Peptides 56 (2014) 77–83

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Soluble expression, purification and functional identification of the framework XV conotoxins derived from different Conus species Yun Wu a,1 , Lei Wang a,1 , Maojun Zhou a,1 , Xiuhua Jiang a , Xiaoyan Zhu a , Yu Chen a , Shaonan Luo a , Yuwen You a , Zhenghua Ren a , Anlong Xu a,b,∗ a State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, National Engineering Research Center of South China Sea Marine Biotechnology, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China b Beijing University of Chinese Medicine, 11 Dong Shan Huan Road, Beijing 100029, People’s Republic of China

a r t i c l e

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Article history: Received 14 August 2013 Received in revised form 14 March 2014 Accepted 17 March 2014 Available online 1 April 2014 Keywords: Bio-activity Conotoxin Cysteine framework Diversity Recombinant expression

a b s t r a c t The conotoxin cysteine framework XV (-C-C-CC-C-C-C-C-), which was named Lt15a, was firstly identified from the cDNA library of Conus litteratus. After that, 18 new framework XV conotoxin sequences were cloned from nine Conus species. Like other conopeptides, the XV-conotoxins have the conserved signal peptide and propeptide, and there are also some conserved residues in their mature peptide. All the framework XV conotoxins were apparently converged into two branches, because of the indel and point mutations occurred in their mature peptides. By fused with thioredoxin and 6 × His tag, six XV-conotoxins were successfully expressed in Escherichia coli and purified. Different framework XV conotoxins have distinct biological activities on mice and frogs, and that may be related to the diversity of the toxin sequences. All the six XV-conotoxins had no obvious effects on the sodium currents of DRG neuron cells of Sprague–Dawley (SD) rats. The identification of this framework of conotoxins enriches our understanding of the structural and functional diversity of conotoxin. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Conotoxins are regarded as the most potential drug leads and diversified molecules in the world. As we know, there are about 500–700 Conus species around the world and the venom of each species contains 100–2000 small highly structured venom peptides [2,13,14]. It is estimated that there are about 500,000 natural conotoxins in the world. Conotoxins are also functionally versatile molecules by selectively targeting different subtypes of neurotransmitter receptors or voltage-gated ion channels [21]. Therefore, conotoxins are usually used as molecular tools in neuroscience research and therapeutic drugs in the clinic [15]. Conotoxins are initially translated as prepropeptide precursors which composed of three regions: a highly conserved signal peptide at the N-terminal end, an intermediate pro-region and the hypervaried mature toxin with conserved cysteine arrangement at C-terminal end [14]. The biologically active conotoxin is produced

∗ Corresponding author at: Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, 135 Xinggangxi Road, Guangzhou 510275, People’s Republic of China. Tel.: +86 20 3933 2990; fax: +86 20 3933 2950. E-mail address: [email protected] (A. Xu). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.peptides.2014.03.018 0196-9781/© 2014 Elsevier Inc. All rights reserved.

by proteolytic cleavage from the prepropeptide, an essential posttranslational step in conotoxin maturation [4]. The mature toxins are a class of small disulfide-rich peptides containing 12–50 amino acids with a high frequency of posttranslational modifications. And the disulfide bridges make the peptides maintain a stable and conserved tertiary structure. It was reported that the hypervaried mature peptide and the posttranslational modifications may result in the molecular diversity of conotoxins [9]. At present, all the conotoxins can be divided into 26 superfamilies based on their conserved signal peptide, or into 26 cysteine frameworks based on the cysteine number, arrangement and disulfide connectivity of the mature peptide [10]. Previously, conotoxins from the same superfamily usually share a characterized cysteine arrangement in their mature peptides and each cysteine arrangement usually corresponds to the specific disulfide connectivity [6,14]. However, since more conotoxins were found afterwards, some new superfamilies and frameworks were reported, this rule has been severely broken. As M superfamily, a superfamily may contain multiple frameworks [25,28]; while the same framework may also exist in more than one superfamily, like framework VI/VII [10,25]. In this paper, we reported the cysteine framework XV (-C-C-CCC-C-C-C-) conotoxins, which were first identified in the venom of

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Fig. 1. The full-length cDNA and putative amino acid sequence of Lt15.1. The signal peptide sequence is in boldface, propeptide sequence is in italics, and mature toxin sequence is underlined. The coding sequence is in uppercase while untranslated region in lowercase letters. Primer1 and Primer2 were designed according to the sequences indicated by the arrow.

