Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

α-RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom

α-RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius... Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 543028, 9 pages http://dx.doi.org/10.1155/2013/543028 Research Article 𝛼 -RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom 1,2 3 3 Maria Cristina Vianna Braga, Arthur Andrade Nery, Henning Ulrich, 4 5 5 Katsuhiro Konno, Juliana Mozer Sciani, and Daniel Carvalho Pimenta CAT/CEPID, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900 Sao ˜ Paulo, SP, Brazil ´ ˆ ˜ ´ ´ Ministerio da Ciencia, Tecnologia e Inovac¸ao, Esplanada dos Ministerios, Bloco E, 70067-900 Brasılia, DF, Brazil Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de Sao ˜ Paulo, Av. Lineu Prestes 748, 05508-900 Sao ˜ Paulo, SP, Brazil Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Laborator ´ io de Bioqu´ımica e Biof´ısica, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900 Sao ˜ Paulo, SP, Brazil Correspondence should be addressed to Daniel Carvalho Pimenta; dcpimenta@butantan.gov.br Received 31 October 2012; Revised 16 January 2013; Accepted 16 January 2013 Academic Editor: Ayman El-Faham Copyright © 2013 Maria Cristina Vianna Braga et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus mayhave50to200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. eTh novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. eTh activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably 𝛼 3𝛽 4and/or𝛼 3𝛽 4𝛼 5 subtype. According to the nomenclature, the new peptide was designated as𝛼 -RgIB. 1. Introduction sequence of the conotoxin precursors (gene superfamilies), the cysteine patterns of conotoxin mature peptide regions Marine mollusks from Conus genus may produce from 50 (cysteine frameworks), and the specificities to pharmacolog- up to 200 biologically active molecules that can be injected ical targets (pharmacological families) [3, 4]. in the prey to capture or be employed as defense and/or Conopeptides of the pharmacological family 𝛼 ,which escape mechanisms to deter competitors. The peptide toxins, acts on neuronal acetylcholine receptor, have been found in called conopeptides, are composed of 10–40 amino acids the A, D, L, M, and S gene superfamilies [5, 6]. (including nonnatural amino acids) and are abundant in the Typically,𝛼 -conotoxins are peptides with 12 to 16 amino venom. Peptides presenting a rigid structure due to more than acid residues and two disulfide bridges, presenting the pattern one disuld fi e bridges are common, being called conotoxins. CC-C-C. These peptides are competitive antagonists of the These peptides act specifically on ionic channels and/or nicotinic acetylcholine receptors (nAChR) and display high neuromuscular receptors [1, 2]. selectivitybysubtypesofthisreceptor[5, 7–9]. After the Conotoxins are classified according to three schemes: blockage of the muscular acetylcholine receptor, the𝛼 -con- the similarities between the endoplasmatic reticulum signal otoxins significantly decrease the amplitude of the motor end 2 International Journal of Peptides plate postsynaptic potentials in vertebrates, paralyzing the a Q-ToF Ultima API (Micromass, Manchester, UK) and/or by prey [10]. MALDI-TOF mass spectrometry on a Ettan MALDI-ToF/Pro In the Brazilian tropical coast, there are approximately System (Amersham Biosciences, Sweden). 18 species of cone snails [11]. Conus regius (Gmelin, 1791) The analysis in the micro-LC-MS Ettan (Amersham is a vermivorous species that inhabits rock and coral deep Biosciences, Sweden) was performed in a 𝜇 RPC C2/C18 waters of Florida (USA), Central America, and the Northeast ST 1.0/150 column (Amersham Biosciences, Sweden), with and East coast of Brazil, including Fernando de Noronha two solvents: (A) formic acid (FA)/H O (1 : 1000) and (B) archipelago [12]. FA/ACN/H O(1:900:100).Thesamplewas eluted at a −1 In this work we described a novel peptide from Conus constant flow rate of 50 𝜇 L⋅min with a5to 65%gradient regius venom, belonging to the 𝛼 -conotoxins family. This of solvent B over 60 min. Q-Tof operated under positive peptide blocks the neuronal acetylcholine receptors on PC12 ionization mode. For MALDI-TOF analyses, a-cyano-4- cells, which comprise𝛼 3𝛽 4 and/or𝛼 3𝛽 4𝛼 5subtypesrecep- hydroxycinnamic acid was used as matrix. tors, probably target of the peptide. 2.5. “De Novo” Peptide Sequencing. Mass spectrometric “de novo” peptide sequencing was carried out in positive ioniza- 2. Material and Methods tion mode on a Q-TOF Ultima API tfi ted with an electro- 2.1. Reagents. All the employed reagents were of analytical spray ion source (Micromass, Manchester, UK). Briefly, the grade and were purchased from Sigma Co (St Louis, MO, amounts of previously lyophilized peptide were dissolved in USA), unless otherwise stated. 50 mM ammonium acetate, reduced with 50 mM DTT, alky- lated by 150 mM iodoacetamide, and hydrolyzed by 25 nM trypsin, according to slight modifications of Westermeier and 2.2. Animals and Venom. Specimens of C. regius were col- Naven [14]. eTh reaction products were then lyophilized and lected at Fernando de Noronha Archipelago, Pernambuco, dissolved in 50% ACN, containing 0.1% FA and injected into Brazil.TheBrazilian EnvironmentalAgency(IBAMA—Insti- −1 thesourceat5𝜇 L⋅min by aHamiltoninfusionpump, or tuto Brasileiro do Meio Ambiente e dos Recursos Naturais directly injected using a Rheodyne 7010 sample loop coupled Renova´veis) license numbers were 030/2000 and 087/2001, −1 to a LC-10A VP Shimadzu pump operating at 20𝜇 L⋅min and the process number was 02001, 000775/00-00. Venom constant flow rate. eTh instrument control and data acquisi- was extracted from the specimens as previously described tion were conducted by MassLynx 4.0 data system (Micro- [13]. The crude venom was obtained by dissection of the mass, Manchester, UK) and experiments were performed by venom duct gland and then freeze-dried and stored at−80 C. scanning a mass-to-charge ratio (m/z)of50–1800 usinga Voucher material is deposited in the malacological col- scan time of 2 s applied during the whole chromatographic lection of Zoology Museum of University of Sao ˜ Paulo, Sao ˜ process. The mass spectra corresponding to each signal from Paulo, Brazil. the total ion current (TIC) chromatogram were averaged, allowing an accurate molecular mass determination. External 2.3. Peptide Fractionation and Purica fi tion. A reversed-phase calibration of the mass scale was performed with NaI. For the binary HPLC system (LC-8A, Shimadzu Co., Japan) was MS/MS analysis, collision energy ranged from 18 to 45 and used for sample fractionation. eTh lyophilized crude venom the precursor ions were selected under a 1-m/z window. powder was solubilized into 0.1% trifluoroacetic acid (TFA) andaliquotswereloadedinaShim-packPrep-ODSC18 ˚ 2.6. Biological Activity. eTh biological activity of 𝛼 -RgIB column (Shimadzu, 3𝜇 m, C18, 300 A, 250 × 20 mm) in wasdeterminedinSwiss Webstermice(5.5to7gbody a two-solvent system: (A) trifluoroacetic acid (TFA)/H O weight) by observation of the behavioral disorders aeft r (1 : 1000) and (B) TFA/Acetonitrile (ACN)/H O(1:900:100). −1 intracranial injection [15] of the peptide diluted in NaCl The sample was eluted at a constant flow rate of 8 mL ⋅min 0.9%, in concentration of 0.1, 0.5 and 1 nmol. Alterations were with a 0 to 60% gradient of solvent B over 60 min. eTh compared to animals injected with NaCl 0.9% (control). All HPLC column eluates were monitored by a Shimadzu SPD- animals were observed by 60 min. 10A detector scanning 220 nm. For𝛼 -RgIB purification, the interest peak was fraction- ated in a Merck C18 column (300× 4.6 mm), in a 19 to 21% B 2.7. Patch Clamp. BC H1 cells, mouse myocytes which −1 gradient over 20 min, at a constant flow rate of 8 mL ⋅min . express nicotinic acetylcholine receptors, were acquired by A subsequent purification step was still necessary to obtain ATCC (CRL-1443) and maintained in culture according to thepeptide.This puricfi ationwas conductedinaMerckC18 Sine and Taylor [16] to electrophysiological experiments. In column (300 × 4.6 mm), in an isocratic elution at 35% B order to verify the subtype of neuronal nicotinic receptor that (TFA/methanol/H O 1:900:100) at a constant flow rate of the peptide acts, PC12 cells were employed and maintained in −1 1mL⋅min . culture according to Greene et al. [17]. Individual cells were subjected to a patch-clamp, at a 2.4. Mass Spectrometry Analysis. Molecular mass analyses whole cell configuration, according to Hamill et al. [ 18]and of thepeaks andthe peptides were performedonamicro- Urlich et al. [19]. Cells were maintained in an extracellular LC-MS Ettan (Amersham Biosciences, Sweden) coupled in solution containing 25 mM HEPES, 5.3 mM KCl,144.8 mM International Journal of Peptides 3 NaCl, 1.2 mM MgCl , 2.38 mM CaCl , and 10 mM glucose 3.3. Sequence Features. A sequence alignment was performed 2 2 (pH 7.4). A recording electrode was filled with intracellular with all𝛼 -conotoxins available at UniProt (supplementary solution containing 25 mM HEPES, 141 mM KCl, 10 mM Table 1 of the supplementary material available online at NaCl, 2 mM MgCl and 1 mM EGTA (pH 7.4). Experiments http://dx.doi.org/10.1155/2013/543028). Basedonthislarge 2, alignment, a phylogeny was constructed (supplementary were carried outatroomtemperature (20–24 