Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels

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Peptides 31 (2010) 384–393

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Identification, by molecular cloning, of a novel type of I2-superfamily conotoxin precursor and two novel I2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of Me´xico Roberto Zamora-Bustillos, Manuel B. Aguilar *, Andre´s Falco´n Laboratorio de Neurofarmacologı´a Marina, Departamento de Neurobiologı´a Celular y Molecular, Instituto de Neurobiologı´a, Universidad Nacional Auto´noma de Me´xico, Campus Juriquilla, Quere´taro 76230, Mexico



Article history: Received 2 September 2009 Received in revised form 3 October 2009 Accepted 6 October 2009 Available online 15 October 2009

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, Me´xico. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I2-conotoxins and three of I2-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I2) or 23- residue signal peptide (I2-like), a 31-residue ‘‘pro’’ region (I2-like), and a 32-residue mature toxin region (I2 and I2-like). In addition, all the precursors have a 13-residue ‘‘post’’ region which contains a g-carboxylation recognition sequence that directs the gcarboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a ‘‘post’’ region has been found in precursors of I-conotoxins that also contain a ‘‘pro’’ region. The ‘‘post’’ peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that g-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I2-like-conotoxins of C. spurius are more related to I2-conotoxins than to I1- and I3-conotoxins from other species, and that they might represent a new subgroup of the I2superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes. ß 2009 Elsevier Inc. All rights reserved.

Keywords: Conoidea Conidae Conus spurius I-conotoxin cDNA cloning Conotoxin precursor

1. Introduction Cone snails or cones (superfamily Conoidea, family Conidae, genus Conus) are marine venomous predators found in tropical habitats. They produce a large number of small, highly structured peptides (‘‘conopeptides’’ if they have zero or one disulfide bridge, or ‘‘conotoxins’’ if they have two or more disulfide bonds). These peptides are essential for prey capture, and they participate in selfdefense and competitive interactions [52]. During Conus evolution, conotoxins and conopeptides have been optimized to bind ion channels and neurotransmitter receptors and transporters with very high affinity and selectivity, and most of them can distinguish between closely related target subtypes [43]. Consequently, conotoxins are useful pharmacological tools for characterizing ion channels and receptor subtypes in

* Corresponding author at: Lab. B-01, Instituto de Neurobiologı´a, Campus UNAMUAQ Juriquilla, Boulevard Juriquilla 3001, Juriquilla, Qro. 76230, Mexico. Tel.: +52 442 238 1043; fax: +52 442 238 1043. E-mail address: [email protected] (M.B. Aguilar). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.10.005

neuroscience research, and they also have considerable potential for developing new drugs [22,34]. It has been calculated that the 500–700 Conus species may express 50,000–70,000 distinct conotoxins [42]. The precursors of conotoxins generally comprise 60–90 amino acids, and they have three well-defined regions: the N-terminal signal sequence, an intervening ‘‘pro’’ region, and the C-terminal, mature toxin region [23,45]. Based mainly on the sequences of the signal peptides, these highly diverse toxins are classified into several gene superfamilies (A, C, D, I, J, L, M, O, P, S, T, and V) [24,26,29,36,42,47,48]. Usually, the members of each superfamily share a highly conserved signal sequence and a particular arrangement of Cys residues in their mature toxins [42,45,52]. Each superfamily can include several pharmacological families, which are defined by the molecular targets and how the toxins affect them [27,38,52]. The I-superfamily of conotoxins was defined by five excitatory peptides purified from the venom of C. radiatus and similar sequences cloned from the venom duct of this species [25]. This superfamily also includes toxins k-BtX from C. betulinus [15] and ViTx from C. virgo [30], which target K+ channels, and precursors