C. litteratus at 2006 [18], and were classified into O2 superfamily according to the signal peptide. We described the cDNA cloning, identification, recombinant expression, functional assay and evolutional study of these conotoxins. These results could expand our understanding of the diversity and function of conotoxins. 2. Materials and methods 2.1. Sample collection and cDNA library construction Specimens of Conus litteratus, Conus betulinus, Conus emaciatus, Conus captitaneus, Conus caracteristicus, Conus rattus, Conus vitulinus, Conus varius, Conus vexillum and Conus miles were collected from reef flats in West Island near Sanya, China. Venom ducts of cone snails were dissected and immediately preserved in liquid nitrogen respectively. Total RNAs were isolated from the homogenized venom duct by using TRIZOL (Invitrogen, Carlsbad, USA) according to the previously described steps [18]. 2.2. Identification of Lt15.1 A cDNA library of the venom duct from C. litteratus was constructed and sequenced as described previously [18]. The fulllength cDNA clone of conotoxin gene was isolated based on the sequences analysis with the BLAST algorithm available at the National Center for Biotechnology Information (NCBI). Five ESTs were found encoding a conotoxin with the cysteine arrangement (-C-C-CC-C-C-C-C-). The full-length cDNA clone was named Lt15.1 and this scaffold was named framework XV (Fig. 1).

Conus species were used respectively as the templates to screen the cDNAs encoding conotoxins with XV scaffold (-C-C-CC-C-CC-C-) as Lt15.1. Because of the highly conserved 5 UTR, signal peptide and 3 UTR of the conotoxins in the same superfamily, two primers, 5 primer (Primer1, 5 -CCTTCATCATGGAGAAACTTAC3 ) designed corresponding to the boundary region between 5 UTR and the signal region of Lt15.1, 3 primer (Primer2, 5 GGTTGTGGCTCTGATGTATTG-3 ) designed according to the 3 UTR region around the stop codon of Lt15.1, were used in the PCR reactions to amplify the XV conotoxin sequences (Fig. 1). The PCR conditions were: 95 ◦ C for 3 min, followed by 30 cycles of 95 ◦ C for 45 s, 55 ◦ C for 45 s and 72 ◦ C for 1 min, the final step was 72 ◦ C for 7 min. The PCR fragments were cloned into pGEM-T Easy Vector (Promega Inc., USA). Ligated vectors were transformed to Escherichia coli DH5␣ competent cells by heat shock. Ampicillin (100 mg/mL) was used for antibiotic resistance selection. Blue/white colony screening was done on LB agar plates using 80 mg/mL X-gal and 0.5 mL/L of 100 mM IPTG to select the positive colonies. The clones were subjected to DNA sequencing (ABI 3730 automatic sequencer, Applied Biosystems, USA) using T7 forward and SP6 reverse primer. 2.4. Sequencing analysis The signal peptide sequences and cleavage sites of conotoxins were predicted using the ConoPrec tool in ConoServer (http://www.conoserver.org) and the SignalP algorithm (http:// www.cbs.dtu.dk/services/SignalP). Nucleotide and amino acid multiple alignments were generated using the ClustalX or GENEDOC, and the alignments were refined manually. Phylogenetic trees were constructed using the neighbor-joining method with MEGA5.