C). Figure 1) and the peptide sequences present at the branch Throughout the experiment, the membrane potential was containing𝛼 -RgIB were realigned (Figure 4). ClustalW stan- clamped at a−60 mV for BC H cells and−70 mV for PC12 3 1 dard annotations consider, as expected, the Cys residues cells, holding potential using an Axon Axopatch amplifier as consensus ( ), and the Glu at the 8th aligned posi- (Molecular Devices, California, USA). Data were recorded tion, as being highly conserved (:). Moreover, according and digitized by Clampex 8.2 software (Molecular Devices, to the algorithm standard notation, the Pro residue at the California, USA) and plots were made using Origin 7.0 13th aligned position is also conserved (.). Among the software (OriginLab Corp., Northampton, MA). UNIPROT database, the SwissModel tool could not identify Control currents were performed with 1.5 mM carbamyl- anysuitabletemplatefor structurepredictionof 𝛼 -RgIB, choline, using the cell-flow technique [ 20]. After the car- therefore an external application was used. Figure 5 presents bamylcholine administration, the peptide (10𝜇 M) was incu- a3Dmodel of 𝛼 -RgIB; created by I-TASSER [23, 24], as batedoncells,and then anotherdoseofthe agonistwas well as three PDB deposited 3D solution structures of 𝛼 - incubated [21]. RgIA (P0C1D0) mutants, a conotoxin that specifically and potently blocks the𝛼 9𝛼 10 nAChR [27]. In spite of𝛼 -RgIB 2.8. Data Fitting, Statistical Analyses, and Sequence Alignment. N- and C-terminal extensions and longer interbridge peptide When data tfi ting was performed, results were presented as sequence, the model and the structures are tridimensionally the calculated value± standard deviation (SD). Otherwise, related, for example, a C-shaped structure, held by the Cys- data correspond to the mean of three individual experiments. bridges. Peptidesequencealignment wasperformed usingClustalW software [ 22]. The 3D model of the peptide 𝛼 -RgIB, as well as three PDB deposited 3D solution structures, was created by 3.4. Biological Activity. The in vivo biological activity of the I-TASSER [23, 24]. peptide was assessed by means of intracranial injection in Swiss Webster mice. Following 1 nmol injection, the animals displayed a hyperactive behavior, defecating and urinating 3. Results all the time, which was not observed for the control group 3.1. Purification. eTh crude venom from C. regius was frac- that received saline solution. Auditory stimuli, for example, a tionated by RP-HPLC, as shown in Figure 1(a).Somepeaks hand-clap or hitting the cage, also triggered the hyperactive could be detected along the profile, and the arrow indicates behavior. Interestingly, the lower doses (0.1 and 0.5 nmol), the peak of interest. Two subsequent chromatographic steps caused the animals to have dicffi ulty in breathing. Although were necessary to purify the peptide (Figures 1(b) and the peptide promoted behavioral disorders, it was not lethal 1(c)), under the conditions described material and methods to the animals. section. After the third step of purification, the purity and the molecular mass of the peptide were assessed by MALDI- TOF/MS (Figure 1(d)). 3.5. Patch Clamp. Whole-cell voltage clamp measurement was used to verify the ion currents on acetylcholine receptors. BC H1 cells, which express the acetylcholine muscle type 3.2. “De Novo” Peptide Sequencing. After cysteine bridge 3 receptors on the surface, and PC12, which terminally differ- reduction and alkylation, the reaction product was digested entiate in neurons and express nicotinic neuronal receptors with trypsin. eTh obtained peptides were submitted to [21] were selected for the experiments. Carbamylcholine, a MS/MS analyses (Figure 2)and ions were selected and stable and well-characterized analogue of acetylcholine, was fragmented by collision with argon (CIF), yielding daughter used as an agonist [28], for it elicits a fast activating current ion spectra (Figure 3)thatwas processedwithBioLynx and that rapidly desensitizes during the application. manually checked for accuracy of interpretation. Since the 10𝜇 M 𝛼 -RgIBwas notabletoinduceany change in digestion allowed peptides with missed cleavage sites, it the ion currents on BC H cells (data not shown), as well was possible to assemble the fragments without the aid of 3 1 as a higher dose (30𝜇 M) of the peptide. d-tubocurarine (a another digestion with a different enzyme. The sequenced classic nicotinic receptor antagonist) was used as a positive peptides, their charge states, and theoretical molecular mass control and successfully to block this channel (data not are presented in Table 1. shown). The peptide sequence was determined to be TWEECCKNPGCRNNHVDRCRGQV. This sequence has After the incubation of the peptide with PC12 cells, 4 cysteine residues with pattern CC-C-C, typical from fast and slow desensitization of the receptor was observed. conotoxins of framework I [25]. This peptide was named Figure 6(b) showsthatonneuronalslowrise-time receptors, 𝛼 -RgIB, according to the guidelines for conotoxins nomen- 𝛼 -RgIB is able to block the ion current by 40%, compared clature ConoServer and has been assigned the following to cells stimulated with carbamylcholine (Figure 6(a)). The UNIPROT accession number: C0HJA8 [3, 6, 26]. blockage was irreversible and persistent, once the current 4 International Journal of Peptides 0 5 10152025303540455055 60 Minutes Detector A (220 nm) HPLC conus regius 10 CVE B.CONC MCris.met (a) 0 15 6 8 10 12 14 16 18 20 22 24 26 28 30 02468 10 12 14 16 18 20 Minutes Minutes Detector A (220 nm) HPLC conus regius A007 B.CONC MCris.met (c) (b) 2703.863 1,150 1,050 1,600 2,000 2,400 2,800 3,200 3,600 𝑚/𝑧 (d) Figure 1: (a) Representative RP-HPLC of the crude C. regius venom. The arrow indicates the peak of interest. (b) Representative RP-HPLC of the selected peak (arrow), indicating the presence of impurities. (c) Isocratic elution of the isolated the selected fraction from chromatogram B. The arrow indicates the peak of interest. (d) MALDI-TOF/MS profile of the purified peptide. does not recover aer ft a new application of the agonist, (gene superfamilies), the cysteine patterns of conotoxin carbamylcholine (Figure 6(c)). mature peptide regions (cysteine frameworks), and the speci- ficities to pharmacological targets [ 3, 4]. This new peptide was termed 𝛼 -Rg-IB because the 4. Discussion peptide acts on neuronal acetylcholine receptors (“𝛼 ”), was extracted from a Conus regius specimen (“Rg”), displays a Conotoxins are classified according to the similarities between the signal sequence of the conotoxin precursors cysteine framework I—CC-C-C (“I”), and was the second Abs Abs Intensity (%) mAU International Journal of Peptides 5 091003AL 27 (0.972) Sm (SG,2×6.00); Sb (3,20.00); Cm (25:48) 335.1610 TOF MS ES+ 1.59𝑒3 377.6908 A D C2 T W E E C C K N P G C R N N H V D R C R G Q V BC E F A2 A3 820.8170 547.5543 821.3205 547.8890 754.3600 548.2238 821.8242 378.6921 755.3594 822.3163 303.1793 548.5588 B2 379.1854 548.8938 822.8320 528.6995 603.2767 823.3126 389.2137 619.3010 396.6663 620.2981 756.3705 839.7988 397.1629 507.2256 576.2546 840.8057 654.3190 1056.3862 415.2154 851.2896 776.3487 655.3229 1115.4175 993.3750 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 𝑚/𝑧 Figure 2: Representative ESI-Q-TOF/MS profile of the trypsin digested purified peptide. eTh deduced sequence is printed above the spectrum, together with the tryptic peptides (A–F). eTh MS profile indicates the tryptic peptides and the charge states. Table 1: eTh oretical and experimental 𝑚/𝑧 values for the tryptic peptides obtained aeft r the enzymatic digestion of 𝛼 -RgIB. + 2+ 3+ [M + H] [M + 2H] [M + 3H] Sequence Ions Exp. eTh or. Exp. eo Th r. Exp. eTh or. 1,2 1,2 1,2 1,2 1,2 1,2 A 1640.63 1639.839 820.82 820.423 547.55 547.282 TWEECCKNPGCR 1,2 1,2 1,2 1,2 B 1056.39 1055.180 528.70 528.094 — x TWEECCK C 754.37 754.359 377.69 377.896 — (252.125) NNHVDR 2 2 3 2,4 D619.30 619.298 — (310.366) —x CRGQV 2 2 E603.28 603.267 — (302.137) —x NPGCR 2 2 5 F335.16 335.147 —x —x CR Acetylation (N-terminal, variable modification). Carbamidomethyl cysteine (fixed modification). Not detected. Not observed. Not expected. peptidediscoveredwithbothbeing from C. regius with a its closest phylogenetic relatives (supplementary material), cysteine framework I (“B”) [25]. besides 𝛼 -RgIA, which was not considered to be similar 𝛼 -RgIA was the rfi st 𝛼 -conotoxin described from C. (according to MEGA5), but was manually inserted in the regius, acting on neuronal nicotinic receptors. This peptide gfi urefor thebeneto fi fsequencecomparison. 𝛼 -RgIA is has been thoroughly characterized in terms of its primary shorter, both in the N- and C-terminal flanking regions, and three-dimensional structures [29], as well as regarding as well as in the inter-Cys-bridge region. Nevertheless, in its biological eeff ct, for example, the blockage of the 𝛼 9𝛼 10 a considerably small universe of possibilities (8 out 12, nAChR [30, 31].