alpha -Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatus That Selectively Targets Neuronal Nicotinic Acetylcholine Receptors*

Marion Loughnan, Trudy Bond, Anne Atkins, Javier CuevasDagger §, David J. AdamsDagger , Natalie M. Broxton, Bruce G. Livett, John G. Down, Alun Jones, Paul F. Alewood, and Richard J. Lewisparallel

From the Centre for Drug Design and Development and Dagger  Dept of Physiology and Pharmacology, The University of Queensland, St. Lucia Queensland 4067, Australia and  The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville Victoria 3052, Australia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have isolated and characterized alpha -conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an alpha -conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described alpha -conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides. The activities of synthetic EpI and its unsulfated analogue [Tyr15]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors). Both EpI and [Tyr15]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia. These results indicate that EpI and [Tyr15]EpI selectively inhibit alpha 3beta 2 and alpha 3beta 4 nicotinic acetylcholine receptors.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cone snail venoms contain complex mixtures of peptides (conotoxins or conopeptides) that bind with high affinity and specificity to a diversity of mammalian macromolecular receptors. Targets currently identified include sodium, calcium, and potassium ion channels and N-methyl-D-aspartate-glutamate, vasopressin and acetylcholine receptors. Most characterized bioactive conopeptides contain post-translational modifications that include carboxylation, hydroxylation, bromination, and C-terminal amidation (1-3) but modifications such as phosphorylation or sulfation have not been demonstrated.

One pharmacological class of conopeptides, the alpha -conotoxins, inhibit acetylcholine action at the nicotinic acetylcholine receptor (nAChR).1 The majority of alpha -conotoxins isolated from fish-hunting cone snails, including GI, GIA, and GII from Conus geographus (4), MI from Conus magus (5), and SI and SII from Conus striatus (6, 7), show extensive structural homology, a conserved cysteine framework, and target the muscle subtype of the nAChR. The alpha -conotoxins PnIA and PnIB from a mollusc-hunting cone snail, Conus pennaceus (8), and ImI from a worm-hunting cone snail, Conus imperialis (9) share a similar cysteine framework to alpha -conotoxins from fish-hunting cones, but have a different sequence pattern and target the neuronal subtypes of the nAChR. The alpha -conotoxins MII and EI from fish-hunting cones resemble the latter group in sequence pattern, but EI targets the muscle-type nAChR (10, 11). The new alpha A-conotoxin class, which includes PIVA isolated from the fish-hunting Conus purpurascens, contains atypical sequences and a novel cysteine framework (12). The sequence, disulfide connectivity, and specificity of alpha -conotoxins for mammalian nAChRs are given in Table I.

The nAChRs are a superfamily of ligand-gated ion channels that is classified into muscle and multiple neuronal subtypes (13). The physiological and pharmacological heterogeneity of neuronal nAChRs is reflected by the diversity of subunit composition of these heteropentameric ion channel complexes (14, 15). Neuronal receptors are composed of several possible alpha  subtypes (alpha 2-alpha 7, alpha 9) and 3 possible beta  subtypes (beta 2-beta 4), whereas muscle receptors are composed of five subunits alpha 12beta epsilon delta or alpha 12beta gamma delta in mature or embryonic tissue, respectively (13). Neuronal nAChRs are distributed in numerous brain regions and on both cell soma and dendrites, but it is the action of nAChRs to modulate synaptic transmission presynaptically that has received much attention recently (14, 16, 17). Selective activation of distinct subtypes of these presynaptic nAChRs by nicotinic agonists can regulate the release of different neurotransmitters, including dopamine, norepinephrine, glutamate, GABA, and acetylcholine (14). To gain a better insight into the role of the various nAChR subtypes in neurotransmission, additional selective nAChR antagonists are required.

In this report we describe EpI, a novel conotoxin from the molluscivorous C. episcopatus that targets the nAChR. The peptide has the cysteine framework typical of alpha -conotoxins and substantial sequence homology with the other alpha -conotoxins isolated from non-piscivorous Conus species (see Table I). However, EpI differs from all previously reported alpha -conotoxins in that it possesses a sulfotyrosine residue.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of EpI from Conus episcopatus

Five specimens of C. episcopatus (Fig. 1) were collected from The Great Barrier Reef, Australia, dissected and an extract of crude venom prepared from the pooled venom duct material as described previously (18). Briefly, ground dried ducts were extracted with 30% acetonitrile/water acidified with 0.1% trifluoroacetic acid, centrifuged, and the supernatants lyophilized (yield 84 mg) and stored at -20 °C prior to HPLC separation.


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Fig. 1.   C. episcopatus, a molluscivorous Conidae (length 8 cm) collected from the Great Barrier Reef, Australia.

Isolation and Purification of EpI

Crude venom (4.4 mg) was fractionated by preparative reversed-phase HPLC (Waters) using a 10-µm Vydac C18 column (1.0 × 25 cm) eluted at 2.5 ml/min with a linear gradient from 0 to 80% B over 80 min: solvent A, 10% acetonitrile, 0.1% trifluoroacetic acid; solvent B, 90% acetonitrile, 0.09% trifluoroacetic acid. Absorbance was monitored at 214 and 280 nm. A major peak eluting at ~20 min was further purified by analytical reversed-phase HPLC using a 5-µm Vydac C18 column (0.46 × 25 cm) eluted at 1 ml/min with a linear gradient of 0 to 50% B over 45 and 90 min. Absorbance was monitored at 214 nm.