2.3. Cloning and sequencing of cDNAs encoding framework XV conotoxins

2.5. Recombinant expression of framework XV conotoxins

In order to investigate the expression of framework XV conotoxins in different Conus species, the cDNA libraries of the nine

In order to express the XV-conotoxins soluble in E. coli, the fusion expressed vector pTRX-XV-conotoxin was constructed according to

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Fig. 2. Schematic representation of the expression vector pTRX-XV-conotoxin.

the procedure of previously described [22] (Table 1). Using this vector, the conotoxin was expressed in E. coli with thioredoxin (TRX) to increase the solubility. The 6-His tag contained by the vector between TRX and conotoxin gene makes the purification of fusion protein easily and simply using Ni2+ affinity chromatography. Protease EK cleavage site was designed between TRX and conotoxin (Fig. 2). The fusion expression vector was transformed into E. coli BL21(DE3) to obtain the engineering strain pTRX-XV-BL21(DE3). The engineering strain was shake-cultured in LB lipid medium with 100 mg/mL ampicillin at 37 ◦ C. Until OD600 comes to 0.6, IPTG was added to a final concentration of 0.1 mM and 20% glucose was added to a final concentration of 0.2%. Then after 10–12 h expression at 18 ◦ C, the cultures were collected by centrifugation. The cell pellet were resuspended in extraction/sonication buffer (500 mM NaCl, 50 mM Tris–HCl, pH 8.0) and extracted by sonication. The cell debris was sedimented by centrifugation and the supernatant was used for purification. The supernatant of the total cell extract was loaded onto Ni2+ chelating sepharose fast flow column (GE Healthcare, USA), which was charged with Ni2+ ions and pre-equilibrated by sonication buffer. The binding protein was eluted by a series of wash buffer (50 mM Tris–HCl, pH 8.0, 500 mM NaCl with gradiently increased concentrations of imidazole). All the elution fractions were collected and analyzed by SDS-PAGE. Fractions containing fusion proteins were applied to a Sephadex G-25 fine column (GE Healthcare, USA) and the buffer was changed to cleavage buffer (50 mM Tris, 200 mM NaCl, and 5 mM CaCl2 , pH 8.0). Fusion protein was cleaved with Protease EK at room temperature for 16 h. The cleavage product was loaded onto a Sephadex G50 Fine column (GE Healthcare, USA) pre-equilibrated with 50 mM NH4 HCO3 . Each elution fraction was analyzed by SDS-PAGE. Fractions containing the target proteins were pooled and condensed by lyophilization (CHRIST BETA 1–8K, Germany). The conotoxins were further purified through a C18 reverse phase column (GL Sciences Inc., Japan) on Waters 600E HPLC system (Waters, USA), and then freeze-drying again [19,23]. 2.6. Biological activity assays The lyophilized peptides were dissolved in normal saline solution. Kunming mice (two weeks old) were injected intracranial (i.c.) with 10 ␮L of the peptide solutions. Control animals were similarly injected with normal saline solution. 2.7. Effects of framework XV conotoxins on the neuromuscular transmission and DRG neurons The mode used to analyze the effect of conotoxin on the neuromuscular transmission was according to the method of Kerr and Yoshikami [11]. The live frogs were stored in the refrigerator at 4 ◦ C to keep their metabolism down. The probe was inserted into the cranial vault and severed the brain and spinal cord. Then move the probe to destroy the brain and isolate the gastrocnemius muscle and the sciatic nerve quickly and gently. The nerve–gastrocnemius muscle preparation was placed in the chamber filled with Ringer’s solution. The sciatic nerve was connected