𝛼 -RgIA and𝛼 -RgIB come from the same since 4 amino acids are necessarily Cys), 𝛼 -RgIA and 𝛼 - animal, belong to the same toxin family, and possess similar RgIB bare considerable similarities: the Pro, at the 13th biological effects; however, their amino acid sequences differ. aligned position, and the charged residues at the 17th and Figure 4 shows the ClustalW alignment of 𝛼 -RgIB and 18th aligned positions. It is noteworthy to mention that, (%) 6 International Journal of Peptides MC 2.3 msms 754 1: TOF MSMS 754.00ES+ 091003AP MaxEnt 3 31 [Ev-102034, It50, En1] (0.050, 200.00, 0.200, 1400.00, 1, Cmp) NN H V R bMax RD H N N V yMax 100 754.42 (𝑀+)𝐻+ 175.14 366.18 y1 755.34 b3 737.36 349.16 526.31 465.25 756.57 640.36 580.27 y4 237.16 367.16 448.24 110.10 290.17 b4 y5 b5 758.08 509.29 720.33 209.16 420.24 563.26 y2 623.31 H 138.08 677.33 332.13 759.27 799.39 252.14 491.26 88.05 694.41 158.11 176.63 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 𝑀/𝑧 Figure 3: MaxEnt3 deconvoluted annotated representative MS/MS profile of the CID spectrum of tryptic peptide C, from Figure 2.yandb series are annotated above the spectrum, as well as other fragments. Clustal 2.1 multiple sequence alignment 𝛼 -RgIB TWEECCKNPGCRNNHVDRCRGQV 23 A6M938 17 NDCCHNAPCRNNHPGIC A1X8C3 GMWDECCDDPPCRQNNMEHCPAS Q2I2R6 ECCDDPPCRQNNMEHCPAS A1X8C2 GVWDECCKDPQCRQNHMQHCPAR P0C8U6 DDCCPDPACRQNHPELCSTR P0C8U7 DDCCPDPACRQNHPEICPSR P0C8U9 NAWLTPEECCAAPACREMILEFCLAGEAFAAALDGFRRLPYR 42 P0C8U8 NAWFTPEECCAAPACRGMILEFCLAGEAFAAALDGFRRLPYR ISEMTWEECCTNPVCRQHYMHYC P0C8V0 23 P01519 ECC NPACGRHYS C 13 ∗∗ ∗ ∗ : . GCCSDPRCRYR C 12 P0C1D0 Figure 4: ClustalW alignment of 𝛼 -RgIB and the closest phylogenetic 𝛼 -conotoxins relatives (calculated according to supplemental Figure 3).( ) Consensus; (:) highly conserved; (.) conserved. The bold underlined amino acid residues of 𝛼 -RgIB were also considered to be conserved. A6M938:𝛼 -conotoxin-like Lp1.10 C. leopardus/homology; A1X8C3:𝛼 -conotoxin-like Lp1.7 C. leopardus/transcript; Q2I2R6: 𝛼 -conotoxin-like Lt1.3 C. litteratus/transcript; A1X8C2:𝛼 -conotoxin-like Lp1.8 C. leopardus/transcript; P0C8U6:𝛼 -conotoxin-like PuSG1.1 C. pulicarius/transcript; P0C8U9:𝛼 -conotoxin-like Pu1.5 C. pulicarius/transcript; P0C8U8:𝛼 -conotoxin-like Pu1.4 C. pulicarius/transcript; P0C8U7/𝛼 -conotoxin-like PuSG1.2 C. pulicarius/trasncritpt; P0C8V0:𝛼 -conotoxin-like Pu1.6 C. pulicarius/transcript; P01519:𝛼 -conotoxin GIA C. geographus/protein; P0C1D0:𝛼 -conotoxin RgIA, C. regius/protein. (Key: UniProt Accesion code: toxin/Conus species/evidence level). in spite of the phylogenetic analyses, all conotoxins listed Moreover, our group has also identified two conotoxins, as in Figure 4 (except𝛼 -RgIA and𝛼 -RgIB) come from other well:Rg11a,belonging to theI superfamily (P84197, [34]); Conus species: C. leopardus (A6 M938, A1X8C2, A1X8C3), and Rg9.1, belonging to the P-superfamily (Q8I6V7; direct C. litteratus (Q2I2R6), C. pulicarius (P0C8U6, P0C8U7, submission). P0C8U8, P0C8U9, and P0C8V0) and C. geographus (P01519). There is no high level of homology between 𝛼 -RgIB Moreover, only P01519 has been detected at the protein and the conopeptides described until the present moment; level and has been characterized as active on the muscular therefore, the identification of a proper 3D structure to serve nicotinic receptors [32]. as a template for homology modeling is deprecated. Instead, Besides𝛼 -RgIA, the following toxins have been isolated a structure was predicted by using I-TASSER server [23, 24]. from C. regius: P85009; P85010; P85011; P85012; and P85013, Figure 5 shows that, in spite of the low homology with𝛼 - all 𝛼 -conotoxin-like peptides belonging to superfamily A; RgIA,𝛼 -RgIB model assumed the same basic shape as the P85016; P85017; P85018; P85019; P85020; P85021 and P85022, NMR determined structures of the𝛼 -RgIA mutants, available all belonging to the M-superfamily of conotoxins [33]. at the PDB database [27]. (%) International Journal of Peptides 7 the pool of PC12 cells expressed 𝛼 3, 𝛼 5, 𝛼 7, 𝛽 2, and 𝛽 4 subunits of neuronal nicotinic receptors, the same pattern NH found by Sargent [40]inPC12cells.However,inspite of𝛼 - NH Rg-IB affinity by the PC12 nicotinic receptors, there are still 2JUR other neuronal nicotinic receptors that may be higher ani ffi ty MODEL COOH targets for these toxins that were not explored in the present COOH work. AuIB, from Conus aulicus,which is also an𝛼 -conotoxin, blocks the 𝛼 3𝛽 4 receptors; however, the currents can be recovered aeft r the toxin washing [ 42]. In our experiments, NH the no-recovery of𝛼 -RgIB is probably due to the irreversible NH 2 action of the peptide on the receptor. Successive applications of the agonist (carbamolycholine), in control experiments, did not cause recovery of the ion currents on slow rise-time 2JUT 2JUS receptors (data not shown). Besides the irreversible action, the peptide may also be able to prolong the desensitization COOH time of the receptor, since the repeated CBC administration COOH on 𝛼 -RgIB-treated PC12 cells was not able to recover the initial current, which is either caused by the irreversible Figure 5: I-TASSER model of 𝛼 -RgIB and PDB NMR superimposed binding of a low affinity toxin or the prolonging of the structures of𝛼 -RgI-A. desensitization time of the receptor (or both). Sudweeks and Yakel [43]showedthat 𝛼 3, 𝛼 7, and 𝛽 2 subunits of nAChR are correlated to fast rise-time receptors. Regarding the rather unique amino acid sequence of𝛼 - The slow desensitization is a characteristic of 𝛼 3𝛽 4receptor, while𝛼 3𝛽 2 receptor is from fast desensitization [44]. The RgIB and thoroughly analyzing our data, we could not rule outthe possibilitythatone of theglutamicacid(Glu) residues fast desensitization receptors, on PC12 cells, contain𝛼 3𝛽 2, of this novel conotoxin would be a gamma-carboxyglutamic 𝛼 3𝛽 2𝛼 5, and𝛼 7 subunits, while slow desensitization recep- tors are formed by subunits𝛼 3𝛽 4and𝛼 3𝛽 4𝛼 5.𝛼 -RgIB was acid residue (Gla). Our suspicions arouse from the slightly higher deviation between the theoretical and calculated able to inhibit the currents elicited by carbamolycholine on molecularmassvaluesfor Aand Bions(Table 1), that PC12 cells, mainly on the slow desensitization component, could reflect that a side chain carboxylation and not an N- which comprise, in our model,𝛼 3𝛽 4and𝛼 3𝛽 4𝛼 5 receptors. terminal acetylation would be present. Moreover, conotoxins The intracranial injection assay was performed to investi- are known for presenting posttranslation modifications, Gla gate whether there would be any direct activity of the toxin included [35–38] and, even though the MALDI data of in the central nervous system (CNS), once peptides can the crude peptide support the proposed peptide sequence, promote behavioral alterations by acting on receptors and ionic channels on CNS. es Th e alterations can indicate activ- MALDI ionization is also a source of facile decarboxyla- tion for Gla residues [39]. Our future experiments with ities on specific ionic channels. For example, 𝜔 -conotoxin C. regius conotoxins (𝛼 -RgIB included) will clarify this GVIA causes trembling on the mice, which indicates an action on calcium ionic channels [45]. The 𝛼 -nicotinic matter. 𝛼 -conotoxins bind to nicotinic acetylcholine receptors. acetylcholine receptor (nAChR) is associated to attention- The subgroup 𝛼 3/5 of𝛼 -conotoxins, from piscivorous Conus, deficit/hyperactivity disorder [ 46]which corroborates our has the motif CCX CX Cand cancause paralysisofthe observations of𝛼 -RgIB-treated hyperactive mice. 3 5 prey by the binding on muscle nicotinic receptors. Another In conclusion, we have isolated a novel conotoxin from subgroup, 𝛼 4/3, that present the motif CCX CX C, bind Conus regius and, by means of a combination of biochemical, 4 3 on neuronal nicotinic receptors. eTh main subgroup of 𝛼 - structural and pharmacological assays were able to classify conotoxins is𝛼 4/7, with motif CCX CX C. These peptides this peptide in the 𝛼 -family and named it 𝛼 -RgIB. There 4 7 are still several peptides to be explored in the C. regius bind in all classes of nicotinic receptors: muscular (e.g., 𝛼 -conotoxin EI), homomeric neuronal (e.g., 𝛼 -conotoxins venom, as our previous qualitative investigations have shown PnIB), and heteromeric neuronal (𝛼 -conotoxins MII and [34] and the current study has focused on the biochemical characterization of one such novel peptide. Further studies AuIB) [9]. Neuronal nicotinic acetylcholine receptors (nAChRs) are still necessary to better characterize the structural and belong to the pentameric superfamily of Cys-loop ligand pharmacological properties of𝛼 -RgIB. gated ionic channels. eTh y are composed of either homo- meric𝛼 or heteromeric𝛼 and𝛽 subunits assembled from a family of 12 distinct neuronal nicotinic subunits (𝛼 2–𝛼 10; Acknowledgments 𝛽 2–𝛽 4) [5]. The combination of subunits 𝛼 2,𝛼 3, and𝛼 4with 𝛽 2and𝛽 4 results in a functional receptor, as well𝛼 7,𝛼 8, This work was supported by Grants from the Brazilian fund- and𝛼 9 homomeric receptors [40, 41]. In our experiments, ing agencies FAPESP and CNPq, including the INCTTOX it was verified by RT-PCR (supplemental Figure 3) that PROGRAM. 