Primary Structure of EpI

A sample of approximately 100 pmol of purified native peptide was fully reduced in the presence of 50 mM dithiothreitol and 50 mM ammonium acetate, pH 8.5, at 37 °C for 1 h, then alkylated in the presence of 100 mM 4-vinylpyridine for 1 h at room temperature in the dark. The alkylated peptide was repurified by HPLC and analyzed by Edman chemistry on an ABI model 470A protein sequencer.

Sulfotyrosine Analysis of EpI

Enzymatic Treatment-- A hydrolysate of EpI was prepared by carboxypeptidase Y digestion. Briefly, a sample of ~2 nmol of native EpI was dissolved in 100 µl of 50 mM ammonium acetate, pH 5.5, and treated with 0.75 µg of carboxypeptidase Y (Boehringer Mannheim, sequencing grade) for 24 h at room temperature (enzyme to protein ratio ~ 1:5). The products of the enzymatic degradation were assessed by analytical C18 HPLC, mass spectrometry (MS), and amino acid analysis.

Preparation of Sulfated Tyrosine Standard-- Sulfated Fmoc-Tyr was treated overnight in mild sodium hydroxide. The reaction mixture was purified on a 5 µM Vydac C18 reversed-phase column (0.46 × 25 cm). With a standard gradient of 0.1% trifluoroacetic acid/acetonitrile, sulfated tyrosine eluted cleanly in the solvent front, while the remaining reaction products were retained on a C18 column. Purity of the sulfated tyrosine (262 Da) was confirmed by MS.

Derivatization of Amino Acids with PITC and Analysis by HPLC-- The free amino acids liberated from the digested peptide and amino acid standards were derivatized with PITC (19). The digests and standards were redried after addition of 40 µl of ethanol/water/triethylamine (2:2:1). The derivatization reagent consisted of ethanol/triethylamine/water/PITC (7:1:1:1) which was made fresh daily. The dried samples were resolubilized with 40 µl of derivatization reagent and the sealed samples allowed to react for 30 min before drying under vacuum. Derivatized amino acids were separated on a Pico-Tag column (Waters) using a linear gradient from 0% B to 46% B over 18 min: solvent A, 0.14 M sodium acetate containing 0.05% (v/v) triethylamine and adjusted to pH 6.35 with glacial acetic acid; solvent B, 60% acetonitrile/water.

Mass Spectrometry (MS)

Mass spectra were acquired on a PE-SCIEX API 111 triple quadrupole mass spectrometer (PE-SCIEX, Ontario, Canada) equipped with an ion spray atmospheric pressure ionization source. Samples (20 µl) were injected into a moving solvent (10 µl/min; 50% acetonitrile, 0.05% trifluoroacetic acid) coupled directly to the ionization source via a fused silica capillary interface (50 µm × 50 cm). Sample droplets were analyzed in both the positive and negative ionization modes. The ions entered the analyzer through an interface plate and subsequently through an orifice (100-120 µm diameter) at declustering potentials from 10 to 90 V in the positive ion mode, and at -50 V in the negative ion mode. Full scan spectra were acquired over m/z 500-2200 in 0.2 Da steps with a total scan time of 3 s.

To help determine if EpI was sulfated, MS spectra of native and synthetic EpI, CCK-8 (a sulfated peptide standard) and MLP111 (a phosphorylated peptide standard) were acquired over m/z 50-100. A high declustering voltage of -220 V and negative ion detection were used to confirm the presence of the sulfate and related ions. The declustering potential is the orifice voltage less the reference voltage (R0), which was 30 or -30 V in positive or negative ion modes, respectively.

HPLC/MS analysis of crude venom (20 µl of a 2 mg/ml solution) was performed with a 5-µm Vydac C18 column (0.21 × 25 cm) eluted at 130 µl/min with 100% A for 2 min and then 0 to 80% B over 80 min: solvent A, 0.05% trifluoroacetic acid; solvent B, 90% acetonitrile, 0.05% trifluoroacetic acid. Mass spectra were acquired over m/z 400-2200 at 0.2 Da steps, with a dwell time of 0.3 ms and a total scan time of 3.2 s.

Determination of Disulfide Connectivity

Native EpI (1 nM) was added to the reaction buffer (25 µl; 0.1 M NH4OAc/acetonitrile, 90/10 (v/v); pH 4) and reacted with the reducing agent Tris(2-carboxyethyl)phosphine (2:1, w/w). An aliquot (1 µl) was injected into the MS to determine the extent of reduction over 2 to 10 min. This partially reduced EpI was reacted with 100 mM of the alkylating reagent maleimide (pH 4.0; 21 °C) over 5 min with 1-µl aliquots injected into the MS to determine the extent of alkylation. A reaction product representing the alkylation of one disulfide bond, as determined by MS, was isolated using reversed-phase gradient HPLC on a 5-µm Vydac C18 column (0.21 × 25 cm). To determine disulfide bond connectivity of EpI, this partially reduced product was subjected to Edman sequencing. Disulfide bond connectivity was also determined by collision-induced dissociation MS. Collision-induced dissociation of the partially reduced and alkylated parent precursor ion was effected by bombardment with ultrapure Argon. Bombardment was carried out in quadrupole-2 with a collision cell gas thickness of 3 × 1014 atoms/cm2 and a collision energy (Q-0 to Q-2 rod offset voltage) of typically 20-40 eV relative to the laboratory frame. The resulting product ion spectra were obtained by scanning quadrupole-3 over m/z 50-2200 at 0.2 Da steps. Spectra were analyzed with MacBioSpec (PE-SCIEX).