to the transducer (Chengdu TME Technology Co., Ltd., China) by a thread and the stimulating electrode was placed on the muscle. The ischiatic nerves are stimulated with a wave pulse of 0.5 ms, 0.5 Hz, 5 v and 4.00 s/div. Test time was 60 min, during which we detected the contractile force of the muscle every 5 min. The conotoxins were dissolved in normal saline solution and added to the preparation by the absorbent cotton. DRG neurons were acutely dissociated from Sprague–Dawley (SD) rats (30-day-old) and maintained in short-term primary culture [24]. Briefly, SD rats of either sex were killed by decapitation and the DRG were isolated quickly and cut into small pieces, then digested with collagenase followed by trypsin. Trypsin inhibitor (1.5 mg/mL, type II-S) was added to inactivate enzyme and the DRG cells were suspended into DMEM and incubated in CO2 incubator at 37 ◦ C for 3 h before patch-clamp experiment. Whole cell recordings of sodium currents were performed using the method as described previously [12,19]. The intracellular buffer contained: 135 mM CsF, 10 mM NaCl, 5 mM N-2-HEPES, 5 mM EGTA, 2 mM Mg-ATP, pH 7.0 adjusted with 1 M CsOH. And the intracellular buffer contained: 30 mM NaCl, 5 mM CsCl, 25 mM d-glucose, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM HEPES, 20 mM TEA chloride, 70 mM TMA chloride, 0.01 mM LaCl, pH 7.4 adjusted with 1 M TEA hydroxide. Whole cell recording was carried out at 25 ◦ C with a patch/whole cell clamp Amplifier CEZ2400 amplifier (Nihon Kohden, Japan). Stimulation and recording were controlled by a pClamp data acquisition system (Axon Instruments, UK). In all voltage-clamp experiments, the membrane potential was held at −80 mV. 3. Results 3.1. Discovery of the framework XV conotoxin The cDNA library of the venom duct from Conus litteratus was constructed successfully and about 42 novel conotoxins were identified by EST sequencing and BLAST analysis [1]. Most of them belong to previously identified superfamilies, such as A, O, T, S, and M superfamily. One small group of clones contain an open reading frame (ORF) of 261 bps encoding an 86 amino acids conotoxin precursor. This precursor was consisted of three parts: signal peptide, propeptide and mature peptide. The cleavage site for the signal peptide was predicted to be between Ser23 and Asp24. The characteristic basic residue Arg49 may act as the processing cleavage site. The mature peptide is consisted of 37 amino acid residues and eight of them are cysteines (Fig. 1). This cDNA clone was named Lt15.1, and “Lt” represents the abbreviation of species C. litteratus, “15” represents the cysteine scaffold (C-C-CC-C-C-C-C) and “1” indicates the number of clone in our experiment. The cysteine pattern was supposed to be designated as framework XV. This class of clones occurred in cDNA library of C. litteratus venom duct at a low frequency. Only five out of 576 ESTs encoded this peptide [18]. The signal peptide region of Lt15a exhibits homology to that of O2 superfamily conotoxins. However, the cysteine arrangement in the mature peptide of Lt15a was distinct with that of O2-conotoxins (C-C-CC-C-C). On the other hand, the cysteine scaffold of Lt15.1 was similar to that of Vi15a belonging to V superfamily, which was obtained from the venom of C. virgo [16], while the precursors of them have totally different signal peptide sequences (Fig. 3).

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Fig. 3. Amino acid sequences alignment of 14 O2-VI/VII-conotoxins, 19 O2-XV-conotoxins and two V superfamily conotoxins. The proteolytic cleavage sites of these peptides were indicated by the arrow.

3.2. Molecular diversity and distribution of framework XV conotoxins To investigate whether there are other conotoxins belonging to framework XV in other Conus species, we screened the venom duct cDNA libraries of other nine species (C. betulinus, C. emaciatus, C. captitaneus, C. caracteristicus, C. rattus, C. vitulinus, C. varius, C. vexillum and C. miles) using the methods of RT-PCR. As a result, 18 conotoxin sequences closely relating to Lt15a were identified from these species. The putative precursors were named Bt15a, Ca15a, Cap15a, Cap15b, Ec15a, Lv15a, Ml15a, Ml15b, Rt15a, Rt15b, Rt15c, S15a, Tx15a, Vr15a, Vr15b, Vt15a, Vt15b and Vx15a respectively (GenBank accession numbers DQ345376, DQ512965, DQ512966, GQ414737–GQ414743, JX293459–JX293467) (Fig. 3). The comparison of 18 precursor sequences of novel XVconotoxins indicated that they have highly conserved signal peptide with only few substitutions to Lt15.1. Based on the similar signal peptide to O2 superfamily, these peptides was classified into O2 superfamily temporarily. The proteolysis of these precursors was all predicted behind the characteristic PR/HR motif to release the mature peptides. All mature peptides have the same cysteine arrangement X1-C-X6-C-X5-CC-X4-C-X1-C-X5-C-X4/5-CX2/3, where X (numeral) represents the number of amino acids between two adjacent cysteines. In addition to the conserved cysteine residues, CKC motif is also conserved among the mature peptide sequences of XV-conotoxins except Tx15a. It is revealed that this class of novel conotoxin widely existed at least in the gene level in Conus species. 3.3. Recombinant expression of framework XV conotoxins The expression and purification of the recombinant framework XV conotoxins (Lt15a, Bt15a, Ec15a, Cap15a, Vr15a and Vx15a)

