Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-type Calcium Channels, from the Chinese Bird Spider Ornithoctonus huwena
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m513542200
ISSN1083-351X
AutoresZhonghua Liu, Jie Dai, Long‐Jun Dai, Meichun Deng, Zhe Hu, Weijun Hu, Songping Liang,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoHuwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I–IV, II—V, and III–VI. Its cDNA, determined by rapid amplification of 3′ and 5′ cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with ω-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA ≈ HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels. Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I–IV, II—V, and III–VI. Its cDNA, determined by rapid amplification of 3′ and 5′ cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with ω-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA ≈ HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels. Ca2+ entry into cells through voltage-gated Ca2+ channels mediates many physiological processes including neurotransmitter release, neurosecretion, neuronal excitation, survival of neurons, and regulation of gene expression. Currently, five main types of Ca2+ channels (T, L, N, P/Q, and R) in vertebrate cells have been defined by their physiological and pharmacological properties (1Catterall W.A. Cell Dev. Biol. 2000; 16: 521-555Crossref Scopus (1921) Google Scholar, 2Hofmann F. Biel M. Flockerzi V. Annu. Rev. Neurosci. 1994; 17: 399-418Crossref PubMed Scopus (452) Google Scholar, 3Olivera B.M. Annu. Rev. Biochem. 1994; 63: 823-867Crossref PubMed Scopus (692) Google Scholar). Among the multiple types of Ca2+ channels, N-type Ca2+ channels are sensitive to ω-conotoxin GVIA that is derived from the sea snail Conus geographus, and are concentrated at presynaptic nerve termini, regulating the influx of Ca2+ necessary for neurotransmitter release in both the central and peripheral nervous systems (4Olivera B.M. Gray W.R. Zeikus R. McIntosh J.M. Varga J. Rivier J. Santos V. Cruz L.J. Science. 1985; 230: 1338-1343Crossref PubMed Scopus (639) Google Scholar, 5Cruz L.J. Olivera B.M. J. Biol. Chem. 1985; 261: 6230-6233Abstract Full Text PDF Google Scholar, 6Olivera B.M. Rivier J. Scottll J.K. Hillyard D.R. Cruz L.J. J. Biol. Chem. 1991; 266: 22067-22070Abstract Full Text PDF PubMed Google Scholar). Several different isoforms of N-type Ca2+ channels have now been cloned and functionally characterized (7Lin Z. Haus S. Edgerton J. Lipscombe D. Neuron. 1997; 18: 153-166Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 8Kaneko S. Cooper C.B. Nishioka N. Yamasaki H. Suzuki A. Jarvis S.E. Akaike A. Satoh M. Zamponi G.W. J. Neurosci. 2002; 22: 82-92Crossref PubMed Google Scholar). Pharmacological and gene knock-out studies implicate N-type Ca2+ channels as key mediators of nociceptive signaling in dorsal root ganglion (DRG) 2The abbreviations used are: DRG, dorsal root ganglion; HWTX-X, huwentoxin-X; RACE, rapid amplification of cDNA ends; RP-HPLC, reverse-phase high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; HVA, high voltage-activated; HOBt/TBTU/NMM, hydroxybenzotriazole monohydrate/2-(1H-benzotriazol-1-yl)-1,2,3,3-tetramethyluronium tetrafluoroborate/N-methylmorpholine. 2The abbreviations used are: DRG, dorsal root ganglion; HWTX-X, huwentoxin-X; RACE, rapid amplification of cDNA ends; RP-HPLC, reverse-phase high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; HVA, high voltage-activated; HOBt/TBTU/NMM, hydroxybenzotriazole monohydrate/2-(1H-benzotriazol-1-yl)-1,2,3,3-tetramethyluronium tetrafluoroborate/N-methylmorpholine. cells, and therefore as potential targets for the development of analgesic drugs (9Altier C. Zamponi G.W. Trends Pharmacol. Sci. 2004; 25: 465-470Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Indeed, ziconotide, a synthetic analog of ω-conotoxin MVIIA that is also a specific N-type Ca2+ channels blocker isolated from the venom of the marine snail Conus magus, has received approval for severe chronic pain resistant to other procedures (10Malmberg A.B. Yaksh T.L. Pain. 1995; 60: 83-90Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 11Garber K. Nat. Biotechnol. 