8 International Journal of Peptides 100 100 0 0 0 3 3 3 Time (s) Time (s) Time (s) (a) (b) (c) Figure 6: PC12 whole-cell characteristic patch clamp currents (expressed as a percentage of response to 1.5 mM carbamoylcholine (CBC)) (a), 1.5 mM CBC + 10𝜇 M𝛼 -RgIB (b), and 1.5 mM CBC (c). Cells were kept at−70 mV. References [12] V. R. Eston, A. E. Migotto, E. C. Oliveira Filho, S. A. Rodrigues, and J. C. Freitas, “Vertical distribution of benthic marine organ- [1] B. M. Olivera and L. J. Cruz, “Conotoxins, in retrospect,” isms on rocky coasts of the Fernando de Noronha archipelago Toxicon,vol.39, no.1,pp. 7–14,2001. (Brazil),” Boletim do Instituto Paulista de Oceanografia ,vol.34, [2] S.R.Woodward,L.J.Cruz, B. M. Olivera, andD.R.Hillyard, pp. 37–53, 1986. “Constant and hypervariable regions in conotoxin propeptides,” [13] L. J. Cruz, G. Corpuz, and B. M. Olivera, “A preliminary study The EMBO Journal ,vol.9,no. 4, pp.1015–1020,1990. of Conus venom protein,” The Veliger ,vol.18, pp.302–308,1976. [3] Q.Kaas, R. Yu,A.H.Jin, S. Dutertre,and D. J. Craik, [14] R. Westermeier and T. Naven, Proteomics in Practice: Labo- “ConoServer: updated content, knowledge, and discovery tools ratory Manual of Proteome Analysis,Wiley-VCH,Weinheim, in the conopeptide database,” Nucleic Acids Research,vol.40,pp. Germany, 2002. D325–D330, 2012. [15] C. Clark, B. M. Olivera, and L. J. Cruz, “A toxin from the [4] Q. Kaas, J. C. Westermann, R. Halai, C. K. L. Wang, and D. J. venom of the marine snail Conus geographus which acts on the Craik, “ConoServer, a database for conopeptide sequences and vertebrate central nervous system,” Toxicon,vol.19, no.5,pp. structures,” Bioinformatics,vol.24, no.3,pp. 445–446, 2008. 691–699, 1981. [5] R.M.Jones,G.E.Cartier,J.M.McIntosh, G. Bulaj, V. E. Farrar, [16] S. M. Sine and P. Taylor, “Functional consequences of agonist- and B. M. Olivera, “Composition and therapeutic utility of mediated state transitions in the cholinergic receptor. Studies in conotoxins from genus Conus: patent status 1996–2000,” Expert cultured muscle cells,” The Journal of Biological Chemistry ,vol. Opinion on er Th apeutic Patents ,vol.11, no.4,pp. 603–623, 2001. 254, no. 9, pp. 3315–3325, 1979. [6] Q.Kaas, J. C. Westerman, andD.J.Craik,“Conopeptide char- [17] L.A.Greene,J.M.Aletta,A.Rukenstein,andS.H.Green,“PC12 acterization and classifications: an analysis using ConoServer,” pheochromocytoma cells: culture, nerve growth factor treat- Toxicon,vol.55, no.8,pp. 1491–1509, 2010. ment, and experimental exploitation,” Methods in Enzymology, [7] O.B.McManus,J.R.Musick,andC.Gonzalez, “Peptideisolated vol. 147, pp.207–216,1987. from the venom of Conus geographus block neuromuscular transmission,” Neuroscience Letters,vol.25,no.1,pp.57–62,1981. [18] O. P. Hamill,A.Marty,E.Neher,B.Sakmann, andF.J.Sigworth, “Improved patch-clamp techniques for high-resolution current [8] B.M.Olivera,W.R.Gray, andL.J.Cruz, “Marinesnail venoms,” recording from cells and cell-free membrane patches,” Pflugers in Marine Toxins and Venoms: Handbook of Natural Toxins,A. Archiv European Journal of Physiology,vol.391,no.2,pp.85–100, T. Tu, Ed., Marcel Dekker, New York, NY, USA, 1989. [9] H. Terlau and B. M. Olivera, “Conus venoms: a rich source of novel ion channel-targeted peptides,” Physiological Reviews,vol. [19] H. Ulrich,J.E.Ippolito, O. R. Paga´n,V.A.Eterovic, ´ R. M. Hann, 84,no. 1, pp.41–68,2004. H. Shi et al., “In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine [10] H. R. Arias and M. P. Blanton, “𝛼 -conotoxins,” International receptor,” Proceedings of the National Academy of Sciences of the Journal of Biochemistry and Cell Biology,vol.32, no.10, pp.1017– United States of America,vol.95, pp.14051–14056,1998. 1028, 2000. [11] E. C. Rios, Brazilian Marine Mollusks Iconography, Fundac¸ao ˜ [20] J. B. Udgaonkar and G. P. Hess, “Chemical kinetic measure- Universidade do Rio Grande, Rio Grande do Sul, Brazil, 1975. ments of a mammalian acetylcholine receptor by a fast-reaction Response (%) Response (%) Response (%) International Journal of Peptides 9 technique,” Proceedings of the National Academy of Sciences of [36] Q. Dai, Z. Sheng, J. H. Geiger, F. J. Castellino, and M. Prorok, the United States of America, vol. 84, no. 24, pp. 8758–8762, 1987. “Helix-helix interactions between homo- and heterodimeric 𝛾 -carboxyglutamate-containing conantokin peptides and their [21] A. A. Nery,R.R.Resende,A.H.Martins,C.A.Trujillo, V. derivatives,” eTh JournalofBiologicalChemistry ,vol.282,no. 17, A. Eterovic, and H. Ulrich, “Alph𝛼 7 nicotinic acetylcholine pp. 12641–12649, 2007. receptor expression and activity during neuronal differentiation of PC12 pheochromocytoma cells,” JournalofMolecular Neuro- [37] K. H. Gowd, V. Twede, M. Watkins et al., “Conantokin-P, an science,vol.41, no.3,pp. 329–339, 2010. unusual conantokin with a long disulfide loop,” Toxicon,vol.52, no. 2, pp. 203–213, 2008. [22] M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, and H. McWilliam, “ClustalW and ClustalX [38] K. Hansson, B. Furie, B. C. Furie, and J. Stenflo, “Isolation version 2,” Bioinformatics,vol.23, pp.2947–2948,2007. and characterization of three novel Gla-containing Conus mar- moreus venom peptides, one with a novel cysteine pattern,” [23] A. Roy, A. Kucukural, and Y. Zhang, “I-TASSER: a unified plat- Biochemical and Biophysical Research Communications,vol.319, form for automated protein structure and function prediction,” no. 4, pp. 1081–1087, 2004. Nature protocols,vol.5,no. 4, pp.725–738,2010. [39] T. Nakamura, Z. Yu, M. Fainzilber, and A. L. Burlingame, “Mass [24] Y. Zhang, “Template-based modeling and free modeling by I- spectrometric-based revision of the structure of a cysteine-rich TASSER in CASP7,” Proteins,vol.69, no.S8, pp.108–117,2007. peptide toxin with𝛾 -carboxyglutamic acid, TxVIIA, from the [25] W. R. Gray, A. Luque, B. M. Olivera, J. Barrett, and L. J. Cruz, sea snail, Conus textile,” Protein Science,vol.5,no. 3, pp.524– “Peptide toxins from Conus geographus venom,” The Journal of 530, 1996. Biological Chemistry,vol.256,no. 10,pp. 4734–4740, 1981. [40] P. B. Sargent, “eTh diversity of neuronal nicotinic acetylcholine [26] B. M. Olivera, G. Bulaj, J. Garrett, H. Terlau, and J. Imperial, receptors,” Annual Review of Neuroscience,vol.16, pp.403–443, “Peptide toxins from the venoms of cone snails and other toxoglossan gastropods,” in Animal Toxins: State of the Art— [41] F. Wang, V. Gerzanich, G. B. Wellst et al., “Assembly of human Perspectives in Health and Biotechnology,M.E.Lima, Ed., neuronal nicotinic receptor𝛼 5 subunits with𝛼 3,𝛽 2, and𝛽 4 Editora UFMG, Belo Horizonte, Brazil, 2009. subunits,” eTh JournalofBiologicalChemistry ,vol.271,no. 30, [27] M.Ellison,C.Haberlandt,M.E.Gomez-Casatietal.,“𝛼 -RgIA: a pp. 17656–17665, 1996. novel conotoxin that specifically and potently blocks the 𝛼 9𝛼 10 [42] S. Luo, J. M. Kulak, G. E. Cartier et al., “𝛼 -conotoxin AuIB nAChR,” Biochemistry,vol.45, no.5,pp. 1511–1517, 2006. selectively blocks𝛼 3𝛽 4 nicotinic acetylcholine receptors and [28] C. Grewer and G. P. Hess, “On the mechanism of inhibition of nicotine-evoked norepinephrine release,” Journal of Neuro- the nicotinic acetylcholine receptor by the anticonvulsant MK- science, vol. 18, no. 21, pp. 8571–8579, 1998. 801 investigated by laser-pulse photolysis in the microsecond- [43] S. N. Sudweeks and J. L. Yakel, “Functional and molecular to-millisecond time region,” Biochemistry,vol.38, no.24, pp. characterization of neuronal nicotinic ACh receptors in rat CA1 7837–7846, 1999. hippocampal neurons,” Journal of Physiology,vol.527,no. 3, pp. [29] R. J. Clark, N. L. Daly, R. Halai, S. T. Nevin, D. J. Adams, and 515–528, 2000. D. J. Craik, “eTh three-dimensional structure of the analgesic [44] S. Bohler,S.Gay,S.Bertrandetal.,“Desensitizationofneuronal 𝛼 -conotoxin, RgIA,” FEBS Letters,vol.582,no. 5, pp.597–602, nicotinic acetylcholine receptors conferred by N-terminal seg- ments of the𝛽 2 subunit,” Biochemistry,vol.40, no.7,pp. 2066– [30] B. Callaghan, A. Haythornthwaite, G. Berecki, R. J. Clark, D. 2074, 2001. J. Craik, and D. J. Adams, “Analgesic𝛼 -conotoxins Vc1.1 and [45] B. M. Olivera, L. J. Cruz, and D. Yashikami, “Eeff cts of Rg1A inhibit N-type calcium channels in rat sensory neurons Conus peptides on the behavior of mice,” Current Opinion in via GABAB receptor activation,” Journal of Neuroscience,vol.28, Neurobiology,vol.9,no. 6, pp.772–777,1999. no.43, pp.10943–10951,2008. [46] T. Dinklo,H.Shaban, J. W. uTh ring et al., “Characteri- [31] M. Ellison, Z. P. Feng, A. J. Park et al., “𝛼 -RgIA, a novel cono- zation of 2-[[4-fluoro-3-(triu fl oromethyl)phenyl]amino]-4-(4- toxin that blocks the𝛼 9𝛼 10 nAChR: structure and identification pyridinyl)-5-thiazolemethanol (JNJ-1930942), a novel positive of key receptor-binding residues,” Journal of Molecular Biology, allosteric modulator of the𝛼 7 nicotinic acetylcholine receptor,” vol. 377, no.4,pp. 1216–1227, 2008. Journal of Pharmacology and Experimental Therapeutics ,vol. [32] D. R. Groebe,W.R.Gray, andS.N.Abramson, “Determinants 336, no. 2, pp. 560–574, 2011. involved in the affinity of 𝛼 -conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors,” Biochemistry,vol. 36,no. 21,pp. 6469–6474, 1997. [33] A. Franco, K. Pisarewicz, C. Moller, D. Mora, G. B. Fields, and F. Mar`ı, “Hyperhydroxylation: a new strategy for neuronal targeting by venomous marine molluscs,” Progress in molecular and subcellular biology, vol. 43, pp. 83–103, 2006. [34] M. C. V. Braga, K. Konno, F. C. V. Portaro et al., “Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin,” Toxicon,vol.45, no.1,pp. 113–122,2005. [35] E. Czerwiec, D. E. Kalume, P. Roepstorff et al., “Novel 𝛾 - carboxyglutamic acid-containing peptides from the venom of Conus textile,” eTh FEBS Journal ,vol.273,no. 12,pp. 2779–2788, 2006. International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Peptides Hindawi Publishing Corporation

α-RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom

Loading next page...