Peptide Synthesis

Chain Assembly-- Two peptides, EpI and [Tyr15]EpI, were each synthesized on a 0.5-mmol scale by manual solid phase peptide synthesis using Fmoc chemistry. The following amino acid side chain protecting groups were used: Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Trt), and Ser(tBu). Met, Gly, and Pro, were used without side chain protection. Sulfated tyrosine was used for the synthesis of EpI and Tyr(tBu) was used for the synthesis of the unsulfated peptide ([Tyr15]EpI). Peptide resin samples (~5 mg) were removed after each coupling step for determination of residual free alpha -amino groups by the quantitative ninhydrin test (20), except for couplings to proline where a coupling efficiency of >99.5% was assumed where the result of a modification of the Isatin test (21, 22) was satisfactory. Double couplings were used to achieve coupling efficiencies of >99.5%. First couplings <95% were obtained only for residues 8 and 9 of [Tyr15]EpI.

Deprotection, Cleavage, and Peptide Oxidation-- The Nalpha -Fmoc group was removed on resin from the fully protected peptide by treatment with piperidine/dimethylformamide (1:1, 2 × 1 min) and the partially deprotected peptide resin was dried under a stream of nitrogen after washing with dimethylformamide and dichloromethane. The peptide was cleaved from the resin and the side chain protecting groups were simultaneously removed by treatment with (i) trifluoroacetic acid/H2O (9:1, v/v) with 50 eq of m-cresol or (ii) trifluoroacetic acid/triisopropylsilane (9:1, v/v) (33), both at 4 °C for 3 to 5 h with similar results. The majority of the trifluoroacetic acid was removed on a rotavapor and the crude peptide precipitated, washed with ether, dissolved in 50% acetonitrile, 0.1% trifluoroacetic acid, diluted with water, and lyophilized. The peptide precipitation, washing, and dissolution steps were carried out under a stream of nitrogen to minimize uncontrolled air oxidation of the peptide. The peptides were purified by reversed-phase HPLC and the purified reduced peptides (100 µM) were oxidized in 100 mM NH4HCO3, pH 8.3, at room temperature for 24-48 h.

Purification of Synthetic EpI and [Tyr15]EpI-- Synthetic peptide oxidation was monitored by analytical reversed-phase HPLC on a 5-µm Vydac C18 column (0.46 × 25 cm). EpI and [Tyr15]EpI were obtained using a preparative 10-µm Vydac C18 column (2.2 × 25 cm) eluted at 8 ml/min with 5 to 50% B over 60 min and 50 to 80% B over 20 min: solvent A, 0.1% trifluoroacetic acid; solvent B, 90% acetonitrile, 0.09% trifluoroacetic acid. The eluant was monitored at 230 nm. Retention times of native and synthetic peptides were compared by analytical reversed-phase HPLC (see above) eluted at 1 ml/min with a linear gradient of 0.7% acetonitrile/min from 0.1% trifluoroacetic acid to 90% acetonitrile, 0.09% trifluoroacetic acid. Coelution was confirmed by co-injection of synthetic and native EpI in a 2:1 ratio.

Materials for Peptide Characterization and Synthesis

Acetonitrile, ethanol, and glacial acetic acid were from BDH Laboratory Supplies, Poole, United Kingdom. High purity water was generated by a Milli-QTM purification system (Millipore, Bedford, MA). Triethylamine (freshly distilled) was from Sigma. Phenylisothiocyanate (PITC) and amino acid standards were from Pierce. Sodium acetate was from Aldrich Chemical Co. 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, piperidine, trifluoroacetic acid, diisopropylethylamine, dimethylformamide (peptide synthesis grade), and Fmoc-sulfotyrosine were purchased from Auspep (Melbourne, Australia). Acetonitrile (HPLC grade) was obtained from Waters Millipore (Milford, MA). Fmoc-aa-resin (Ramage Amide-Resin, tricyclic amide linker) was purchased from BAChem and Fmoc-L-amino acids were purchased from BAChem, Nova Biochem, and Auspep. Stock solutions (100 mM) of Tris(2-carboxyethyl)phosphine (Strem Chemicals), maleimide (Aldrich), 4-vinylpyridine and dithiothreitol were prepared in 90:10 (v/v) 0.1 M NH4OAc/acetonitrile, pH 4.0, and stored at -20 °C. Other reagents and solvents were ACS analytical reagent grade.

Measurement of Catecholamine Secretion from Chromaffin Cells

Chromaffin cells were isolated from bovine adrenal medullary tissue and cultured as described previously (23). The protocol employed for the catecholamine release studies was that detailed by Livett et al. (24). Experiments were conducted on 24-well culture plates containing 0.4 ml of medium/well at 1.25 × 106 cells/ml. Following a 3-day incubation at 37 °C in an atmosphere of 10% carbon dioxide, the culture was allowed to equilibrate to room temperature for 5 min. To remove the incubation medium, the culture was washed with modified Locke's buffer of the following composition in mM: NaCl, 154; KCl, 2.6; CaCl2, 2.2; K2HPO4, 2.15; KH2PO4, 0.85; MgSO4, 1.18; D-glucose, 10; and bovine serum albumin, 0.5%. The cells were preincubated in the presence or absence of synthetic conotoxin (0-10 µM) for 5 min at room temperature and then incubated for 5 min with 4 µM nicotine ± conotoxin. Cells treated with buffer alone, 4 µM nicotine alone, or conotoxin alone served as controls. The incubation medium containing released catecholamines was removed and acidified with 2 M perchloric acid. To determine total cell catecholamines, the cells in each culture plate well were treated for 5 min with 0.01 M perchloric acid to lyse the cells and acidified to a final concentration of 0.4 M perchloric acid. The released catecholamines were quantified by electrochemical oxidation, after separation by reverse-phase HPLC (24). For K+-induced catecholamine release experiments, 56 mM KCl was used as the agonist instead of nicotine. In the nicotine/conotoxin competition experiments, the cells were incubated with synthetic EpI or [Tyr15]EpI at 1 µM and varying concentrations of nicotine (0-100 µM).