2005; 23: 399Crossref PubMed Scopus (37) Google Scholar). Another more selective blocker of N-type Ca2+ channels, ω-conotoxin CVID isolated from C. magus, is currently in Phase II clinical trials in Australia. It is hoped that this peptide may overcome some of the side effects associated with MVIIA use (12Adams D.J. Smith A.B. Schroeder C.I. Yasuda T. Lewis R.J. J. Biol. Chem. 2003; 278: 4057-4062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 13Lewis R.J. Nielsen K.J. Craik D.J. Loughnan M.L. Adams D.A. Sharpe I.A. Luchian T. Adams D.J. Bond T. Thomas L. Jones A. Matheson J.-L. Drinkwater R. Andrews P.R. Alewood P.F. J. Biol. Chem. 2000; 275: 35335-35344Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 14Smith M.T. Cabot P.J. Ross F.B. Robertson A.D. Lewis R.J. Pain. 2002; 96: 119-127Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 15Penna R.D. Paice J.A. Pain. 2000; 85: 291-296Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).The most specific blockers of N-type Ca2+ channels known to date are isolated from the venoms of cone snails. Only a few peptide toxins from other animals have been reported to be able to act specifically on this type of channels, such as Ptu1, isolated from the venom of assassin bugs Peirates turpis (16Corzoa G. Adachi-Akahane S. Nagao T. Kusui Y. Nakajima T. FEBS Lett. 2001; 499: 256-261Crossref PubMed Scopus (45) Google Scholar), and HWTX-I, isolated from the venom of spider Ornithoctonus huwena (17Peng K. Chen X.D. Liang S.P. Toxicon. 2001; 39: 491-498Crossref PubMed Scopus (51) Google Scholar). It is well known that spiders are among the oldest animals on the earth. There are 38,000 described spider species, with at least a similar number uncharacterized. A very conservative estimate of 20 pharmacologically distinct peptides per species leads to an estimated total of ∼1.5 million spider venom peptides, which is much larger than the ∼50,000 peptides estimated to be present in venoms of cone snails (18Tedford H.W. Sollod B.L. Maggio F. King G.F. Toxicon. 2004; 43: 601-618Crossref PubMed Scopus (117) Google Scholar). Thus it is reasonable to believe that the spider venoms might contain a number of peptide probes that are more specific for isoforms of N-type Ca2+ channels. These peptides might aid to discriminate among their isoforms and would become ideal drug candidates for treatment of N-type Ca2+ channel-related disorders.The Chinese bird spider, O. huwena, is found mainly in the hilly areas of Yunnan and Guangxi provinces in the south of China (19Liang S. Toxicon. 2004; 43: 575-585Crossref PubMed Scopus (106) Google Scholar). More than 10 peptide toxins have been isolated from this spider venom, including the N-type Ca2+ channel inhibitor (HWTX-I) (17Peng K. Chen X.D. Liang S.P. Toxicon. 2001; 39: 491-498Crossref PubMed Scopus (51) Google Scholar), insecticidal neurotoxins (HWTX-II, HWTX-VII, and HWTX-VIII) (20Shu Q. Liang S.P. J. Pept. Res. 1999; 53: 486-491Crossref PubMed Scopus (40) Google Scholar, 21Shu Q. Lu S.Y. Gu X.C. Liang S.P. Protein Sci. 2002; 11: 245-252Crossref PubMed Scopus (36) Google Scholar), tetrodotoxin-sensitive Na+ channel blocker (HWTX-IV) (22Peng K. Shu Q. Liu Z. Liang S.P. J. Biol. Chem. 2002; 277: 47564-47571Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), the smallest lectin-like peptide (SHL-I) (23Lu S.Y. Liang S.P. Gu X.C. J. Protein Chem. 1999; 18: 609-617Crossref PubMed Scopus (37) Google Scholar), and others. In the present study, we report the isolation and characterization of huwentoxin-X (HWTX-X) from the venom of O. huwena, a