were carried out as the method described above. The fusion proteins were eluted from the Ni2+ chelating sepharose column with 200 mM imidazole in the sonication buffer. After cleaved with protease EK at room temperature for 16 h, the fusion proteins were purified by Sephadex G-50 column and the toxins was lyophilized for further verification. To analyze the characterization of recombinant framework XV conotoxins, the lyophilized protein was resolved in ddH2 O and analyzed using reverse-phase HPLC, and only a single peak was eluted. This peak was further analyzed using MALDI-TOF–TOF–MS. The molecular weights of these conotoxins were shown in Table 1. The SDS-PAGE, HPLC profiles and MS data were shown in supplementary data. In average, 5 mg of recombinant framework XV conotoxins were obtained from l L culture. 3.4. Biological activity To assess the effects of recombinant framework XV conotoxins on mice, two-week-old Kunming mice were injected intracranially with XV-conotoxins at a dose of 1 nmol/g (Table 2A). When Lt15a was injected, the mice turned into coma status with body crouched. Bt15a exhibited excitatory effect on mice. The mice tumbled, and then turned into seizure symptom. Ec15a elicited hyperactive in mice, followed by slight lethargy for 20 min. Vx15a also elicited excitatory effect on mice. The tested mice moved fast and jumped within 5 min. All the tested mice gradually recovered from the inhibitory impacts of XV-conotoxins within 60 min. Cap15a and Vr15a had no detectable effects when injected into mice. Thus, different XV-conotoxins showed different physiology activities. Framework XV conotoxins also had different effects on the neuromuscular transmission of frogs (Table 2B). Ec15a can inhibit the muscle contraction to 50% within 5 min, completely inhibited within 45 min. The muscle contraction recovered to 60% after washed out. Vx15a caused a muscle arrhythmic tremor which

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Table 1 The six framework XV conotoxins for the recombinant expression. Conotoxin

Mature peptide

Conus species

Molecular weight

GenBank number

Lt15a Bt15a Ec15a Cap15a Vx15a Vr15a

ECTTKHRRCEKDEECCPNLECKCLTSPDCQSGYKCKP QCTPRNQRCEGDAECCPNLVCKCFTRPDCQSGYKCDTS QCTPKDAPCDDNNQCCSGLECKCFNMPDCQSGSTCRV QCTQQGYGCDETEECCSNLSCKCSGSPLCTSSYCRP QCIPQNVSCEEDDECCSNLECKCTSAPDCNFPKCRA QCTGAGFECEETPECCPNLTCKCSGSPLCARYRCKP

C. litteratus C. betulinus C. emaciatus C. captitaneus C. vexillum C. varius

4256.707 4250.488 3992.243 3862.329 3974.249 3876.494

DQ345376 DQ512965 DQ512966 GQ414740 GQ414742 JX293464

Table 2 Biological assays of framework XV conotoxins. Sample

Dose (nmol/g)

A. Effects on mice Lt15a 1 1 Bt15a Ec15a

1

Cap15a Vx15a

1 1

Vr15a

1

Sample

Dose (␮mol/L)

Symptoms (n = 5)

Coma state, crouched. Recovered in 1 h. Tumbled, then seizure. Recovered in 1 h. Hyperactive after injection for 5 min, followed by slight lethargy for 20 minutes. Recovered in 30–60 min. No detectable effects. Move fast and jump in 5 min. Recovered in 30 min. No detectable effects. Sample amounts (␮L)