Rat Phrenic Nerve-Hemidiaphragm Studies

Male Buffalo rats (150-250 g, 3-4 months old) were sacrificed by asphyxiation with carbon dioxide and exsanguinated. The left hemi-diaphragm and phrenic nerve were isolated as described previously (25) and mounted under 1 g of resting tension in a 25-ml organ bath containing Krebs-Henseleit solution aerated with carbogen (95% O2, 5% CO2). The nerve was stimulated via a bipolar electrode with pulses of 0.5-ms duration and supramaximal voltage using a Grass S48 stimulator with an SIU5 isolation unit. Single twitches were produced by continuous stimulation at 0.1 Hz, interspersed at 5-min intervals by train-of-four twitches (2 Hz for 1.9 s) or tetanic contractions (50 Hz for 2 s), measured alternately. Responses were detected using a Grass FT03 isometric force-displacement transducer connected to a Neotrace DC amplifier and recorded via a Mac Lab/4S using the application Chart (v 3.4/s) on a Macintosh LC630 computer. The tissue was treated with synthetic conotoxin (1-10 µM) in buffer for 60 min, washed several times with buffer alone, and then allowed a 30-min recovery time prior to treatment with the next higher toxin concentration. Data analyses were performed as described previously (26).

Acetylcholine-evoked Currents in Rat Parasympathetic Neurons

Preparation-- Membrane currents were studied in isolated parasympathetic neurons of neonatal rat intracardiac ganglia. The procedures for isolation of these neurons have been described in detail previously (27). Briefly, neonatal rats (3-8 days old) were sacrificed by decapitation, and the heart was excised and placed in a saline solution containing (mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 7.7 glucose, and 10 HEPES (pH adjusted to 7.2 with NaOH). Atria were removed and incubated for 1 h at 37 °C in saline solution containing 1 mg/ml collagenase (Type 2; Worthington Biochemical Corp., Freehold, NJ). Following enzymatic treatment, clusters of ganglia were dissected from the epicardial ganglion plexus, transferred to a sterile culture dish containing high glucose culture medium (Dulbecco's modified Eagle's medium), 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin, and triturated using a fine-bore Pasteur pipette. The dissociated neurons were then plated onto 18-mm glass coverslips coated with laminin, and incubated at 37 °C for 24-72 h under 95% air, 5% CO2, in a humid atmosphere. At the time of the experiments, neurons were transferred to a recording chamber (0.5-ml volume) and viewed at 400 × magnification using an inverted phase-contrast microscope.

Electrical Recordings and Data Analysis-- Membrane currents were recorded from isolated intracardiac neurons under voltage clamp conditions using the whole-cell patch recording configuration (28). In whole-cell experiments, electrical access was achieved either by rupturing the membrane patch and dialyzing the cell, or through the use of the perforated patch method (29). The perforated patch method allows the intracellular integrity of the neurons to be maintained to confirm that any effects are independent of possible cell dialysis effects which can alter the functional response of these neurons (30).

For perforated patch experiments, a stock solution of 60 mg/ml amphotericin B in dimethyl sulfoxide was prepared the day of the experiment and kept on ice, in the dark. Immediately prior to use, the amphotericin B stock solution was diluted in pipette solution (see below) to yield a final concentration of 360 µg/ml amphotericin B in 0.6% dimethyl sulfoxide. Following gigaseal formation, the neurons were held at -70 mV and voltage pulses (20 ms) to -80 mV were applied at 1 Hz. Amphotericin B incorporation into the membrane patch resulted in a fast capacitive transient, the appearance of a slow capacitive transient and a decrease in the series resistance (RS). Experiments were continued only if RS decreased to <= 10 megohm within 10 min of seal formation. Final series resistance was usually 5-8 megohm, and approximately 50% RS compensation was used.

Electrodes for whole-cell experiments were pulled from thin-walled borosilicate glass (TF150; Clark Electromedical Inst., Reading, United Kingdom) and the final pipette resistance was 1-3 megohm for conventional and perforated patch whole-cell recording. Membrane currents evoked by agonist application were amplified and filtered (5 kHz) using and Axopatch 200A patch clamp amplifier (Axon Instruments Inc., Foster City, CA) and recorded on DAT tape using an analog to digital tape recorder (DT-1204; Biologic, Claix, France). Membrane currents were continuously monitored on a digital oscilloscope and a chart recorder. For computer analysis, acetylcholine (ACh)-evoked currents were played back from digital tape, filtered, and digitized (Digidata 1200A; Axon Instruments Inc.). Whole-cell currents were filtered at 1 kHz (-3 dB; 4-pole Bessel filter), sampled at 3 kHz and acquired to a PC Pentium/100 MHz computer using the Axoscope 1.0 and pClamp 6 acquisition programs (Axon Instruments Inc.). Data are expressed as mean ± S.E.