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cloned from the venom ducts of C. vexillum, C. miles, C. striatus, C. capitaneus, C. imperialis, and C. textile [3,31]. The arrangement of the eight Cys residues within the I-conotoxins (C–C–CC–CC–C–C, where ‘‘–’’ denotes one or more non-Cys residue) has been assigned framework # 11 or XI [44]. The I-superfamily has been subdivided into gene superfamilies I1 and I2 based largely on the signal peptide sequence and the presence or absence of the canonical pro-peptide region in the precursor [4]. Recently, five I1-conotoxin-like precursors with a novel signal sequence have been proposed to define the I3-superfamily [55]. Here we describe the sequences of novel I-conopeptide precursors from the venom duct of the worm-hunting cone snail Conus spurius, which were identified by cDNA cloning based on conserved elements in the 50 -untranslated regions (UTRs) of previously reported I-conotoxins. 2. Materials and methods 2.1. Biological material One specimen of C. spurius was collected near Sand Island, State of Campeche, in the Gulf of Me´xico in May, 2008. The venom duct was dissected from the living snail, immediately added to RNAlater (Qiagen, Hilden, Germany), incubated overnight in the reagent at 6 8C, and then stored at 70 8C. 2.2. Isolation of total RNA Total RNA from a half of the venom duct from a single C. spurius snail (about 10 mg) was isolated and purified using an SV Total RNA Isolation System Kit (Promega, Madison WI) according to the instruction manual. 2.3. cDNA cloning and sequencing Approximately 200 ng of total RNA from the venom duct was used to generate cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad CA) with a 30 -RACE Adapter Primer: 50 GGCCACGCGTCGACTAGTAC-(dT)17-30 (Sigma-Genosys, The Woodlands TX). For PCR amplification of cDNAs encoding I-superfamily conotoxins, forward primer ISF-1 (50 -AGAGAAGTGACGGAGATCAA-30 ) and reverse adapter primer ISR-1 (50 -GGCCACGCGTCGACTAGTAC-30 ) were employed. Primer ISF-1 was designed on the basis of conserved elements in 50 -untranslated regions (UTRs) of known I-superfamily conotoxins, whereas ISR-1 is a shorter version of the 30 -RACE Adapter Primer without the poly dT tail. PCR amplification was carried out under the following conditions: a total volume of 50 ml contained 2 ml of cDNA, 1 colorless reaction buffer, 2 mM MgCl2, each primer at 0.2 mM, 0.24 mM dNTP Mix, and 0.04 U of GoTaq1 DNA Polymerase (Promega). The PCR amplifications were performed on a Thermal cycler 2720 (Applied Biosystems, Foster City, CA) programmed for 3 min at 95 8C for initial denaturation, followed by 35 cycles of 95 8C for 30 s, 58 8C for 30 s, 72 8C for 30 s, and a final step of 72 8C for 5 min. The PCR products were analyzed by electrophoresis on 1.5% lowmelting point agarose gels (Promega). A prominent DNA band of 500 bp was excised from the gel and purified using the PureLink Quick Gel Extraction Kit (Invitrogen). The PCR products were cloned into the pGEM-T-Easy vector (Promega) via TA cloning, and transformed into electro-competent E. coli cells XL1-Blue (Stratagene, La Jolla CA). Thirty clones were randomly selected for cDNA purification and sequencing. Plasmids were purified employing the Wizard Plus SV Minipreps DNA Purification Systems (Promega), and they were sequenced in both directions with M13 primers at the Laboratorio Nacional de Geno´mica para la Biodiversidad, CINVESTAV-Irapuato (Irapuato, Gto., Me´xico), using the dideoxy