novel specific blocker of N-type Ca2+ channels in rat DRG cells. cDNA sequencing by rapid amplification of the 3′ and 5′ cDNA ends (RACE) method indicates HWTX-X is initially expressed as a prepropeptide, an expression pattern similar to other spider peptide toxins and conotoxins. The determination of the solution structure of HWTX-X by two-dimensional 1H NMR with distance geometry and simulated annealing reveals that HWTX-X adopts the same inhibitor cystine knot motif seen in ω-conotoxins (e.g. GVIA or MVIIA).EXPERIMENTAL PROCEDURESMaterials and Animals—Kunming albino mice and Sprague-Dawley rats were purchased from the Xiangya School of Medicine, Central South University. Cockroaches were from our laboratory stock colonies. All sequencing reagents were purchased from Applied Biosystems (Foster City, CA). The 3′ and 5′ RACE kits and TRIzol reagent were purchased from Invitrogen. Restriction enzymes, Taq DNA polymerase, and pGEMT Easy Vector system were from Promega. All synthesis reagents were purchased from Chemassist Corp. Trifluoroacetic acid and α-cyano-4-hydroxycinnamic acid were from Sigma. All other reagents are analytical grade.Toxin Purification—The venom was obtained by electrical stimulation of female spiders, and the freeze-dried crude venom was stored at -20 °C prior to analysis. Lyophilized venom, dissolved in double-distilled water, was applied onto a reverse-phase high performance liquid chromatography (RP-HPLC) Vydac C18 column (300 Å, 4.6 × 250 mm) using a Waters Alliance system. Venom components were eluted using a linear acetonitrile gradient (0–60% acetonitrile, 0.1% trifluoroacetic acid in 60 min) at a flow rate of 1.0 ml/min. Elution of peptides was monitored at 215 nm.Mass Spectrometry—The molecular masses of peptides were determined using MALDI-TOF MS (Applied Biosystems, Voyager-DE STR Biospectometry work station). Ionization was achieved by irradiation with a nitrogen laser (337 nm), with a 20-kV acceleration voltage. α-Cyano-4-hydroxycinnamic acid was used as matrix. Prior to each analysis in the reflection mode, the masses were calibrated internally using HWTX-I (MH+, 3751.45 Da).Amino Acid Sequencing by Automated Edman Degradation—The native or carboxymethylated peptide was submitted to automatic N-terminal sequencing on an Applied Biosystems model 491 gas-phase sequencer. Edman degradation was performed with a normal automatic cycle program.HWTX-X cDNA Isolation and Characterization—The full-length HWTX-X cDNA was obtained using the RACE method as described previously (24Diao J. Lin Y. Tang J. Liang S. Toxicon. 2003; 42: 715-723Crossref PubMed Scopus (35) Google Scholar). Briefly, 5 μg of mRNA, extracted from the venomous glands of the spider O. huwena, was taken to convert mRNA into cDNA by using the 3′ RACE kit supplied with Superscript II reverse transcriptase and a universal adapter primer (5′-GGCCACGCGTCGACTAGTAC(dT)-3′). The cDNA was then used as a template for PCR amplification using the abridged universal adapter primer (5′-CGAAGCTTGGCCACGCGTCGACTAGTAC-3′) and the gene specific primer (5′-GG(A/G/C/T)AA(A/G)CC(A/G/C/T)TG(C/T)TA(C/T)GG(A/G/C/T)-3′) designed corresponding to the N-terminal residues (Gly6-Lys-Pro-Cys-Tyr-Gly11) of HWTX-X. Based on the partial cDNA sequence of HWTX-X determined by 3′ RACE, an antisense primer was designed and synthesized for 5′ RACE. With the strategy described by the RACE kit supplier, the 5′-end cDNA of HWTX-X was cloned by using the gene-specific primer (5′-GTGAACACACTCCGCAGCAC-3′) and nested primer, respectively. The amplified products were then precipitated and cloned into the pGEM-T easy vector for sequencing.Peptide Synthesis, Folding, and Purification—HWTX-X was synthesized using an Fmoc (N-(9-fluorenyl)methoxycarbonyl)/tert-butyl strategy and HOBt/TBTU/NMM coupling method on an automatic peptide synthesizer (PerSeptive Biosystems) (25Li D. Xiao Y. Xu X. Xiong