The control external solution for dialyzed and perforated whole-cell recordings was physiological saline solution containing (mM): 140 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 glucose, and 10 HEPES-NaOH, pH 7.2. The pipette solution for conventional whole-cell recordings consisted of (mM): 140 CsCl, 2 MgATP, 0.2 guanosine 5'-triphosphate sodium salt (GTP), 2 Cs4BAPTA, 10 HEPES-CsOH, pH 7.2. The perforated patch pipette solution contained (mM): 75 K2SO4, 55 KCl, 5 MgSO4, 10 HEPES (pH to 7.2 with N-methyl-d-glucamine). The recording chamber was continuously perfused (2 ml/min) with the indicated solutions at room temperature (22-23 °C). ACh-evoked responses were elicited during whole-cell recordings by focal and rapid application of 500 µM ACh ± synthetic EpI or [Tyr15]EpI in physiological saline solution containing 100 nM atropine from a multibarrel flow pipe mounted on a piezoelectric positioning system (PZS-200; Burleigh Instruments, Inc., Fishers, NY). Atropine (0.1-1 µM) was added to both the bath and solutions through the multibarrel flow pipes to inhibit muscarinic receptor-mediated currents observed in perforated-patch whole-cell recordings (30). The flow pipe constructed from theta -tubing was positioned 50 µm from the cell soma to evoke maximal responses to ACh. The osmotic activities of the solutions were monitored with a vapor pressure osmometer (Wescor 5500).

Materials for Chromaffin Cell Culture and Parasympathetic Neuron Studies

For chromaffin cells, culture media, penicillin (G, sodium, NF grade), streptomycin sulfate, and fetal calf serum were obtained from Life Technologies, Inc. Collagenase A was from Boehringer-Mannheim and Percoll from Pharmacia. Tissue culture plates were from Sarstedt. Bovine serum albumin was from HÄMOSAN, perchloric acid and buffer salts were from BDH Chemicals Ltd., and all stock solutions were prepared in Locke's buffer and stored at -20 °C. For rat parasympathetic neuron studies, all chemical reagents were analytical grade. ACh, atropine sulfate, and mecamylamine hydrochloride were from Sigma and alpha -bungarotoxin was from Calbiochem (La Jolla, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation and Chemical Characterization of EpI

Identification of EpI-- Most alpha -conotoxins identified to date have two disulfide bonds, fall within the mass range 1300-2000 Da, and elute at relatively low concentrations of organic solvent on reversed-phase chromatography. HPLC/MS (Fig. 2A) and HPLC (not shown) of a crude extract from C. episcopatus revealed a major, early eluting peak (at 22 or 19 min by analytical or preparative HPLC, respectively) that was identified as a new conopeptide we named EpI. EpI was present in the crude venom at 1-5% of the A214 absorbing material.


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Fig. 2.   Identification of EpI. A, base peak chromatogram obtained from a HPLC/MS of the crude venom of C. episcopatus (m/z 400-2200, declustering potential 50 V). B, MS spectrum of EpI from HPLC/MS at a declustering potential 15 V, with an inset showing the reconstructed spectrum. C, MS spectrum of EpI from A at a declustering potential 50 V, with an inset showing the reconstructed spectrum.

Selected scans from two HPLC/MS runs at different declustering potentials of 15 and 50 V are shown in Fig. 2, B and C. At the lower declustering potential, two coeluting masses differing by 80 Da were observed, while at higher declustering potentials the lower mass predominated. These spectra, together with the observation of a reduced tendency to lose 80 Da when analyzed in negative ion detection mode (31), suggested EpI was a sulfated peptide of average molecular mass 1866.6 Da. Further confirmation of the presence of a sulfated amino acid being present in EpI was obtained using a high declustering potential (-220 V) and negative ion detection. The resultant mass spectrum revealed the presence of characteristic sulfate ions, SO3- and SO4- at m/z 80 and 96, respectively (data not shown). Comparison of the spectra of EpI to those of CCK-8 (a sulfated peptide) and MLP 111 (a phosphorylated peptide) confirmed that EpI was sulfated (data not shown).

Primary and Secondary Structure of EpI-- Purified EpI was reduced, alkylated, and its sequence determined by Edman sequencing to be GCCSDPRCNMNNPDYC. This sequence gave the predicted relative molecular mass (Mr) of 1787.02 Da for the oxidized, COOH-terminally amidated form, 80 Da less than molecular mass 1867.08 Da predicted for the sulfated peptide. Disulfide connectivity was determined by partial reduction, alkylation, and Edman sequencing. EpI was found to have the conserved alpha -conotoxin framework [1-3, 2-4] with disulfide linkages between Cys2-Cys8 and Cys3-Cys16. MS indicated that the carboxyl terminus was amidated, assuming one residue was sulfated (data not shown). MS also showed that residues at positions 2 and 8 had been selectively alkylated and that EpI possessed two disulfide bonds (data not shown).

Identifying the Post-translational Modification-- Reduced and alkylated EpI was cleaved with trypsin to generate two fragments which were analyzed by MS and further fragmented by MS/MS to help locate the site of the +80 Da modification. This showed that the modification was in the COOH-terminal fragment, most likely at Tyr15 (data not shown). EpI was also digested with carboxypeptidase Y to generate an enzymic hydrolysate and, after treatment of the hydrolysate with PITC, amino acid analysis of this material showed that the tyrosine residue was sulfated (Fig. 3).


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Fig. 3.   Modified amino acid analysis of sulfated tyrosine (PTC derivatives detected). A, native EpI hydrolysate generated by carboxypeptidase Y digestion; B, analysis blank; C, sulfated tyrosine standard.