Symptoms (n = 3)

B. Effects on the neuromuscular transmission of frogs 10 100 No detectable effects. Lt15a 10 100 No detectable effects. Bt15a 10 100 Inhibited muscle contraction to 50% in Ec15a 5 min, completely inhibited in 45 min. Recovered to 60% after washed out. Cap15a 10 100 No detectable effects. 10 100 Caused a muscle arrhythmic tremor Vx15a which retained 20 min, and disappeared after washed out. 10 100 No detectable effects. Vr15a

did not affect the contraction, and could retain for 20 min. The arrhythmic tremor disappeared after washed out. The other four conotoxins had no detectable effects on muscle contraction. The effects of recombinant framework XV conotoxins on sodium currents in dissociated SD rats DRG neurons were characterized. Unfortunately, all the six conotoxins had no obvious effects on the sodium currents (n = 3). 4. Discussion Conotoxins have long been known for their high sequence, structure and bioactivity diversity. At present, there are 2354 conotoxin nucleotides and 5448 proteins reported by Conoserver, a famous database of conotoxin [10]. Framework XV conotoxins had been first isolated from the cDNA library of venom duct of C. litteratus at 2006 [18]. We cloned 18 new framework XV sequences from nine Conus species with three different feeding types at this work. This is confirmed that framework XV conotoxins widely existed in cone snails. According to the nomenclature, XV-conotoxins were classified into O2 superfamily because of the high similarity between their signal peptides. But the cysteine arrangement of framework XV was the same with V superfamily. We made an alignment of conotoxins from O2 superfamily, framework XV and V superfamily (Fig. 3). XV-conotoxins are obviously longer than the other two. And the proteolytic cleavage sites of signal peptide and propeptide are different among the three groups. The signal peptide of framework XV is similar to O2 superfamily, but they also have some differences. The signal peptide of framework XV has 23 residues, while

O2 superfamily only has 19 residues. And there are more differences between their propeptides, including the sequence length and base composition. The gene structures of the framework XV and O2 superfamily conotoxins are also different [25]. Thus the two frameworks belonging to O2 superfamily were not so close to each other. It is not the same case with A superfamily, of which also included different cysteine frameworks (framework I, II, VI), but have highly conserved signal peptides and the same proteolytic cleavage sites [20]. V superfamily has the same cysteine scaffold with framework XV. This is like the framework VI/VII in O1, O2 and O3 superfamilies [5]; the framework XI in I1, I2 and I3 superfamilies [8,27]; or the framework XIV in J and L superfamilies [7,17]. All of them are different superfamilies with different signal peptide but the same cysteine scaffold. So maybe framework XV conotoxins can define a new superfamily, which can be named “V2”, referring to the nomenclature of O and I superfamilies, rather than classified into O2 superfamily. It is worth noting that the XV-conotoxins not only have the conserved signal peptide and propeptide, but also have some conserved sites in mature peptide. We constructed a phylogenetic tree using the 19 framework XV conotoxin sequences by the N–J method. All the toxins were apparently converged into two clades (Fig. 4). There are five loops within the mature region of XVconotoxins. The toxins in clade1 have five residues in loop5, while there are only four residues in clade2 (Fig. 3). Cysteine codon usage (TGC or TGT) analysis of framework XV conotoxins reveals codon conservation in specific cysteine positions (Fig. 5). The preferred codon for Cys1, Cys4, Cys6 and Cys7 in these conotoxins is TGC, whereas Cys2, Cys3 and Cys8 are preferentially encoded by TGT, and Cys5 shows a less-biased ratio of TGC/TGT. In order to check if position-specific codon conservation applies to other residues, we analyzed codon usages of nine conserved amino acids (Thr52, Glu59, Glu60, Glu63, Pro66, Asn67, Leu68, Lys71 and Pro76) that located in the mature peptide (Fig. 5). Most of them are preferentially encoded by a specific codon, except the two pralines (Pro66 and Pro76), which show a less-biased ratio of two codons like Cys5. Then we carefully analyzed the codon usage of Cys5, an interesting phenomenon was discovered. That all the framework XV conotoxins in clade1 encoded Cys5 by TGT, and all the clade2 toxins used TGC. The two prolines (Pro66 and Pro76) also followed this rule. Pro66 is encoded by CCA in clade1 toxins, and encoded by CCG in clade2. Pro76 is encoded by CCT in clade1, and CCA in clade2. Such as Lt15a, which belonged to clade1, Cys5 is encoded by TGT, Pro66 CCA, and Pro76 CCT (Fig. 1). The differences of codon usages of these residues correspond to the two phylogenetic clades of XV-conotoxins. Thus after the two clades separated long before, the toxins retained specific codon usages of some conserved amino acids, while also formed their own special codon usages of some other sites. As mentioned above, the number of residues in loop5 is different between the two clades. Considering the conserved residues and codon usages within the mature region, a triple-nucleotides insertion/deletion (indel) should be occurred in loop5. And point mutations can be found in all the five loops of the mature peptide. It can be assumed that all the framework XV conotoxins were probably originated from the same ancestral gene of the Conus ancestors.