 
/lp/hindawi-publishing-corporation/rgib-a-novel-antagonist-peptide-of-neuronal-acetylcholine-receptor-DMl0MV41SQ
Publisher
Hindawi Publishing Corporation
Copyright
Copyright © 2013 Maria Cristina Vianna Braga et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1687-9767
DOI
10.1155/2013/543028
Publisher site
See Article on Publisher Site

Abstract

Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 543028, 9 pages http://dx.doi.org/10.1155/2013/543028 Research Article 𝛼 -RgIB: A Novel Antagonist Peptide of Neuronal Acetylcholine Receptor Isolated from Conus regius Venom 1,2 3 3 Maria Cristina Vianna Braga, Arthur Andrade Nery, Henning Ulrich, 4 5 5 Katsuhiro Konno, Juliana Mozer Sciani, and Daniel Carvalho Pimenta CAT/CEPID, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900 Sao ˜ Paulo, SP, Brazil ´ ˆ ˜ ´ ´ Ministerio da Ciencia, Tecnologia e Inovac¸ao, Esplanada dos Ministerios, Bloco E, 70067-900 Brasılia, DF, Brazil Departamento de Bioqu´ımica, Instituto de Qu´ımica, Universidade de Sao ˜ Paulo, Av. Lineu Prestes 748, 05508-900 Sao ˜ Paulo, SP, Brazil Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Laborator ´ io de Bioqu´ımica e Biof´ısica, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900 Sao ˜ Paulo, SP, Brazil Correspondence should be addressed to Daniel Carvalho Pimenta; dcpimenta@butantan.gov.br Received 31 October 2012; Revised 16 January 2013; Accepted 16 January 2013 Academic Editor: Ayman El-Faham Copyright © 2013 Maria Cristina Vianna Braga et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus mayhave50to200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. eTh novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. eTh activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably 𝛼 3𝛽 4and/or𝛼 3𝛽 4𝛼 5 subtype. According to the nomenclature, the new peptide was designated as𝛼 -RgIB. 1. Introduction sequence of the conotoxin precursors (gene superfamilies), the cysteine patterns of conotoxin mature peptide regions Marine mollusks from Conus genus may produce from 50 (cysteine frameworks), and the specificities to pharmacolog- up to 200 biologically active molecules that can be injected ical targets (pharmacological families) [3, 4]. in the prey to capture or be employed as defense and/or Conopeptides of the pharmacological family 𝛼 ,which escape mechanisms to deter competitors. The peptide toxins, acts on neuronal acetylcholine receptor, have been found in called conopeptides, are composed of 10–40 amino acids the A, D, L, M, and S gene superfamilies [5, 6]. (including nonnatural amino acids) and are abundant in the Typically,𝛼 -conotoxins are peptides with 12 to 16 amino venom. Peptides presenting a rigid structure due to more than acid residues and two disulfide bridges, presenting the pattern one disuld fi e bridges are common, being called conotoxins. CC-C-C. These peptides are competitive antagonists of the These peptides act specifically on ionic channels and/or nicotinic acetylcholine receptors (nAChR) and display high neuromuscular receptors [1, 2]. selectivitybysubtypesofthisreceptor[5, 7–9]. After the Conotoxins are classified according to three schemes: blockage of the muscular acetylcholine receptor, the𝛼 -con- the similarities between the endoplasmatic reticulum signal otoxins significantly decrease the amplitude of the motor end 2 International Journal of Peptides plate postsynaptic potentials in vertebrates, paralyzing the a Q-ToF Ultima API (Micromass, Manchester, UK) and/or by prey [10]. MALDI-TOF mass spectrometry on a Ettan MALDI-ToF/Pro In the Brazilian tropical coast, there are approximately System (Amersham Biosciences, Sweden). 18 species of cone snails [11]. Conus regius (Gmelin, 1791) The analysis in the micro-LC-MS Ettan (Amersham is a vermivorous species that inhabits rock and coral deep Biosciences, Sweden) was performed in a 𝜇 RPC C2/C18 waters of Florida (USA), Central America, and the Northeast ST 1.0/150 column (Amersham Biosciences, Sweden), with and East coast of Brazil, including Fernando de Noronha two solvents: (A) formic acid (FA)/H O (1 : 1000) and (B) archipelago [12]. FA/ACN/H O(1:900:100).Thesamplewas eluted at a −1 In this work we described a novel peptide from Conus constant flow rate of 50 𝜇 L⋅min with a5to 65%gradient regius venom, belonging to the 𝛼 -conotoxins family. This of solvent B over 60 min. Q-Tof operated under positive peptide blocks the neuronal acetylcholine receptors on PC12 ionization mode. For MALDI-TOF analyses, a-cyano-4- cells, which comprise𝛼 3𝛽 4 and/or𝛼 3𝛽 4𝛼 5subtypesrecep- hydroxycinnamic acid was used as matrix. tors, probably target of the peptide. 2.5. “De Novo” Peptide Sequencing. Mass spectrometric “de novo” peptide sequencing was carried out in positive ioniza- 2. Material and Methods tion mode on a Q-TOF Ultima API tfi ted with an electro- 2.1. Reagents. All the employed reagents were of analytical spray ion source (Micromass, Manchester, UK). Briefly, the grade and were purchased from Sigma Co (St Louis, MO, amounts of previously lyophilized peptide were dissolved in USA), unless otherwise stated. 50 mM ammonium acetate, reduced with 50 mM DTT, alky- lated by 150 mM iodoacetamide, and hydrolyzed by 25 nM trypsin, according to slight modifications of Westermeier and 2.2. Animals and Venom. Specimens of C. regius were col- Naven [14]. eTh reaction products were then lyophilized and lected at Fernando de Noronha Archipelago, Pernambuco, dissolved in 50% ACN, containing 0.1% FA and injected into Brazil.TheBrazilian EnvironmentalAgency(IBAMA—Insti- −1 thesourceat5𝜇 L⋅min by aHamiltoninfusionpump, or tuto Brasileiro do Meio Ambiente e dos Recursos Naturais directly injected using a Rheodyne 7010 sample loop coupled Renova´veis) license numbers were 030/2000 and 087/2001, −1 to a LC-10A VP Shimadzu pump operating at 20𝜇 L⋅min and the process number was 02001, 000775/00-00. Venom constant flow rate. eTh instrument control and data acquisi- was extracted from the specimens as previously described tion were conducted by MassLynx 4.0 data system (Micro- [13]. The crude venom was obtained by dissection of the mass, Manchester, UK) and experiments were performed by venom duct gland and then freeze-dried and stored at−80 C. scanning a mass-to-charge ratio (m/z)of50–1800 usinga Voucher material is deposited in the malacological col- scan time of 2 s applied during the whole chromatographic lection of Zoology Museum of University of Sao ˜ Paulo, Sao ˜ process. The mass spectra corresponding to each signal from Paulo, Brazil. the total ion current (TIC) chromatogram were averaged, allowing an accurate molecular mass determination. External 2.3. Peptide Fractionation and Purica fi tion. A reversed-phase calibration of the mass scale was performed with NaI. For the binary HPLC system (LC-8A, Shimadzu Co., Japan) was MS/MS analysis, collision energy ranged from 18 to 45 and used for sample fractionation. eTh lyophilized crude venom the precursor ions were selected under a 1-m/z window. powder was solubilized into 0.1% trifluoroacetic acid (TFA) andaliquotswereloadedinaShim-packPrep-ODSC18 ˚ 2.6. Biological Activity. eTh biological activity of 𝛼 -RgIB column (Shimadzu, 3𝜇 m, C18, 300 A, 250 × 20 mm) in wasdeterminedinSwiss Webstermice(5.5to7gbody a two-solvent system: (A) trifluoroacetic acid (TFA)/H O weight) by observation of the behavioral disorders aeft r (1 : 1000) and (B) TFA/Acetonitrile (ACN)/H O(1:900:100). −1 intracranial injection [15] of the peptide diluted in NaCl The sample was eluted at a constant flow rate of 8 mL ⋅min 0.9%, in concentration of 0.1, 0.5 and 1 nmol. Alterations were with a 0 to 60% gradient of solvent B over 60 min. eTh compared to animals injected with NaCl 0.9% (control). All HPLC column eluates were monitored by a Shimadzu SPD- animals were observed by 60 min. 10A detector scanning 220 nm. For𝛼 -RgIB purification, the interest peak was fraction- ated in a Merck C18 column (300× 4.6 mm), in a 19 to 21% B 2.7. Patch Clamp. BC H1 cells, mouse myocytes which −1 gradient over 20 min, at a constant flow rate of 8 mL ⋅min . express nicotinic acetylcholine receptors, were acquired by A subsequent purification step was still necessary to obtain ATCC (CRL-1443) and maintained in culture according to thepeptide.This puricfi ationwas conductedinaMerckC18 Sine and Taylor [16] to electrophysiological experiments. In column (300 × 4.6 mm), in an isocratic elution at 35% B order to verify the subtype of neuronal nicotinic receptor that (TFA/methanol/H O 1:900:100) at a constant flow rate of the peptide acts, PC12 cells were employed and maintained in −1 1mL⋅min . culture according to Greene et al. [17]. Individual cells were subjected to a patch-clamp, at a 2.4. Mass Spectrometry Analysis. Molecular mass analyses whole cell configuration, according to Hamill et al. [ 18]and of thepeaks andthe peptides were performedonamicro- Urlich et al. [19]. Cells were maintained in an extracellular LC-MS Ettan (Amersham Biosciences, Sweden) coupled in solution containing 25 mM HEPES, 5.3 mM KCl,144.8 mM International Journal of Peptides 3 NaCl, 1.2 mM MgCl , 2.38 mM CaCl , and 10 mM glucose 3.3. Sequence Features. A sequence alignment was performed 2 2 (pH 7.4). A recording electrode was filled with intracellular with all𝛼 -conotoxins available at UniProt (supplementary solution containing 25 mM HEPES, 141 mM KCl, 10 mM Table 1 of the supplementary material available online at NaCl, 2 mM MgCl and 1 mM EGTA (pH 7.4). Experiments http://dx.doi.org/10.1155/2013/543028). Basedonthislarge 2, alignment, a phylogeny was constructed (supplementary were carried outatroomtemperature (20–24 C). Figure 1) and the peptide