Synthesis of EpI and [Tyr15]EpI-- Solid phase chemical synthesis of EpI was undertaken to confirm the proposed structure of EpI and to provide sufficient material for further biological (present study) and structural characterization.2 The cleavage conditions selected to remove reduced EpI from resin were a compromise between achieving adequate removal of the side chain protecting groups and preventing undesirable desulfation. Problems were encountered with the incomplete removal of the tBu and Pmc protecting groups. Unfortunately, the use of 1,2-ethanedithiol to scavenge tBu and thioanisole to accelerate Arg(Pmc) deprotection by trifluoroacetic acid is incompatible with sulfopeptide synthesis (33). Oxidation of pure reduced EpI resulted in the production of one major isomer and two minor isomers. The major oxidized product, which eluted earlier than the reduced form, was purified by reversed-phase HPLC and compared with native EpI. MS results were consistent with the amidated form (synthetic EpI calculated Mr 1867.08 Da, observed molecular mass 1866.6 Da). Oxidized synthetic EpI coeluted with the native EpI peptide (Fig. 4). HPLC chromatograms (Fig. 4) and MS analyses (data not shown) confirmed the purity of native and synthetic EpI and synthetic [Tyr15]EpI.


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Fig. 4.   Analytical reverse phase HPLC comparison of native and synthetic EpI and [Tyr15]EpI. A, native EpI; B, synthetic EpI; C, co-injection of synthetic plus native EpI in a 2:1 ratio; D, synthetic [Tyr15]EpI. Detection at 214 nm and retention time in minutes.

Using a similar protocol, the analogue [Tyr15]EpI yielded one predominant isomer and two minor isomers. The purified unsulfated analogue [Tyr15]EpI eluted slightly later than the sulfopeptide EpI. MS gave a result consistent with [Tyr15]EpI (calculated Mr 1787.02 Da, observed molecular mass 1786.2 Da).

Mode of Action of EpI

Muscle-type Nicotinic Acetylcholine Receptor Response-- Synthetic EpI and [Tyr15]EpI were tested in the rat phrenic nerve-hemidiaphragm preparation (26). Neither peptide significantly inhibited single twitches, train-of-four twitches, or tetanic contractions after incubation for 1 h over the concentration range 1-10 nM (data not shown).

Neuronal-type Nicotinic Acetylcholine Receptor Response-- Synthetic EpI and [Tyr15]EpI inhibited nicotine-induced catecholamine release in bovine chromaffin cells (Fig. 5). EpI inhibited 50% of nicotine-induced adrenaline and noradrenaline release at 8.4 × 10-8 M and 2.1 × 10-7 M (pIC50 ± S.E. values of 7.1 ± 0.12 M and 6.7 ± 0.06 M), respectively. [Tyr15]EpI was less potent, inhibiting nicotine-induced adrenaline and noradrenaline release at 8.5 × 10-7 M and 1.0 × 10-6 M (pIC50 values of 6.1 ± 0.1 M and 6.0 ± 0.08 M), respectively. Hill slopes obtained for these inhibitory dose-response curves ranged from -0.6 to -0.7, with 95% confidence intervals ranging from -0.8 to -0.4, indicating slopes significantly different from -1. Both EpI and [Tyr15]EpI appear to be full functional antagonists of nicotine-evoked noradrenaline and adrenaline release (Fig. 5A). K+-induced catecholamine release was not inhibited by either peptide up to 10 µM (data not shown), indicating that the inhibition by EpI of nicotine-evoked catecholamine secretion was via inhibition at the nAChRs, rather than by block of the voltage-gated ion channels.


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Fig. 5.   Synthetic EpI and [Tyr15]EpI inhibition of the nicotine-evoked catecholamine release in bovine chromaffin cells. A, concentration-dependent conotoxin inhibition of 4 µM nicotine-stimulated adrenaline (Ad) and noradrenaline (NA) release over 5 min (n = 3-4). Neither conotoxin inhibited 56 mM K+-induced release (data not shown). B, effect of EpI on the concentration-dependent nicotine-induced release of noradrenaline, expressed as a percentage of total cell noradrenaline (n = 3-4). EpI caused a parallel shift in the response indicative of functional competitive antagonism. Similar results were obtained for noradrenaline release and for the effect of [Tyr15]EpI on both adrenaline and noradrenaline release (data not shown).

Addition of EpI (1 µM) with various concentrations of nicotine (0-100 µM) resulted in a parallel shift of the nicotine dose-response curve to the right (Fig. 5B). [Tyr15]EpI caused a similar shift in the nicotine dose-response curve (data not shown). These observations are consistent with EpI and [Tyr15]EpI acting as functional competitive inhibitors of nicotine action at the nAChR.

ACh-evoked Membrane Currents in Rat Parasympathetic Neurons-- To further investigate the effects of synthetic EpI on neuronal AChRs, whole-cell recording of membrane currents evoked in response to rapid application of ACh was carried out in isolated parasympathetic neurons from rat intracardiac ganglia. ACh-evoked currents obtained in response to 500 µM ACh in a neuron held at -60 mV are shown in Fig. 6 (control traces). ACh-evoked peak current amplitude was substantially larger than previously reported in these neurons (27, 30, 34) due to the rapid application of ACh minimizing nicotinic ACh receptor desensitization (35). ACh-evoked currents were completely and reversibly inhibited by bath application of 1 µM mecamylamine. In the presence of 1 nM EpI, the ACh-evoked current amplitude was reversibly decreased by 24% but the kinetics of the response appeared unchanged (Fig. 6A). The percentage inhibition of ACh-evoked current observed over the voltage range -120 to -20 mV was similar, indicating that the block at the nicotinic AChR was not voltage dependent. Bath application of 300 pM [Tyr15]EpI similarly reduced the amplitude of the ACh-evoked current by 27% and its effect was reversible upon washout (Fig. 6B). Fig. 6C shows inhibition of ACh-evoked currents obtained in the presence of a maximal effective concentration of EpI (300 nM) or 300 nM [Tyr15]EpI in the same neuron. The EpI-resistant current obtained in the presence of both EpI and [Tyr15]EpI exhibited an increased rate of decay and was abolished by mecamylamine (1 µM) or alpha -bungarotoxin (1 µM). Dose-response relations obtained for the inhibition of ACh-evoked current amplitude at -60 mV in the presence of EpI and [Tyr15]EpI are shown in Fig. 6D. The dose-response relations were fitted with a single site adsorption isotherm with half-maximal inhibitory concentrations (IC50) of 1.6 and 0.4 nM for EpI and [Tyr15]EpI, respectively. Maximal inhibition obtained with EpI and [Tyr15]EpI concentrations up to 1 µM was 60% of the peak ACh-evoked current. The EpI-resistant current was observed in the majority (>80%) although not all neurons studied, and was blocked by alpha -bungarotoxin.