X. Lu S. Liu Z. Zhu Q. Wang M. Gu X. Liang S. J. Biol. Chem. 2004; 279: 37734-37740Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The crude linear peptide was diluted to a final concentration of 30 μm by 0.1 m Tris-HCl solution (pH 8.0) containing 5 mm reduced glutathione and 0.5 mm oxidized glutathione. The solution was stirred slowly at room temperature for 24 h and the folding reaction was monitored by RP-HPLC and MALDI-TOF MS. The oxidized product was purified by semipreparative RP-HPLC using a 40-min linear acetonitrile gradient (10–30% acetonitrile, 0.1% trifluoroacetic acid) on a column (C18, 1.0 × 25 cm) at 3 ml/min flow rate. The purity of the synthetic HWTX-X was confirmed by analytical RP-HPLC and MALDI-TOF MS.Biological Assays—The toxicity of HWTX-X was qualitatively assayed by intraperitoneal injection into 18–20-g mice of both sexes and intra-abdominal injection into adult male cockroaches (Periplaneta americana) with body weights of 0.3–0.5 g using 20-μl solutions (in 0.9% (w/v) normal saline). Vas deferens assays were performed according to the method of Liang (26Liang S.P. Chen X.D. Shu Q. Zhang Y. Peng K. Toxicon. 2000; 38: 1237-1246Crossref PubMed Scopus (26) Google Scholar). Briefly, adult male Sprague-Dawley rats were killed by CO2 anesthesia followed by decapitation. Vas deferentia were mounted in 5-ml organ baths, with the top of each tissue attached to an isometric force transducer and the bottom attached to a movable support and straddled with platinum stimulating electrodes. The vasa were immersed in Krebs solution and stretched by a passive force of about 10 millinewtons. After an equilibration period of 30 min with frequent changes of medium, the vasa were stimulated with single electrical field pulses (30 V, 0.1 ms duration) every 20 s. The resulting twitch responses mediated by sympathetic nerves were recorded on a chart recorder (RM6240B).Electrophysiological Assays—Acutely dissociated DRG cells were prepared from 4-week-old Sprague-Dawley rats and maintained in short-term primary culture using the method described by Hu et al. (27Hu H.Z. Li Z.W. J. Physiol. 1997; 501: 67-75Crossref PubMed Scopus (61) Google Scholar). The dissociated cells were suspended in essential Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 μg/ml streptomycin. The cells were plated on a poly-l-lysine-coated dish (35 × 10 mm) and incubated at 37 °C in an atmosphere of 5% CO2. Cells in culture for 3–24 h were used in the patch experiments. Experiments were conducted at room temperature (20–25 °C).Cell current recordings were made with the whole cell patch clamp technique using an EPC-9 patch clamp amplifier (HEKA Electronid, Lambrecht, German). Voltage steps and data acquisition were controlled using a PC computer with software Pulsefit+Pulse 8.0 (HEKA Electronics, Lambrecht, Germany). The patch pipettes with DC resistances of 2–3 MΩ were fabricated from borosilicate glass tubing (VWR micropipettes, 100 μl, VWR Company) using a two-stage vertical microelectrode puller (PC-10, Narishige, Japan) and fire-polished by a heater (Narishige, Japan). Data were sampled at 10 KHz and filtered at 3 KHz. Under the voltage clamp 70–80% series resistance compensation was applied.Ca2+ channel currents (ICa2+) were measured using Ba2+ as a charge carrier (IBa2+). For these experiments, external solutions contained (in mm): 160 triethanolamine-Cl, 10 HEPES, 2 BaCl2, 10 glucoses, and 200 nm tetrodotoxin, adjusted to pH 7.4 with triethanolamine-OH. The internal solution contained (in mm): 120 CsCl2, 5 Mg-ATP, 0.4 Na2-GTP, 10 EGTA, 20 HEPES-CsOH (pH 7.2) (13Lewis R.J. Nielsen K.J. Craik D.J. Loughnan M.L. Adams D.A. Sharpe I.A. Luchian T. Adams D.J. Bond T. Thomas L. Jones A. Matheson J.