sequences present at the branch Throughout the experiment, the membrane potential was containing𝛼 -RgIB were realigned (Figure 4). ClustalW stan- clamped at a−60 mV for BC H cells and−70 mV for PC12 3 1 dard annotations consider, as expected, the Cys residues cells, holding potential using an Axon Axopatch amplifier as consensus ( ), and the Glu at the 8th aligned posi- (Molecular Devices, California, USA). Data were recorded tion, as being highly conserved (:). Moreover, according and digitized by Clampex 8.2 software (Molecular Devices, to the algorithm standard notation, the Pro residue at the California, USA) and plots were made using Origin 7.0 13th aligned position is also conserved (.). Among the software (OriginLab Corp., Northampton, MA). UNIPROT database, the SwissModel tool could not identify Control currents were performed with 1.5 mM carbamyl- anysuitabletemplatefor structurepredictionof 𝛼 -RgIB, choline, using the cell-flow technique [ 20]. After the car- therefore an external application was used. Figure 5 presents bamylcholine administration, the peptide (10𝜇 M) was incu- a3Dmodel of 𝛼 -RgIB; created by I-TASSER [23, 24], as batedoncells,and then anotherdoseofthe agonistwas well as three PDB deposited 3D solution structures of 𝛼 - incubated [21]. RgIA (P0C1D0) mutants, a conotoxin that specifically and potently blocks the𝛼 9𝛼 10 nAChR [27]. In spite of𝛼 -RgIB 2.8. Data Fitting, Statistical Analyses, and Sequence Alignment. N- and C-terminal extensions and longer interbridge peptide When data tfi ting was performed, results were presented as sequence, the model and the structures are tridimensionally the calculated value± standard deviation (SD). Otherwise, related, for example, a C-shaped structure, held by the Cys- data correspond to the mean of three individual experiments. bridges. Peptidesequencealignment wasperformed usingClustalW software [ 22]. The 3D model of the peptide 𝛼 -RgIB, as well as three PDB deposited 3D solution structures, was created by 3.4. Biological Activity. The in vivo biological activity of the I-TASSER [23, 24]. peptide was assessed by means of intracranial injection in Swiss Webster mice. Following 1 nmol injection, the animals displayed a hyperactive behavior, defecating and urinating 3. Results all the time, which was not observed for the control group 3.1. Purification. eTh crude venom from C. regius was frac- that received saline solution. Auditory stimuli, for example, a tionated by RP-HPLC, as shown in Figure 1(a).Somepeaks hand-clap or hitting the cage, also triggered the hyperactive could be detected along the profile, and the arrow indicates behavior. Interestingly, the lower doses (0.1 and 0.5 nmol), the peak of interest. Two subsequent chromatographic steps caused the animals to have dicffi ulty in breathing. Although were necessary to purify the peptide (Figures 1(b) and the peptide promoted behavioral disorders, it was not lethal 1(c)), under the conditions described material and methods to the animals. section. After the third step of purification, the purity and the molecular mass of the peptide were assessed by MALDI- TOF/MS (Figure 1(d)). 3.5. Patch Clamp. Whole-cell voltage clamp measurement was used to verify the ion currents on acetylcholine receptors. BC H1 cells, which express the acetylcholine muscle type 3.2. “De Novo” Peptide Sequencing. After cysteine bridge 3 receptors on the surface, and PC12, which terminally differ- reduction and alkylation, the reaction product was digested entiate in neurons and express nicotinic neuronal receptors with trypsin. eTh obtained peptides were submitted to [21] were selected for the experiments. Carbamylcholine, a MS/MS analyses (Figure 2)and ions were selected and stable and well-characterized analogue of acetylcholine, was fragmented by collision with argon (CIF), yielding daughter used as an agonist [28], for it elicits a fast activating current ion spectra (Figure 3)thatwas processedwithBioLynx and that rapidly desensitizes during the application. manually checked for accuracy of interpretation. Since the 10𝜇 M 𝛼 -RgIBwas notabletoinduceany change in digestion allowed peptides with missed cleavage sites, it the ion currents on BC H cells (data not shown), as well was possible to assemble the fragments without the aid of 3 1 as a higher dose (30𝜇 M) of the peptide. d-tubocurarine (a another digestion with a different enzyme. The sequenced classic nicotinic receptor antagonist) was used as a positive peptides, their charge states, and theoretical molecular mass control and successfully to block this channel (data not are presented in Table 1. shown). The peptide sequence was determined to be TWEECCKNPGCRNNHVDRCRGQV. This sequence has After the incubation of the peptide with PC12 cells, 4 cysteine residues with pattern CC-C-C, typical from fast and slow desensitization of the receptor was observed. conotoxins of framework I [25]. This peptide was named Figure 6(b) showsthatonneuronalslowrise-time receptors, 𝛼 -RgIB, according to the guidelines for conotoxins nomen- 𝛼 -RgIB is able to block the ion current by 40%, compared clature ConoServer and has been assigned the following to cells stimulated with carbamylcholine (Figure 6(a)). The UNIPROT accession number: C0HJA8 [3, 6, 26]. blockage was irreversible and persistent, once the current 4 International Journal of Peptides 0 5 10152025303540455055 60 Minutes Detector A (220 nm) HPLC conus regius 10 CVE B.CONC MCris.met (a) 0 15 6 8 10 12 14 16 18 20 22 24 26 28 30 02468 10 12 14 16 18 20 Minutes Minutes Detector A (220 nm) HPLC conus regius A007 B.CONC MCris.met (c) (b) 2703.863 1,150 1,050 1,600 2,000 2,400 2,800 3,200 3,600 𝑚/𝑧 (d) Figure 1: (a) Representative RP-HPLC of the crude C. regius venom. The arrow indicates the peak of interest. (b) Representative RP-HPLC of the selected peak (arrow), indicating the presence of impurities. (c) Isocratic elution of the isolated the selected fraction from chromatogram B. The arrow indicates the peak of interest. (d) MALDI-TOF/MS profile of the purified peptide. does not recover aer ft a new application of the agonist, (gene superfamilies), the cysteine patterns of conotoxin carbamylcholine (Figure 6(c)). mature peptide regions (cysteine frameworks), and the speci- ficities to pharmacological targets [ 3, 4]. This new peptide was termed 𝛼 -Rg-IB because the 4. Discussion peptide acts on neuronal acetylcholine receptors (“𝛼 ”), was extracted from a Conus regius specimen (“Rg”), displays a Conotoxins are classified according to the similarities between the signal sequence of the conotoxin precursors cysteine framework I—CC-C-C (“I”), and was the second Abs Abs Intensity (%) mAU International Journal of Peptides 5 091003AL 27 (0.972) Sm (SG,2×6.00); Sb (3,20.00); Cm (25:48) 335.1610 TOF MS ES+ 1.59𝑒3 377.6908 A D C2 T W E E C C K N P G C R N N H V D R C R G Q V BC E F A2 A3 820.8170 547.5543 821.3205 547.8890 754.3600 548.2238 821.8242 378.6921 755.3594 822.3163 303.1793 548.5588 B2 379.1854 548.8938 822.8320 528.6995 603.2767 823.3126 389.2137 619.3010 396.6663 620.2981 756.3705 839.7988 397.1629 507.2256 576.2546 840.8057 654.3190 1056.3862 415.2154 851.2896 776.3487 655.3229 1115.4175 993.3750 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 𝑚/𝑧 Figure 2: Representative ESI-Q-TOF/MS profile of the trypsin digested purified peptide. eTh deduced sequence is printed above the spectrum, together with the tryptic peptides (A–F). eTh MS profile indicates the tryptic peptides and the charge states. Table 1: eTh oretical and experimental 𝑚/𝑧 values for the tryptic peptides obtained aeft r the enzymatic digestion of 𝛼 -RgIB. + 2+ 3+ [M + H] [M + 2H] [M + 3H] Sequence Ions Exp. eTh or. Exp. eo Th r. Exp. eTh or. 1,2 1,2 1,2 1,2 1,2 1,2 A 1640.63 1639.839 820.82 820.423 547.55 547.282 TWEECCKNPGCR 1,2 1,2 1,2 1,2 B 1056.39 1055.180 528.70 528.094 — x TWEECCK C 754.37 754.359 377.69 377.896 — (252.125) NNHVDR 2 2 3 2,4 D619.30 619.298 — (310.366) —x CRGQV 2 2 E603.28 603.267 — (302.137) —x NPGCR 2 2 5 F335.16 335.147 —x —x CR Acetylation (N-terminal, variable modification). Carbamidomethyl cysteine (fixed modification). Not detected. Not observed. Not expected. peptidediscoveredwithbothbeing from C. regius with a its closest phylogenetic relatives (supplementary material), cysteine framework I (“B”) [25]. besides 𝛼 -RgIA, which was not considered to be similar 𝛼 -RgIA was the rfi st 𝛼 -conotoxin described from C. (according to MEGA5), but was manually inserted in the regius, acting on neuronal nicotinic receptors. This peptide gfi urefor thebeneto fi fsequencecomparison. 𝛼 -RgIA is has been thoroughly characterized in terms of its primary shorter, both in the N- and C-terminal flanking regions, and three-dimensional structures [29], as well as regarding as well as in the inter-Cys-bridge region. Nevertheless, in its biological eeff ct, for example, the blockage of the 𝛼 9𝛼 10 a considerably small universe of possibilities (8 out 12, nAChR [30, 31].