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Fig. 6.   Synthetic EpI and [Tyr15]EpI inhibition of nicotinic ACh-evoked currents in parasympathetic neurons from rat intracardiac ganglia. A, superimposed traces of whole-cell currents evoked in response to rapid application of 500 µM ACh in the absence (Control) and presence of 1 nM EpI (open circle ). ACh-evoked currents were obtained at a holding potential of -60 mV under perforated patch whole-cell recording conditions at 22 °C. B, superimposed traces of whole-cell ACh-evoked currents in a neuron held at -60 mV in the absence and presence of 300 pM [Tyr15]EpI (bullet ). C, superimposed traces of ACh-evoked currents obtained from a dialyzed neuron held at -60 mV in the absence (Control) and presence of 300 nM EpI (open circle ) or 300 nM [Tyr15]EpI (bullet ). D, dose-dependent inhibition of ACh-evoked peak current by EpI (open circle ) and [Tyr15]EpI (bullet ) normalized to the peak current amplitude of the ACh-evoked current obtained at -60 mV in the absence of EpI. The dose-response relations were fit with a single site adsorption isotherm giving IC50 values of 1.6 and 0.4 nM for EpI and [Tyr15]EpI, respectively. The data points represent the mean ± S.E. of 14 cells.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have isolated and characterized a new alpha -conotoxin, EpI, from the venom of the molluscivorous snail C. episcopatus collected from the Great Barrier Reef, Australia. The peptide was classified as an alpha -conotoxin based on sequence, disulfide connectivity, and pharmacology. alpha -Conotoxin EpI is the first sulfated conotoxin characterized. The sulfate, which is on Tyr15 of EpI, is almost completely lost at declustering potentials used routinely for the MS analysis of peptides (>= 40 V), making its detection difficult. However, the sulfotyrosine could be identified by amino acid analysis and MS at low declustering potentials or in negative ion detection mode (31). The coelution of native and synthetic EpI support our direct evidence that EpI was sulfated at Tyr15. Interestingly, no [Tyr15]EpI was detected in the venom of C. episcopatus.

While extensive post-translational modifications are characteristic of conopeptides, phosphorylation or sulfation in conotoxins has not previously been demonstrated. However, the possible presence of sulfated and/or phosphorylated derivatives of PnIB has been suggested on the basis of a mass difference of 80 Da in unconfirmed observations of Fainzilber et al. (36). Tyrosine sulfation has been recognized as a widespread post-translational modification of proteins and consensus features of tyrosine sulfation sites have been described (37, 38). The known tyrosine-sulfated proteins can be classified into discrete functional classes including blood clotting factors, neuropeptides, extracellular matrix proteins, and immune system proteins (37). A recent revision of the consensus features for tyrosine O-sulfation based on mutational analysis experiments found, contrary to previous findings (i) that neighboring residues contribute only moderately to sulfation and (ii) that extensive sulfation can be obtained despite dramatic changes in the charge distribution of the neighboring residues, with basic residues not only being permissible near the site of sulfation but perhaps enhancing sulfation (39). The single critical position for tyrosine O-sulfation is -1, which should contain a neutral or acidic residue. EpI fulfils these requirements. Another suggested consensus feature for tyrosine sulfation is the absence of disulfide-bonded cysteine residues from -7 to +7 (37, 38). EpI clearly does not fit this criterion, possessing a disulfide-bonded cysteine residue at -7 and +1. However, there is no direct evidence that disulfide bonds necessarily prevent the sulfation of tyrosine (38). EpI may be the first example of this motif. The functional role of sulfation in this alpha -conotoxin is less clear. Aside from any effect on potency or selectivity, sulfation may have evolved as a strategy to improve the in vivo stability of conotoxins. However, reports in the literature are equivocal. Differential behavior of sulfated and unsulfated peptides has been observed in studies of the degradation of human gastrin and cholecystokinin by endopeptidase, where the presence of a sulfate group reduced the rate of hydrolysis of gastrin by endopeptidase, yet enhanced cholecystokinin degradation by the same enzyme (40).