-L. Drinkwater R. Andrews P.R. Alewood P.F. J. Biol. Chem. 2000; 275: 35335-35344Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 14Smith M.T. Cabot P.J. Ross F.B. Robertson A.D. Lewis R.J. Pain. 2002; 96: 119-127Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 15Penna R.D. Paice J.A. Pain. 2000; 85: 291-296Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). IBa2+ was evoked at -50 or 0 mV from a holding potential of -90 or -40 mV. HWTX-X, ω-conotoxin GVIA, MVIIA, nifedipine, and NiCl2 were applied to the extracellular environment by low pressure ejection from a blunt pipette positioned about 50–100 μm away from the cell being recorded. Data were given as mean ± S.E. and statistical significance (p < 0.05) was determined using a paired or independent Student's t test as appropriate.NMR Spectroscopy of HWTX-X—The NMR sample was prepared by dissolving HWTX-X in 450 μl of 20 mm deuterium sodium acetate buffer (H2O/D2O, 9:1, v/v) containing 0.002% NaN3 and 0.01 mm EDTA with a final concentration of 6 mm HWTX-X and a pH of 4.0. Sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 was added to a final concentration of 200 μm as an internal chemical shift reference. For experiments in D2O, the sample used in H2O experiments was lyophilized and then redissolved in 450 μl of 99.996% D2O (Cambridge Isotope Laboratories). All NMR spectra were observed on a 500-MHz Bruker DRX-500 spectrometer with a sample temperature of 298 K. Several sets of two-dimensional spectra were recorded in a phase-sensitive mode by the time-proportional phase increment method following standard pulse sequences and phase cycling. TOCSY spectra were obtained with a mixing time of 85 ms. NOESY spectra were recorded in D2O with a mixing time of 200 ms and in H2O with mixing times of 100, 200, and 400 ms. Solvent suppression was achieved by the presaturation method. All two-dimensional measurements were recorded with 1024–512 frequency data points and were zero-filled to yield 2048–1024 data matrices except for the high resolution DQF-COSY spectrum. The DQF-COSY spectrum was recorded with 2048–1024 data points in the t2 and t1 dimensions, respectively, and zero-filled to 4096–2048 points to measure the coupling constants. All spectra were processed and analyzed using Felix 98.0 (Biosym Technologies) software running on a Silicon Graphics O2 work station. Before Fourier transformation, the signal was multiplied by a sine bell or sine bell square window functions with a π/2 phase shift.Structure Calculations—Distance constraints were obtained from the intensities of cross-peaks in NOESY spectra with a mixing time of 200 ms. All NOE data were classified into four distance ranges, 1.8–2.5, 1.8–3.0, 1.8–4.0, and 1.8–5.0 Å, corresponding to strong, medium, weak, and very weak NOE values, respectively. According to the method (28Hertz A. Hernandez J.F. Gagnon J. Hong T.T. Pham T.T. Nguyen T.M. Le-Nguyen D. Chiche L. Biochemistry. 2001; 40: 7973-7983Crossref PubMed Scopus (148) Google Scholar), if the connectivity involved side chain protons, 3.0-, 4.0-, and 5.0-Å upper bounds were used instead to account for higher mobility. For sequential dαN and dNN connectivities, we used bounds of 2.5-, 3.0-, and 4.0-Å, respectively. Pseudo-atom corrections were applied to non-stereospecifically assigned methyl and methylene protons according to the method of Wüthrich (38Wüthrich K. NMR of Protein and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Ten dihedral angle restraints derived from 3JNH-CαH coupling constants were restrained to -120 ± 30° for 3JNH-CαH ≥ 8.80 Hz and -65 ± 25° for 3JNH-CαH ≤ 5.5Hz. Three distance constraints were added to each disulfide bridge that was mainly determined from NMR data. The corresponding distances were 2.02 ± 0.02, 2.99 ± 0.5, and 2.99 ± 0.5 Å for S(i) - S(j), S(i) - Cβ(j), and S(j) - Cβ(i), respectively. Structure calculations of HWTX-X were run on a Silicon Graphics work station using the standard protocol of the X-PLOR-NIH 2.9.6 program (29Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Res. 