𝛼 -RgIA and𝛼 -RgIB come from the same since 4 amino acids are necessarily Cys), 𝛼 -RgIA and 𝛼 - animal, belong to the same toxin family, and possess similar RgIB bare considerable similarities: the Pro, at the 13th biological effects; however, their amino acid sequences differ. aligned position, and the charged residues at the 17th and Figure 4 shows the ClustalW alignment of 𝛼 -RgIB and 18th aligned positions. It is noteworthy to mention that, (%) 6 International Journal of Peptides MC 2.3 msms 754 1: TOF MSMS 754.00ES+ 091003AP MaxEnt 3 31 [Ev-102034, It50, En1] (0.050, 200.00, 0.200, 1400.00, 1, Cmp) NN H V R bMax RD H N N V yMax 100 754.42 (𝑀+)𝐻+ 175.14 366.18 y1 755.34 b3 737.36 349.16 526.31 465.25 756.57 640.36 580.27 y4 237.16 367.16 448.24 110.10 290.17 b4 y5 b5 758.08 509.29 720.33 209.16 420.24 563.26 y2 623.31 H 138.08 677.33 332.13 759.27 799.39 252.14 491.26 88.05 694.41 158.11 176.63 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 𝑀/𝑧 Figure 3: MaxEnt3 deconvoluted annotated representative MS/MS profile of the CID spectrum of tryptic peptide C, from Figure 2.yandb series are annotated above the spectrum, as well as other fragments. Clustal 2.1 multiple sequence alignment 𝛼 -RgIB TWEECCKNPGCRNNHVDRCRGQV 23 A6M938 17 NDCCHNAPCRNNHPGIC A1X8C3 GMWDECCDDPPCRQNNMEHCPAS Q2I2R6 ECCDDPPCRQNNMEHCPAS A1X8C2 GVWDECCKDPQCRQNHMQHCPAR P0C8U6 DDCCPDPACRQNHPELCSTR P0C8U7 DDCCPDPACRQNHPEICPSR P0C8U9 NAWLTPEECCAAPACREMILEFCLAGEAFAAALDGFRRLPYR 42 P0C8U8 NAWFTPEECCAAPACRGMILEFCLAGEAFAAALDGFRRLPYR ISEMTWEECCTNPVCRQHYMHYC P0C8V0 23 P01519 ECC NPACGRHYS C 13 ∗∗ ∗ ∗ : . GCCSDPRCRYR C 12 P0C1D0 Figure 4: ClustalW alignment of 𝛼 -RgIB and the closest phylogenetic 𝛼 -conotoxins relatives (calculated according to supplemental Figure 3).( ) Consensus; (:) highly conserved; (.) conserved. The bold underlined amino acid residues of 𝛼 -RgIB were also considered to be conserved. A6M938:𝛼 -conotoxin-like Lp1.10 C. leopardus/homology; A1X8C3:𝛼 -conotoxin-like Lp1.7 C. leopardus/transcript; Q2I2R6: 𝛼 -conotoxin-like Lt1.3 C. litteratus/transcript; A1X8C2:𝛼 -conotoxin-like Lp1.8 C. leopardus/transcript; P0C8U6:𝛼 -conotoxin-like PuSG1.1 C. pulicarius/transcript; P0C8U9:𝛼 -conotoxin-like Pu1.5 C. pulicarius/transcript; P0C8U8:𝛼 -conotoxin-like Pu1.4 C. pulicarius/transcript; P0C8U7/𝛼 -conotoxin-like PuSG1.2 C. pulicarius/trasncritpt; P0C8V0:𝛼 -conotoxin-like Pu1.6 C. pulicarius/transcript; P01519:𝛼 -conotoxin GIA C. geographus/protein; P0C1D0:𝛼 -conotoxin RgIA, C. regius/protein. (Key: UniProt Accesion code: toxin/Conus species/evidence level). in spite of the phylogenetic analyses, all conotoxins listed Moreover, our group has also identified two conotoxins, as in Figure 4 (except𝛼 -RgIA and𝛼 -RgIB) come from other well:Rg11a,belonging to theI superfamily (P84197, [34]); Conus species: C. leopardus (A6 M938, A1X8C2, A1X8C3), and Rg9.1, belonging to the P-superfamily (Q8I6V7; direct C. litteratus (Q2I2R6), C. pulicarius (P0C8U6, P0C8U7, submission). P0C8U8, P0C8U9, and P0C8V0) and C. geographus (P01519). There is no high level of homology between 𝛼 -RgIB Moreover, only P01519 has been detected at the protein and the conopeptides described until the present moment; level and has been characterized as active on the muscular therefore, the identification of a proper 3D structure to serve nicotinic receptors [32]. as a template for homology modeling is deprecated. Instead, Besides𝛼 -RgIA, the following toxins have been isolated a structure was predicted by using I-TASSER server [23, 24]. from C. regius: P85009; P85010; P85011; P85012; and P85013, Figure 5 shows that, in spite of the low homology with𝛼 - all 𝛼 -conotoxin-like peptides belonging to superfamily A; RgIA,𝛼 -RgIB model assumed the same basic shape as the P85016; P85017; P85018; P85019; P85020; P85021 and P85022, NMR determined structures of the𝛼 -RgIA mutants, available all belonging to the M-superfamily of conotoxins [33]. at the PDB database [27]. (%) International Journal of Peptides 7 the pool of PC12 cells expressed 𝛼 3, 𝛼 5, 𝛼 7, 𝛽 2, and 𝛽 4 subunits of neuronal nicotinic receptors, the same pattern NH found by Sargent [40]inPC12cells.However,inspite of𝛼 - NH Rg-IB affinity by the PC12 nicotinic receptors, there are still 2JUR other neuronal nicotinic receptors that may be higher ani ffi ty MODEL COOH targets for these toxins that were not explored in the present COOH work. AuIB, from Conus aulicus,which is also an𝛼 -conotoxin, blocks the 𝛼 3𝛽 4 receptors; however, the currents can be recovered aeft r the toxin washing [ 42]. In our experiments, NH the no-recovery of𝛼 -RgIB is probably due to the irreversible NH 2 action of the peptide on the receptor. Successive applications of the agonist (carbamolycholine), in control experiments, did not cause recovery of the ion currents on slow rise-time 2JUT 2JUS receptors (data not shown). Besides the irreversible action, the peptide may also be able to prolong the desensitization COOH time of the receptor, since the repeated CBC administration COOH on 𝛼 -RgIB-treated PC12 cells was not able to recover the initial current, which is either caused by the irreversible Figure 5: I-TASSER model of 𝛼 -RgIB and PDB NMR superimposed binding of a low affinity toxin or the prolonging of the structures of𝛼 -RgI-A. desensitization time of the receptor (or both). Sudweeks and Yakel [43]showedthat 𝛼 3, 𝛼 7, and 𝛽 2 subunits of nAChR are correlated to fast rise-time receptors. Regarding the rather unique amino acid sequence of𝛼 - The slow desensitization is a characteristic of 𝛼 3𝛽 4receptor, while𝛼 3𝛽 2 receptor is from fast desensitization [44]. The RgIB and thoroughly analyzing our data, we could not rule outthe possibilitythatone of theglutamicacid(Glu) residues fast desensitization receptors, on PC12 cells, contain𝛼 3𝛽 2, of this novel conotoxin would be a gamma-carboxyglutamic 𝛼 3𝛽 2𝛼 5, and𝛼 7 subunits, while slow desensitization recep- tors are formed by subunits𝛼 3𝛽 4and𝛼 3𝛽 4𝛼 5.𝛼 -RgIB was acid residue (Gla). Our suspicions arouse from the slightly higher deviation between the theoretical and calculated able to inhibit the currents elicited by carbamolycholine on molecularmassvaluesfor Aand Bions(Table 1), that PC12 cells, mainly on the slow desensitization component, could reflect that a side chain carboxylation and not an N- which comprise, in our model,𝛼 3𝛽 4and𝛼 3𝛽 4𝛼 5 receptors. terminal acetylation would be present. Moreover, conotoxins The intracranial injection assay was performed to investi- are known for presenting posttranslation modifications, Gla gate whether there would be any direct activity of the toxin included [35–38] and, even though the MALDI data of in the central nervous system (CNS), once peptides can the crude peptide support the proposed peptide sequence, promote behavioral alterations by acting on receptors and ionic channels on CNS. es Th e alterations can indicate activ- MALDI ionization is also a source of facile decarboxyla- tion for Gla residues [39]. Our future experiments with ities on specific ionic channels. For example, 𝜔 -conotoxin C. regius conotoxins (𝛼 -RgIB included) will clarify this GVIA causes trembling on the mice, which indicates an action on calcium ionic channels [45]. The 𝛼 -nicotinic matter. 𝛼 -conotoxins bind to nicotinic acetylcholine receptors. acetylcholine receptor (nAChR) is associated to attention- The subgroup 𝛼 3/5 of𝛼 -conotoxins, from piscivorous Conus, deficit/hyperactivity disorder [ 46]which corroborates our has the motif CCX CX Cand cancause paralysisofthe observations of𝛼 -RgIB-treated hyperactive mice. 3 5 prey by the binding on muscle nicotinic receptors. Another In conclusion, we have isolated a novel conotoxin from subgroup, 𝛼 4/3, that present the motif CCX CX C, bind Conus regius and, by means of a combination of biochemical, 4 3 on neuronal nicotinic receptors. eTh main subgroup of 𝛼 - structural and pharmacological assays were able to classify conotoxins is𝛼 4/7, with motif CCX CX C. These peptides this peptide in the 𝛼 -family and named it 𝛼 -RgIB. There 4 7 are still several peptides to be explored in the C. regius bind in all classes of nicotinic receptors: muscular (e.g., 𝛼 -conotoxin EI), homomeric neuronal (e.g., 𝛼 -conotoxins venom, as our previous qualitative investigations have shown PnIB), and heteromeric neuronal (𝛼 -conotoxins MII and [34] and the current study has focused on the biochemical characterization of one such novel peptide. Further studies AuIB) [9]. Neuronal nicotinic acetylcholine receptors (nAChRs) are still necessary to better characterize the structural and belong to the pentameric superfamily of Cys-loop ligand pharmacological properties of𝛼 -RgIB. gated ionic channels. eTh y are composed of either homo- meric𝛼 or heteromeric𝛼 and𝛽 subunits assembled from a family of 12 distinct neuronal nicotinic subunits (𝛼 2–𝛼 10; Acknowledgments 𝛽 2–𝛽 4) [5]. The combination of subunits 𝛼 2,𝛼 3, and𝛼 4with 𝛽 2and𝛽 4 results in a functional receptor, as well𝛼 7,𝛼 8, This work was supported by Grants from the Brazilian fund- and𝛼 9 homomeric receptors [40, 41]. In our experiments, ing agencies FAPESP and CNPq, including the INCTTOX it was verified by RT-PCR (supplemental Figure 3) that PROGRAM. 8 International Journal of Peptides 100 100 0 0 0 3 3 3 Time (s) Time (s) Time (s) (a) (b) (c) Figure 6: PC12 whole-cell characteristic patch clamp currents (expressed as a percentage of response to 1.5 mM carbamoylcholine (CBC)) (a), 1.5 mM CBC + 10𝜇 M𝛼 -RgIB (b), and 1.5 mM CBC (c). Cells were kept at−70 mV. References [12] V. R. Eston, A. E. Migotto, E. C. Oliveira Filho, S. A. Rodrigues, and J. C. Freitas, “Vertical distribution of benthic marine organ- [1] B. M. Olivera and L. J. Cruz, “Conotoxins, in retrospect,” isms on rocky coasts of the Fernando de Noronha archipelago Toxicon,vol.39, no.1,pp. 7–14,2001. (Brazil),” Boletim do Instituto Paulista de Oceanografia ,vol.34, [2] S.R.Woodward,L.J.Cruz, B. M. Olivera, andD.R.Hillyard, pp. 37–53, 1986. “Constant and hypervariable regions in conotoxin propeptides,” [13] L. J. Cruz, G. Corpuz, and B. M. Olivera, “A preliminary study The EMBO Journal ,vol.9,no. 4, pp.1015–1020,1990. of Conus venom protein,” The Veliger ,vol.18, pp.302–308,1976. [3] Q.Kaas, R. Yu,A.H.Jin, S. Dutertre,and D. J. Craik, [14] R. Westermeier and T. Naven, Proteomics in Practice: Labo- “ConoServer: updated content, knowledge, and discovery tools ratory Manual of Proteome Analysis,Wiley-VCH,Weinheim, in the conopeptide database,” Nucleic Acids Research,vol.40,pp. Germany, 2002. D325–D330, 2012. [15] C. Clark, B. M. Olivera, and L. J. Cruz, “A toxin from the [4] Q. Kaas, J. C. Westermann, R. Halai, C. K. L. Wang, and D. J. venom of the marine snail Conus geographus which acts on the Craik, “ConoServer, a database for conopeptide sequences and vertebrate central nervous system,” Toxicon,vol.19, no.5,pp. structures,” Bioinformatics,vol.24, no.3,pp. 445–446, 2008. 