EpI is constructed with 4 residues in loop one and 7 residues in loop two and was anticipated to have a structure similar to those reported for PnIA and PnIB (41, 42). Compared with other 4/7 framework alpha -conotoxins, EpI has homology to both loop one and loop two of PnIA and PnIB and to loop one of MII (see Table I). The alpha -conotoxins EpI, MII (10, 43, 44), PnIA, PnIB (36), and ImI (9, 43, 45) all contain 4 amino acid residues in loop one, the first of which is serine, suggesting that this construct may be important for neuronal nAChR selectivity. The crystal structure of [Tyr15]EpI and a detailed comparison with PnIA and PnIB are reported elsewhere.2

                              
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Table I
alpha -Conotoxin sequence, loop size species of origin, prey preference, and mammalian nAChR target

The activities of synthetic EpI and its unsulfated analogue [Tyr15]EpI were similar. Both caused competitive inhibition of nicotine-induced catecholamine release in bovine adrenal chromaffin cells (predominantly neuronal alpha 3beta 4 nAChRs (46)) but neither had an effect on the rat phrenic nerve-hemidiaphragm (muscle nAChRs). Thus EpI and [Tyr15]EpI are selective inhibitors of neuronal nAChRs. At nanomolar concentrations, both peptides partially inhibited ACh-evoked currents in a dose-dependent manner in isolated parasympathetic neurons of rat intracardiac ganglia. The EpI block of nAChRs was reversible and did not exhibit any voltage dependence. The residual current that remained after saturating doses of EpI or [Tyr15]EpI was found to be sensitive to inhibition by alpha -bungarotoxin, indicating that EpI has little, if any, effect on neuronal alpha 7 nAChRs of rats. Given that the remainder of the ACh-evoked current in rat parasympathetic neurons arises from nAChRs constructed primarily of alpha 3beta 2 and/or alpha 3beta 4 subunits (47), it appears that EpI selectively inhibits neuronal alpha 3beta 2 and alpha 3beta 4 nAChRs. The difference in the rank order of potency between EpI and [Tyr15]EpI at bovine chromaffin cells and rat parasympathetic neurons may indicate that sulfation has an effect on selectivity across rat and bovine nAChRs. It is possible that EpI may have evolved to selectively target molluscan forms of the nAChR as part of the prey capture strategy of C. episcopatus. The more than 50-fold difference in absolute potency between EpI and [Tyr15]EpI at bovine chromaffin cells and rat parasympathetic neurons is not unexpected given results of other investigators with the more sensitive patch-clamp technique. For example, alpha -conotoxin ImI has an IC50 of 0.22 µM in patch-clamp studies on frog oocytes (45) compared with 3.0 µM in bovine chromaffin cells (43). Species differences in the susceptibility of muscle- and neuronal-nicotinic receptors to alpha -conotoxins have also been noted for alpha -conotoxins MI (44) and ImI (9).

With respect to its ability to inhibit nAChRs in bovine chromaffin cells, EpI is the most potent of the alpha -conotoxins tested to date, being 5-fold more potent than PnIB (IC50 = 0.7 µM) and 20-fold more potent than ImI (IC50 = 3 µM) (43). It is unlikely that EpI targets bovine alpha 7 receptors, given that EpI inhibits both adrenaline and noradrenaline secretion in bovine chromaffin cells but only the adrenaline cells contain alpha 7 (49). Furthermore, studies in the rat parasympathetic intracardiac ganglia indicate that EpI has little if any effect on rat neuronal alpha 7 nAChRs. It has previously been shown that alpha -conotoxin MII specifically targets rat alpha 3beta 2 nAChRs (10, 44), whereas we show here that EpI and [Tyr15]EpI likely target rat alpha 3beta 2 and alpha 3beta 4. On the basis that Campos-Caro et al. (46) have shown that there is no beta 2 subunit expressed in bovine chromaffin cells (which contain neuronal alpha 3, alpha 5, alpha 7, and beta 4 subunits) it appears that EpI targets bovine alpha 3beta 4 receptors. However, there is pharmacological evidence that alpha 3beta 4 in bovine chromaffin cells behaves similarly to alpha 3beta 2 in the rat (46, 50).

Our findings with EpI reinforce those of Groebe et al. (48) with MI who suggest that caution should be exercised when alpha -conotoxins are used to pharmacologically define the nicotinic receptor subtypes across different species. A radioligand binding assay utilizing [125I-Tyr15]EpI to more precisely define its site of binding in different species is under development. The recent finding of Kulak et al. (14) shows that alpha -conotoxins with appropriate nAChR-subtype specificity have potential as novel therapeutics. Given that its structure is now well defined,2 EpI provides a useful template for the design of selective small molecule inhibitors of neuronal nAChRs.

    ACKNOWLEDGEMENTS

We thank John Gehrmann for helpful discussions, Jon-Paul Bingham and Ian Loch for assistance and advice concerning cone collection, and Ian Loch for identification of C. episcopatus. A specimen of C. episcopatus, voucher number C.203165, has been lodged with the Australian Museum, 6 College St., Sydney.

    FOOTNOTES

* This work was supported in part by grants from the Department of Industry, Science and Tourism and AMRAD Operations, Australia (to P. F. A. and R. J. L.) and the Australian Research Council (to B. G. L. and J. G. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Biology, University of California, San Diego, La Jolla, CA 92093.

parallel To whom correspondence should be addressed: Centre for Drug Design and Development, The University of Queensland, Brisbane 4072, Australia. Tel.: 61-7-3365-1924; Fax: 61-7-3365-1990.

1 The abbreviations used are: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; tBu, t-butyl; CTX, conotoxin; EpI, alpha -conotoxin EpI; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PITC phenylisothiocyanate; PMC, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; HPLC, high performance liquid chromatography; Trt, triphenylmethyl.

2 S.-H. Hu, M. Loughnan, R. Miller, C. M. Weeks, R. H. Blessing, P. F. Alewood, R. J. Lewis, and J. L. Martin, unpublished data.

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Abstract
Introduction
Materials & Methods
Results
Discussion
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