2003; 160: 66-74Crossref Scopus (1843) Google Scholar).RESULTSPurification Characterization and Synthesis of HWTX-X—Crude venom from the spider O. huwena was fractionated by RP-HPLC on a Vydac C18 column (Fig. 1A). More than 20 peaks were observed in the chromatogram. The sharp peak labeled HWTX-X, eluted at 13 min at a point in the gradient of about 18% acetonitrile, 0.1% trifluoroacetic acid, was found to block N-type Ca2+ channels in rat DRG cells. As assessed by mass spectrometry, the purified fraction contained one component, whose average molecular mass (M + H)+ was determined as 2,931.34 Da. The purity of HWTX-X was over 98%, as judged by RP-HPLC and N-terminal sequence analysis. The complete amino acid sequence of HWTX-X was derived by Edman degradation (fig. 2), revealing it as a 28-amino acid polypeptide containing six cysteine residues. The molecular mass of HWTX-X determined by mass spectrometry was 6 Da less than that calculated from its amino acid sequence, assuming all 6 cysteine residues are involved in disulfide bridges. Primary structure analysis of HWTX-X highlights that a basic peptide with four basic residues (Lys1, Lys7, Lys15, and Lys24) but no acidic residues, HWTX-X, is also a rather polar peptide containing only seven hydrophobic residues, which is consistent with its relatively short retention time in the RP-HPLC.FIGURE 2Sequence alignment of HWTX-X with some other toxins active on vertebrate HVA Ca2+ channels. Identical amino acids are shown in gray and the two well conserved Gly6 and Lys26/Arg26 are in black. The sequence length, net charge, and the percentage identity (I %) are shown to the right of the sequences. The disulfide linkage and the four loops are indicated above and below the sequences, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The amino acid sequence of HWTX-X was used to search the protein data base for possible homologues using an online BLAST search. HWTX-X has low sequence homology with other huwentoxins, but it shares ∼50% homology with two Ca2+ channel blockers, namely Ptu1 (34 residues) isolated from the venom of assassin bugs P. turpis (16Corzoa G. Adachi-Akahane S. Nagao T. Kusui Y. Nakajima T. FEBS Lett. 2001; 499: 256-261Crossref PubMed Scopus (45) Google Scholar, 30Bernard C. Corzo G. Mosbah A. Nakajima T. Darbon H. Biochemistry. 2001; 40: 12795-12800Crossref PubMed Scopus (32) Google Scholar) and ω-conotoxin SVIA (24 residues) isolated from the venom of Conus striatus (Fig. 2) (31Ramilo C. Zafaralla G.C. Nadasdi L. Hammerland L.G. Yoshikami D. Gray W.R. Kristipati R. Ramachandran J. Miljanich G. Olivera B.M. Cruz L.J. Biochemistry. 1992; 31: 9919-9926Crossref PubMed Scopus (155) Google Scholar). When the amino acid sequences of HWTX-X and ω-conotoxins GVIA, MVIIA, and CVID were aligned, it was found that sequence homology between HWTX-X and these ω-conotoxins is low (<32%), but they indeed share some similarities. The four peptides are basic and they all have six conserved Cys residues that are arranged to give a four-loop Cys framework (C-C-CC-C-C). Moreover, among other amino acid residues, they contain the well conserved neutral residue Gly6 and the basic residues Lys26 or Arg26 (HWTX-X numbering) (Fig. 2), which are also conserved throughout all ω-conotoxins. These similarities represent substantial clues for investigating the bioactivity of HWTX-X in Ca2+ channels.Because of the low amount of HWTX-X in the crude venom of O. huwena, this peptide was synthesized and then renatured, which yielded a major product as revealed by RP-HPLC and MALDI-TOF spectrometry (Fig. 1, inset). The purified product was homogeneous on the analysis by reverse-phase HPLC, and its mass (2931.45 Da) was in good accordance with the theoretical mass for the oxidized analog. Furthermore, the synthetic HWTX-X was able to block N-type Ca2+ channels in DRG cells similarly to the native