incubated with 0.7 nm [3H]citalopram (70–87 Ci/mmol). Reactions were carried out in 50 mmTris-HCl (pH 7.4) containing 120 mm NaCl and 5 mm KCl at 25 °C for 60 min. Imipramine (10 μm) was used to define nonspecific binding (50Brown N.L. Sirugue O. Worcel M. Eur. J. Pharmacol. 1986; 123: 161-165Crossref PubMed Scopus (20) Google Scholar, 51D'Amato R.J. Largent B.L. Snowman A.M. Snyder S.H. J. Pharmacol. Exp. Ther. 1987; 242: 364-371PubMed Google Scholar). Guinea pig striatal membranes were incubated with 12.0 μm[3H]WIN,35,428 (60–87 Ci/mmol). Reactions were carried out in 50 mm Tris-HCl (pH 7.4) containing 120 mm NaCl at 0–4 °C for 2 h. GBR-12909 (0.1 μm) was used to define nonspecific binding (52Javitch J.J. Blaustein R.O. Snyder S.H. Mol. Pharmacol. 1984; 26: 35-44PubMed Google Scholar, 53Madras B.K. Spealman R.D. Fahey M.A. Neumeyer J.L. Saha J.K. Milius R.A. Mol. Pharmacol. 1989; 36: 518-524PubMed Google Scholar). Extracted crude venom from C. marmoreus was initially size-fractionated using a Sephadex G-25 column (see Fig. 1). Column fractions eluting in a range corresponding to small peptides were further purified using reverse phase HPLC utilizing conditions described under “Methods and Methods” and in Fig. 1. Throughout subsequent purification, HPLC fractions were assayed by means of intracerebral ventricular injection into mice (54Clark C. Olivera B.M. Cruz L.J. Toxicon. 1981; 19: 691-699Crossref PubMed Scopus (48) Google Scholar). Intracerebral ventricular injection of fractions containing mr10a produced hypokinetic “sluggish” symptoms. The venom fraction was initially purified using a trifluoroacetic/acetonitrile gradient system. To obtain further purification, the new fractions were lyophilized and resuspended in 0.05% heptafluorobutyric acid (HFBA) and run on a reverse phase C18 column using a 0.05% HFBA, acetonitrile gradient. Further purification was obtained using strong cation exchange chromatography. The fraction was then desalted using reverse phase HPLC. Final purified product is shown in Fig.2, panel B. The disulfide bonds of the purified peptide were reduced, and Cys residues were carboxymethylated. The alkylated peptide was then chemically sequenced and yielded NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. Mass spectrometry of the peptide verified the sequence and indicated that Cys residues are present as disulfides and the C terminus is the free carboxyl (monoisotopic MH+ (Da): calculated, 1408.5; observed, 1408.5). The sequence of the peptide was independently confirmed by preparation of synthetic peptide as described under “Experimental Procedures.” The mr10a peptide has four Cys residues and, therefore, three possible disulfide linkages. All three disulfide bond linkages were synthesized to unequivocally identify the native configuration. Peptides were initially synthesized in linear form using pairwise protection of Cys residues (see “Experimental Procedures”). Acid cleavage from resin removed trityl-protecting groups, and ferricyanide oxidation was used to close the first disulfide bridge. Iodine oxidation was subsequently used to remove S-acetamidomethyl protection groups and close the second bridge. Using this method, each possible disulfide arrangement was synthesized, i.e. Cys1-Cys2, Cys3-Cys4; Cys1-Cys3, Cys2-Cys4, and Cys1-Cys4, Cys2-Cys3. Final purified yields of each peptide were 12.5, 5.2, and 12.4%, respectively. Synthesis of each isomer was confirmed with mass spectrometry (calculated monoisotopic MH+, 1408.5; observed, 1408.6, 1408.7, and 1408.6, respectively). The three forms of the peptide were distinguishable using reverse phase HPLC based on elution time. In addition, they were distinguishable by peak width, with the (Cys1-Cys4, Cys2-Cys3) form having the narrowest peak width (Fig. 2). Both the elution time and peak shape of the (Cys1-Cys4, Cys2-Cys3) disulfide form match that of the native peptide. Additionally, co-injection of each synthetic form indicates that native mr10a co-elutes only with synthetic peptide of the (Cys1-Cys4, Cys2-Cys3) configuration, providing unambiguous evidence for this disulfide linkage being native (Fig. 2). The mr10a peptide sequence was used to design degenerate polymerase chain reaction primers for 3′ and 5′ RACE (rapid amplification of cDNA ends) amplification of the complete mr10a precursor cDNA. The polymerase chain reaction primers were designed to yield overlapping 3′ and 5′ RACE products, allowing the complete cDNA sequence to be assembled from the two sequences. Amplification of C. marmoreus cDNA gave specific products of 610 base pairs in the 3′ RACE and 300 base pairs in the 5′ RACE reactions. These polymerase chain reaction products were cloned, and multiple clones of both the 5′ and 3′ RACE products were isolated and sequenced. For both the 5′ and 3′ RACE products, the multiple clones all represented the same sequence, and the appropriate segments of the mr10a peptide sequence were represented by the cloned sequence. The 5′ and 3′ RACE product sequences were assembled to give the complete mr10a prepropeptide precursor cDNA sequence (Fig.3). The first ATG start codon encountered from the 5′ end of the cDNA initiates an open reading frame of 61 amino acids, encoding a protein with a structure typical of a conotoxin prepropeptide. The first 24 N-terminal amino acids compose a hydrophobic signal sequence region. The mature mr10a peptide sequence is located at the C-terminal end of the precursor sequence, immediately preceded by a basic amino acid (Arg) signaling proteolytic peptide processing. The stop codon is immediately downstream of the last cysteine residue of the mr10a peptide. A 3′-untranslated region of ∼500 base pairs is terminated by a typical poly(A)+addition signal (AATAAA) and a poly(A) tail. The mr10a precursor exhibits significant sequence homology to a previously identified family of conotoxin genes, the T-superfamily, although the mature mr10a peptide is distinct from any of the previously isolated T-superfamily conotoxins (Fig.4). Previously isolated T-superfamily conotoxins all share the cysteine framework CC–CC (7Walker C. Steel D. Jacobsen R.B. Lirazan M.B. Cruz L.J. Hooper D. Shetty R. DelaCruz R.C. Nielsen J.S. Zhou L. Bandyopadhy" @default.
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• W2030309070 title "Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity" @default.
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