chain termination method on a 3730xl DNA Analyzer (Applied Biosystems). 2.4. Sequence analysis The nucleotide sequences yielded by the automated sequencer were first edited by removing vector and/or adapter sequences. The open reading frames (ORFs) corresponding to the precursors of the cloned cDNA sequences were predicted with the ORF Finder server [40]. Signal peptides were predicted, using the neural network method and hidden Markov models, with the SignalP 3.0 server [12,14]. Physicochemical parameters such as theoretical isoelectric point [2] and grand average of hydropathicity [32] were calculated by means of the ProtParam server [19]. For sequence comparison and phylogenetic analysis of precursors and mature toxins, we relied on the ConoServer database [8,29]. The search for protein I1-, I2- and I3-superfamily precursors yielded six, 29, and seven entries, respectively (August 25, 2009). For our analysis, we removed six entries: three lacking the signal peptide, and three containing Cys arrangements distinct from that of the I-conotoxins. The search for protein I1-, I2- and I3superfamily wild type toxins yielded 32, 29 and seven entries, respectively; we removed three entries containing a Cys arrangement distinct from that of the I-conotoxins. We kept the names annotated in this database. The ClustalW2 [33] server at EMBL-EBI [13] was employed for multiple amino acid sequence alignment and calculation of percent sequence identities, using the default settings. Multiple sequence alignments were entered into MEGA 4.0 [51], and the phylogenetic analyses were performed with the neighbor-joining method [49] using the Jones–Taylor–Thornton (JTT) matrix [28] and pair-wise deletion of gaps. Bootstrap values [16] were estimated from 2000 replicates (95% confidence) [20] with a random seed. For the phylogenetic analysis, the mature toxin regions of the I2-like-precursors from C. spurius were considered to start at the Cside of the last basic residue of the ‘‘pro’’ region preceding the first Cys residue of the C-end of the precursor; the mature regions of the I2-precursors from this species were considered to start at the Cside of the predicted signal peptides, which is in agreement with the chemically determined amino acid sequences of toxins sr11a [1] and sr11b (unpublished results); in both cases, Gly and the other residues of the ‘‘post’’ region were removed from our sequence dataset. Consensus sequences were identified by the graphical method ‘‘Sequence logos’’ [50], as implemented in the WebLogo server and employing the default essential (non-graphic) settings [11]. 2.5. Nomenclature The ten distinct precursor sequences obtained in this work are named as Sr11.1 to Sr11.10, where ‘‘Sr’’ refers to the species C. spurius, ‘‘11’’ specifies the type of Cys framework (C–C–CC–CC–C– C), and ‘‘1’’ to ‘‘10’’ are numbers arbitrarily assigned to the different precursor sequences. In order to save space, only the sequences corresponding to the ORFs are displayed. 3. Results 3.1. Identification of cDNA clones encoding I-conotoxin precursors PCR amplification of cDNA from the venom duct from C. spurius with primers F1 and R1 (based on conserved sequences in the 50 UTRs of I-conotoxins and the adapter sequence of a 30 -RACE Adapter Primer, respectively) yielded one prominent PCR product band with 500 bp, which was amplified in E. coli cells. With this


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Fig. 1. (A) Nucleotide sequences and deduced amino acid sequences of the open reading frames of the most frequent PCR products corresponding to I-superfamily cDNAs from Conus spurius. The names of the encoded mature toxins are in parentheses. The predicted ‘‘pro’’ regions are underlined, the mature toxins are shaded, and the ‘‘post’’ regions are doubly underlined. The Cys residues of the mature toxins are in bold type. (B) Comparison of the ten I-conotoxin pre-pro-peptide sequences found in C. spurius. The figures in parentheses are the numbers of clones obtained. The ‘‘pro’’ peptides are underlined, the predicted mature toxins are shaded, and the ‘‘post’’ peptides are doubly underlined. The Cys residues of the predicted mature toxins are in bold type. The asterisk before the N-terminal Gly residues of the ‘‘post’’ regions indicates probable amidation of the preceding amino acid. Identical residues (*), conserved substitutions (:), and semi-conserved substitutions (.) among all the precursors are shown along the bottom. Refer to GeneBank accession numbers GU013531, GU013532, GU013533, GU013534, GU013535, GU013536, GU013537, GU013538, GU013539, and GU013540.

strategy, we obtained 30 cDNAs coding for whole precursors of Iconotoxins. After deducing the encoded amino acid sequences (Fig. 1A), ten distinct precursors were identified: Seven precursors (Sr11.1–Sr11.7) have the structure: signal peptide-mature toxin‘‘post’’ region, characteristic of the I2-conotoxins, whereas three precursors (Sr11.8–Sr11.10) have the organization: signal peptide‘‘pro’’ region-mature toxin-‘‘post’’ region, typical of the I1- and I3conotoxins, except for the presence of the ‘‘post’’ region (Fig. 1B). These precursors contain three different mature toxins: sr11a (clones Sr11.1 and Sr11.2), sr11b (clones Sr11.3–Sr11.7), and sr11c (clones Sr11.8–Sr11.10) (Fig. 1A). Given that precursors Sr11.8– Sr11.10 share two features with I2-conotoxins (similar signal sequence and the ‘‘post’’ region) but only one (the ‘‘pro’’ region) with the I1- and I3-conotoxins (Fig. 2), we will tentatively refer to them as the I2-like precursors from C. spurius. In the case of the I2-precursors, the signal peptides, the mature toxins, and the ‘‘post’’ regions all have 26, 32, and 13 residues, respectively. In the case of the I2-like-precursors the signal