691–699, 1981. [5] R.M.Jones,G.E.Cartier,J.M.McIntosh, G. Bulaj, V. E. Farrar, [16] S. M. Sine and P. Taylor, “Functional consequences of agonist- and B. M. Olivera, “Composition and therapeutic utility of mediated state transitions in the cholinergic receptor. Studies in conotoxins from genus Conus: patent status 1996–2000,” Expert cultured muscle cells,” The Journal of Biological Chemistry ,vol. Opinion on er Th apeutic Patents ,vol.11, no.4,pp. 603–623, 2001. 254, no. 9, pp. 3315–3325, 1979. [6] Q.Kaas, J. C. Westerman, andD.J.Craik,“Conopeptide char- [17] L.A.Greene,J.M.Aletta,A.Rukenstein,andS.H.Green,“PC12 acterization and classifications: an analysis using ConoServer,” pheochromocytoma cells: culture, nerve growth factor treat- Toxicon,vol.55, no.8,pp. 1491–1509, 2010. ment, and experimental exploitation,” Methods in Enzymology, [7] O.B.McManus,J.R.Musick,andC.Gonzalez, “Peptideisolated vol. 147, pp.207–216,1987. from the venom of Conus geographus block neuromuscular transmission,” Neuroscience Letters,vol.25,no.1,pp.57–62,1981. [18] O. P. Hamill,A.Marty,E.Neher,B.Sakmann, andF.J.Sigworth, “Improved patch-clamp techniques for high-resolution current [8] B.M.Olivera,W.R.Gray, andL.J.Cruz, “Marinesnail venoms,” recording from cells and cell-free membrane patches,” Pflugers in Marine Toxins and Venoms: Handbook of Natural Toxins,A. Archiv European Journal of Physiology,vol.391,no.2,pp.85–100, T. Tu, Ed., Marcel Dekker, New York, NY, USA, 1989. [9] H. Terlau and B. M. Olivera, “Conus venoms: a rich source of novel ion channel-targeted peptides,” Physiological Reviews,vol. [19] H. Ulrich,J.E.Ippolito, O. R. Paga´n,V.A.Eterovic, ´ R. M. Hann, 84,no. 1, pp.41–68,2004. H. Shi et al., “In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine [10] H. R. Arias and M. P. Blanton, “𝛼 -conotoxins,” International receptor,” Proceedings of the National Academy of Sciences of the Journal of Biochemistry and Cell Biology,vol.32, no.10, pp.1017– United States of America,vol.95, pp.14051–14056,1998. 1028, 2000. [11] E. C. Rios, Brazilian Marine Mollusks Iconography, Fundac¸ao ˜ [20] J. B. Udgaonkar and G. P. Hess, “Chemical kinetic measure- Universidade do Rio Grande, Rio Grande do Sul, Brazil, 1975. ments of a mammalian acetylcholine receptor by a fast-reaction Response (%) Response (%) Response (%) International Journal of Peptides 9 technique,” Proceedings of the National Academy of Sciences of [36] Q. Dai, Z. Sheng, J. H. Geiger, F. J. Castellino, and M. Prorok, the United States of America, vol. 84, no. 24, pp. 8758–8762, 1987. “Helix-helix interactions between homo- and heterodimeric 𝛾 -carboxyglutamate-containing conantokin peptides and their [21] A. A. Nery,R.R.Resende,A.H.Martins,C.A.Trujillo, V. derivatives,” eTh JournalofBiologicalChemistry ,vol.282,no. 17, A. Eterovic, and H. Ulrich, “Alph𝛼 7 nicotinic acetylcholine pp. 12641–12649, 2007. receptor expression and activity during neuronal differentiation of PC12 pheochromocytoma cells,” JournalofMolecular Neuro- [37] K. H. Gowd, V. Twede, M. Watkins et al., “Conantokin-P, an science,vol.41, no.3,pp. 329–339, 2010. unusual conantokin with a long disulfide loop,” Toxicon,vol.52, no. 2, pp. 203–213, 2008. [22] M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, and H. McWilliam, “ClustalW and ClustalX [38] K. Hansson, B. Furie, B. C. Furie, and J. Stenflo, “Isolation version 2,” Bioinformatics,vol.23, pp.2947–2948,2007. and characterization of three novel Gla-containing Conus mar- moreus venom peptides, one with a novel cysteine pattern,” [23] A. Roy, A. Kucukural, and Y. Zhang, “I-TASSER: a unified plat- Biochemical and Biophysical Research Communications,vol.319, form for automated protein structure and function prediction,” no. 4, pp. 1081–1087, 2004. Nature protocols,vol.5,no. 4, pp.725–738,2010. [39] T. Nakamura, Z. Yu, M. Fainzilber, and A. L. Burlingame, “Mass [24] Y. Zhang, “Template-based modeling and free modeling by I- spectrometric-based revision of the structure of a cysteine-rich TASSER in CASP7,” Proteins,vol.69, no.S8, pp.108–117,2007. peptide toxin with𝛾 -carboxyglutamic acid, TxVIIA, from the [25] W. R. Gray, A. Luque, B. M. Olivera, J. Barrett, and L. J. Cruz, sea snail, Conus textile,” Protein Science,vol.5,no. 3, pp.524– “Peptide toxins from Conus geographus venom,” The Journal of 530, 1996. Biological Chemistry,vol.256,no. 10,pp. 4734–4740, 1981. [40] P. B. Sargent, “eTh diversity of neuronal nicotinic acetylcholine [26] B. M. Olivera, G. Bulaj, J. Garrett, H. Terlau, and J. Imperial, receptors,” Annual Review of Neuroscience,vol.16, pp.403–443, “Peptide toxins from the venoms of cone snails and other toxoglossan gastropods,” in Animal Toxins: State of the Art— [41] F. Wang, V. Gerzanich, G. B. Wellst et al., “Assembly of human Perspectives in Health and Biotechnology,M.E.Lima, Ed., neuronal nicotinic receptor𝛼 5 subunits with𝛼 3,𝛽 2, and𝛽 4 Editora UFMG, Belo Horizonte, Brazil, 2009. subunits,” eTh JournalofBiologicalChemistry ,vol.271,no. 30, [27] M.Ellison,C.Haberlandt,M.E.Gomez-Casatietal.,“𝛼 -RgIA: a pp. 17656–17665, 1996. novel conotoxin that specifically and potently blocks the 𝛼 9𝛼 10 [42] S. Luo, J. M. Kulak, G. E. Cartier et al., “𝛼 -conotoxin AuIB nAChR,” Biochemistry,vol.45, no.5,pp. 1511–1517, 2006. selectively blocks𝛼 3𝛽 4 nicotinic acetylcholine receptors and [28] C. Grewer and G. P. Hess, “On the mechanism of inhibition of nicotine-evoked norepinephrine release,” Journal of Neuro- the nicotinic acetylcholine receptor by the anticonvulsant MK- science, vol. 18, no. 21, pp. 8571–8579, 1998. 801 investigated by laser-pulse photolysis in the microsecond- [43] S. N. Sudweeks and J. L. Yakel, “Functional and molecular to-millisecond time region,” Biochemistry,vol.38, no.24, pp. characterization of neuronal nicotinic ACh receptors in rat CA1 7837–7846, 1999. hippocampal neurons,” Journal of Physiology,vol.527,no. 3, pp. [29] R. J. Clark, N. L. Daly, R. Halai, S. T. Nevin, D. J. Adams, and 515–528, 2000. D. J. Craik, “eTh three-dimensional structure of the analgesic [44] S. Bohler,S.Gay,S.Bertrandetal.,“Desensitizationofneuronal 𝛼 -conotoxin, RgIA,” FEBS Letters,vol.582,no. 5, pp.597–602, nicotinic acetylcholine receptors conferred by N-terminal seg- ments of the𝛽 2 subunit,” Biochemistry,vol.40, no.7,pp. 2066– [30] B. Callaghan, A. Haythornthwaite, G. Berecki, R. J. Clark, D. 2074, 2001. J. Craik, and D. J. Adams, “Analgesic𝛼 -conotoxins Vc1.1 and [45] B. M. Olivera, L. J. Cruz, and D. Yashikami, “Eeff cts of Rg1A inhibit N-type calcium channels in rat sensory neurons Conus peptides on the behavior of mice,” Current Opinion in via GABAB receptor activation,” Journal of Neuroscience,vol.28, Neurobiology,vol.9,no. 6, pp.772–777,1999. no.43, pp.10943–10951,2008. [46] T. Dinklo,H.Shaban, J. W. uTh ring et al., “Characteri- [31] M. Ellison, Z. P. Feng, A. J. Park et al., “𝛼 -RgIA, a novel cono- zation of 2-[[4-fluoro-3-(triu fl oromethyl)phenyl]amino]-4-(4- toxin that blocks the𝛼 9𝛼 10 nAChR: structure and identification pyridinyl)-5-thiazolemethanol (JNJ-1930942), a novel positive of key receptor-binding residues,” Journal of Molecular Biology, allosteric modulator of the𝛼 7 nicotinic acetylcholine receptor,” vol. 377, no.4,pp. 1216–1227, 2008. Journal of Pharmacology and Experimental Therapeutics ,vol. [32] D. R. Groebe,W.R.Gray, andS.N.Abramson, “Determinants 336, no. 2, pp. 560–574, 2011. involved in the affinity of 𝛼 -conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors,” Biochemistry,vol. 36,no. 21,pp. 6469–6474, 1997. [33] A. Franco, K. Pisarewicz, C. Moller, D. Mora, G. B. Fields, and F. Mar`ı, “Hyperhydroxylation: a new strategy for neuronal targeting by venomous marine molluscs,” Progress in molecular and subcellular biology, vol. 43, pp. 83–103, 2006. [34] M. C. V. Braga, K. Konno, F. C. V. Portaro et al., “Mass spectrometric and high performance liquid chromatography profiling of the venom of the Brazilian vermivorous mollusk Conus regius: feeding behavior and identification of one novel conotoxin,” Toxicon,vol.45, no.1,pp. 113–122,2005. [35] E. Czerwiec, D. E. Kalume, P. Roepstorff et al., “Novel 𝛾 - carboxyglutamic acid-containing peptides from the venom of Conus textile,” eTh FEBS Journal ,vol.273,no. 12,pp. 2779–2788, 2006. International Journal of Peptides Advances in International Journal of BioMed Stem Cells Virolog y Research International International Genomics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Nucleic Acids International Journal of Zoology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com The Scientific Journal of Signal Transduction World Journal Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Advances in Genetics Anatomy Biochemistry Research International Research International Microbiology Research International Bioinformatics Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Enzyme Journal of International Journal of Molecular Biology Archaea Research Evolutionary Biology International Marine Biology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal

International Journal of PeptidesHindawi Publishing Corporation

Published: Feb 27, 2013

References