peptide as determined by the patch clamp technique. Because the synthetic HWTX-X seemed to be structurally and functionally identical to the native toxin, it might be used for the further studies.The cDNA Sequence of HWTX-X—The full-length cDNA sequence of HWTX-X was obtained by overlaying two fragments from the 3′ and 5′ RACE. As shown in Fig. 3, the oligonucleotide sequence of the cDNA is 486 bp comprising the 5′-untranslated region, the open reading frame, and the 3′-untranslated region. The open reading frame encodes a 65-residue peptide corresponding to the precursor of HWTX-X, which contains a signal peptide of 20 residues, a propeptide of 17 residues, and a mature peptide of 28 residues. The prepropeptide of the HWTX-X precursor shows limited sequence identity with those of other reported precursors. Different from most ω-conotoxins, HWTX-X has no extra amino acid Gly or Gly + Arg/Lys residues at its C terminus, known to allow "post-modification" of α-amidation at the C-terminal residue (49Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2004; 279: 32453-32463Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), implying that the C-terminal residue of mature toxin is not amidated.FIGURE 3The oligonucleotide sequence of HWTX-X cDNA. The amino acid composition of the precursor reading from the cDNA is below the nucleotide sequence. The potential endoproteolytic sites are indicated with down arrows. The signal sequence is shown in gray, the propeptide is boxed, and the mature peptide is underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biological Assays—HWTX-X elicited no toxic symptoms in either vertebrates or invertebrates during a period of 48 h post-injection, when it was assayed in vivo by direct injection into mice and cockroaches. ω-Conotoxins (e.g. GVIA, MVIIA, and CVID) are known to block the twitch response to low frequency electrical nerve stimulation in rat vas deferens (3Olivera B.M. Annu. Rev. Biochem. 1994; 63: 823-867Crossref PubMed Scopus (692) Google Scholar, 32Lew M.J. Flinn J.P. Pallaghy P.K. Murphy R. Whorlow S.L. Wright C.E. Norton R.S. Angus J.A. J. Biol. Chem. 1997; 272: 12014-12023Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Because of the similarities between HWTX-X and these ω-conotoxins, similar pharmacological experiments were carried out to test the effect of HWTX-X on this tissue. In all the experiments conducted (n = 10), addition of 10 μm HWTX-X to the bath had no effect on the twitch response of vas deferens during 30 min, but 1 μm GVIA or MVIIA caused a rapid reduction of the twitch response, in agreement with previous studies (3Olivera B.M. Annu. Rev. Biochem. 1994; 63: 823-867Crossref PubMed Scopus (692) Google Scholar, 32Lew M.J. Flinn J.P. Pallaghy P.K. Murphy R. Whorlow S.L. Wright C.E. Norton R.S. Angus J.A. J. Biol. Chem. 1997; 272: 12014-12023Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) (data not shown).Effect of HWTX-X on Ca2+ Channels in Rat DRG Cells—It is widely accepted that rat DRG cells exhibit two categories of voltage-gated Ca2+ channels: low voltage-activated channels (T-type) and high voltage-activated (HVA) channels (N-, L-, P/Q-, and R-types), which can be discriminated by their voltage dependence and kinetics. Low voltage-activated currents can be elicited by a 100-ms step depolarization to -50 mV from a holding potential (Vh)of -90 mV, whereas only HVA currents are activated if the cell is depolarized from a Vh of -40 to 0 mV (33Scroggs R.S. Fox A.P. J. Physiol. 1992; 445: 639-658Crossref PubMed Scopus (308) Google Scholar, 34Miller R.J. Science. 1987; 235: 46-52Crossref PubMed Scopus (1102) Google Scholar, 35Miller R.J. J. Biol. Chem. 1992; 267: 1403-1406Abstract Full Text PDF PubMed Google Scholar, 36Nowychy M.C. Fox A.P. Tsien R.W. Nature. 1985; 316: 440-443Crossref PubMed Scopus (16
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