peptides, the ‘‘pro’’ regions, the mature toxins, and the ‘‘post’’ regions all have 23, 31, 32 and 13 residues, respectively. The mature toxins of all the precursors have the expected Cys pattern that characterizes the I-conotoxins (C–C–CC–CC–C–C) [25] (Fig. 1B). In all cases, the ‘‘post’’ regions are followed by single TGA stop codons (Fig. 1A). Generation of the mature toxins sr11a and sr11b from the I2precursors requires proteolytic cleavage at the C-side of the Ala residue in the sequence-TNA located at the C-terminal end of the signal peptide, whereas in the case of the I2-like-precursors, biosynthesis of the mature toxin sr11c involves proteolytic cleavage at the C-side of the Arg residue in the sequence-LSR located at the C-terminal end of the ‘‘pro’’ region of the precursors (Fig. 1B). According to their cDNA sequences, the three distinct mature toxins (sr11a, sr11b, and sr11c) derived from all these precursors should have an amidated Pro at their C-termini due to standard processing of the Gly residue at the C-end of the mature toxin regions [18,54] from -FGPG to -FGP-nh2 (clones

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Fig. 2. Comparison of the three most frequent I-conotoxin precursors from C. spurius with the other known I-conotoxin precursors. Identical residues (*), conserved substitutions (:), and semi-conserved substitutions (.) among all the precursors are shown along the bottom. Residues in the multiple sequence alignment (by ClustalW2) identical to the consensus sequence (the most frequent residues in the ‘‘sequence logo’’) (by WebLogo) are highlighted in black background. The figures to the right are the number of amino acid residues that comprise each precursor. The names of the precursors are those annotated in the ConoServer database [28]. The first one or two letters refer to the Conus species. Piscivorous species: Sx, C. striolatus; S, C. striatus; R, C. radiatus. Molluscivorous species: Ep, C. episcopatus; Tx, C. textile. Vermivorous species: Sr, C. spurius; Be, Bt, C. betulinus; Lt, C. litteratus; Eb, C. eburneus; Im, C. imperialis; Em, C. emaciatus; Mi, C. miles; Cp, C. capitaneus; Vx, C. vexillum; Vi, C. virgo (ViTx) or C. vitulinus (Vi11.3 and Vi11.7); Ar, C. arenatus; Ca, C. caracteristicus; Pu, C. pulicarius.

Sr11.1–Sr11.7) and -LVPG to -LVP-nh2 (clones Sr11.8–Sr11.10) (Fig. 1B). This processing has been confirmed in the case of toxin sr11a [1], and it implies that the removal of the ‘‘post’’ region, which contains a g-carboxylation recognition site (g-CRS) [3], is a prerequisite for amidation to occur. It should be noted that there are two precursors (differing at positions four and nine of the signal peptide) encoding toxin sr11a, whereas there are five precursors (differing at positions four, nine and 21 of the signal peptide, and at position four of the ‘‘post’’ region) that code for toxin sr11b, and three precursors (differing at positions 15 of the ‘‘pro’’ region, and at position five of the ‘‘post’’ region) that encode toxin sr11c (Fig. 1B). The origin of these differences is not clear; given that all the clones were obtained from a single specimen, they may be due to allelic variation or one event of gene duplication and mutation in the case of the two precursors of sr11a; in the case of sr11c, two events of gene duplication and mutation, or one gene duplication and allelic

variation might explain the presence of three precursors; the five precursors of sr11b might imply up to four events of gene duplication and mutation, or two gene duplications and allelic variations. Since all the clones were obtained with the primer pair F1-R1 (based on conserved elements in the I-conotoxin 50 -UTRs that are very close to the initiation codon, and the adapter sequence of the universal 30 -RACE Adapter Primer, respectively), we have no additional information about the 50 -UTRs of these precursors; however, we found short (160 bp) 30 -UTRs followed by poly-A tails of variable length (16–92 bp) (data not shown). 3.2. Sequence characterization of the I-conotoxin precursors The signal peptides of the I-conotoxin precursors of C. spurius are highly conserved, with the eight distinct signal peptides having 18 identical residues (the signal peptides of the three I2-like