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Fig. 4. Phylogeny reconstruction of 19 framework XV conotoxins using neighbor-joining method with MEGA5. Bootstrap scores for branches are shown at nodes.

And a triple-nucleotides indel occurred in the ancestral gene, thus a new conotoxin gene was generated. These two genes would then undergo various point mutations during their following evolution, while some sites still maintained a strict conservation. And a new series of homologous toxin genes would be generated. Finally a large number of new conotoxins have come out. In order to obtain the pure conotoxins for biological activity research, we chose six framework XV conotoxins to recombinant expression. TRX was used as the protein fusion partner for

maximizing the correct folding and solubility of recombinant conotoxins. And the low temperature (∼20 ◦ C) and low IPTG concentration (0.1 mM) also can greatly improve the solubility of recombinant protein. Two O-conotoxins Lt7a and Lt6c also had been producted by recombinant expression, which used the same vector and similar conditions. Both of them were proved blocking the voltagesensitive sodium channels [19,23]. Omega-conotoxin MVIIA has been expressed through the fusion of glutathione-S-transferase (GST), and possessed strong analgesic activity [26]. Another

Fig. 5. Position-specific codon conservation in the mature peptide of framework XV conotoxins. Codon usages of eight cysteines and other nine conserved residues were analyzed. Amino acids refer to the sequence of Ca15a.

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O1-conotoxin TxVIA from Conus textile, was producted as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host, also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae [3]. The biological activities of the six framework XV conotoxins are different to each other. This may be related to the diversity of the toxin sequences. Lt15a, Ec15a, Bt15a and Vx15a can cause physiological responses on mice, moreover, Ec15a and Vx15a have effects on the neuromuscular transmission of frogs. In order to identify the target of framework XV conotoxins, electrophysiological studies were carried out. But none of them showed obvious effects on the currents of sodium channels. The physiological target of XV-conotoxins is still to be identified. Further studies of the conotoxin-target interaction at the molecular level would be needed to give more information about the mechanisms of their bioactivities. In summary, we have described the cDNA cloning, identification, recombinant expression, functional assay and evolutional study of a novel cysteine framework conotoxin, framework XV conotoxins. These toxins probably derived from a common ancestor, but later evolved into two branches. Different XV conotoxins have different biological activities on mice and frogs, but their physiological target is still unknown. The identification of this new framework of conotoxins expands our understanding of the diversity of conotoxin, and lays a foundation for further studies. Ethics statement The Conus specimens were collected from public water bodies in China, and no specific permissions were required. We have confirmed that our studies did not involve endangered or protected species. All animal procedures were carried out according to the approved protocol of the Institutional Animal Care and Use Committee at the Sun Yat-sen University. Acknowledgments This work was supported by the State National High-Tech Development Program (863 Program) (2008AA09Z401), National Science and Technology Support Program (2012BAD117B02) and Shenzhen Science and Technology Industry and Information Committee (SW201110155). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.peptides.2014.03.018. References [1] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.

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