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precursors are identical) (Fig. 1B). However, this region is longer in the I2-precursors (26 residues) than in the I2-like- (23 residues), and clear differences are observed at the C-terminus of this region, starting at position 21: -(V/F)VLTNA (I2-) vs. -SPG (I2-like-) (Fig. 1B); there are other differences at positions four and nine, but in these cases the residues present in the three I2-likeprecursors (Arg and Gly, respectively) also occur in four (Arg) and three (Gly) I2-precursors (Fig. 1B). The ‘‘pro’’ regions of the three I2like-precursors all have 31 residues, and they are almost absolutely conserved; one of them differs from the others only at position 15 (Fig. 1B). In both the I2- and the I2-like-precursors, the ‘‘post’’ regions comprise 13 amino acid residues, of which eight are conserved (Fig. 1B). The three different mature I-conotoxins described in this work have the same number of amino acids between the Cys residues, including three amino acids between the two pairs of adjacent Cys residues (Fig. 1B); this latter feature has been found in all I2- and I3conotoxins and in most group-B I1-conotoxins described thus far [3,6,15,25,30,31,55]. However, compared with toxin sr11a purified from the venom of C. spurius [1], the novel toxins sr11b and sr11c have many amino acid substitutions and only 13 (including eight Cys) residues are shared by the three, 32-residue mature toxins (Fig. 1B). The Cys codons of the mature toxins from C. spurius are highly conserved: in the thirty clones coding for the ten distinct precursors, Cys-1, Cys-3, Cys-5, and Cys-7 are encoded by the TGC triplet, whereas Cys-4, Cys-6 and Cys-8 are coded by the TGT codon; interestingly Cys-2 is encoded by TGC in the precursors of sr11a (Sr11.1–Sr11.2) and sr11c (Sr11.8–Sr11-10), but it is coded by TGT in those of sr11b (Sr11.3–Sr11.7) (Fig. 1A). 4. Discussion In this work, we found ten different precursors encoding three distinct mature I-conotoxins from the vermivorous snail C. spurius collected off the coast of Campeche, Me´xico. Two of these toxins are novel (sr11b and sr11c), whereas one (sr11a) was purified and chemically sequenced from the venom of specimens collected off the coast of Yucata´n, Me´xico [1]. Given that we conducted this study from a single Conus spurius specimen, we cannot conclude that all the individuals belonging to this species express the particular conotoxins we found. The I-conotoxins have been subdivided into the gene superfamilies I1 and I2 based on the sequence of the signal peptide, the presence (I1) or absence (I2) of the ‘‘pro’’ region, and the absence (I1) or presence (I2) of the ‘‘post’’ region [4,39]; five I1-like precursors with a novel signal sequence have been recently proposed to define the I3-superfamily [55]. Here, we found seven precursors with the typical structure of I2-conotoxins, but we also identified three precursors having the organization that characterizes the I1- and the I3-superfamilies except that they also contain a ‘‘post’’ region, which had only been found in the I2 superfamily (Figs. 1B and 2). Thus, C. spurius is the first species in which ‘‘post’’ peptides have been identified in I1/I3-like precursors, which, as explained in Section 3.1, we have tentatively named as I2like precursors. In order to gain insight into the relationship between the I2 and the I2-like precursors of C. spurius, and between the latter and the I1-, I2-, and I3-superfamilies, we conducted a phylogenetic analysis of the precursors reported to date. For this analysis, we only included the most frequent precursors from C. spurius, Sr11.1, Sr11.4, and Sr11.9 (Fig. 1), each encoding one of the three distinct mature toxins, sr11a, sr11b, and sr11c, respectively. The overall mean p-distance (calculated by MEGA 4.0) of the 39-sequence ClustalW2 multiple alignment (Fig. 2) is 0.564 (pair-wise deletion), which is within the acceptable range (
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