O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents

Abstract Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3’ UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.


Background
Conotoxins are small marine peptides typically comprising 10-50 amino acids and 1-5 disulfide bridges, which are produced by marine mollusks known as cone snails [1]. There are over 700 species in the genera of Conus [2,3]. Each Conus species may contain up to 1000 conotoxins, which makes the library of bioactive conotoxin peptides very large [4,5], perhaps over 140,000 different peptides in total. Conotoxins act potently and selectively on a lot of membrane receptors and ion channels, thus showing great potential as neuropharmacology tools and therapeutic candidates, and one calcium channel inhibitor, ω-conotoxin MVIIA (Prialt TM ), was developed as a drug for treating neuropathic pain [6][7][8].
According to the similarities of signal region sequences, conotoxins can be divided into 30 gene superfamilies [9]. The O1 superfamily which was first named in 1995, mainly contains the cysteine framework VI/VII (-C-C-CC-C-C-) and can be divided into the κ, δ, ω and μO pharmacological families [10]. At present, the δ and ω families have been found only in the O1 gene superfamily, which is the most abundant superfamily and contains more than 700 conotoxin sequences [11]. κ-PVIIA targeting voltage-gated potassium channels is the only O1-conotoxin belonging to the κ family [12]. O1-conotoxins targeting voltage-gated sodium channels belong to the δ and μO families [13]. The μO family conotoxins are sodium channel antagonists, while the δ family conotoxins are sodium channel agonists that delay the inactivation of sodium channels. The ω family conotoxins, such as ω-conotoxin MVIIA (Prialt TM ), are blockers of voltage-gated calcium channels [14], suggesting that obtaining novel O1 superfamily conotoxins is important for the development of new analgesic drugs.
In a previous study, we presented the gene structures of nine conotoxin superfamilies, which have similar architectures with two introns and three exons [15]. For conotoxins in the same superfamily, the beginning and ending portions of the intronic sequences are conserved [16,17], so it becomes possible to clone novel conotoxin sequences from the genome using primers corresponding to the 3' UTR and 3' end of the intron. In this study, we report a novel O1-conotoxin Tx6.7, which was directly cloned from the genomic DNA of Conus textile. The effects of Tx6.7 were tested on sodium, potassium and calcium currents in DRG neurons and ion channel subtypes (hNa V 1.3, hNa V 1.4, hNa V 1.7, hNa V 1.8, hCa V 1.2 and hCa V 2.2) in HEK293 cells or ND7/23 cells. Tx6.7 was also tested for cytotoxic activities on ND7/23 cells and analgesic activities by the mouse hot plate assay.

Specimen collection and genomic DNA cloning
Specimens of Conus textile were collected from reef flats on West Island near Sanya, China. Genomic DNA of Conus textile was prepared from 30 mg of frozen tissue (muscular foot) using the E.Z.N.A. TM Mollusc DNA Kit (OMEGA, America) according to the manufacturer's standard protocol. Genomic DNA PCR was performed to obtain the gene sequences of O1conotoxins. The primers were designed based on the 3' end of the intron preceding the mature peptide region and the 3' UTR elements conserved in the O1 superfamily (forward primer: 5'-CGATCCATCTGTCCATCCATC-3'; reverse primer: 5'-GAKGGGAGTAGAACACATCACTA-3'). The amplified PCR products were extracted and cloned. DNA sequencing was carried out using an ABI 3730 automated DNA Analyzer (Thermo Fisher Scientific, America).

Peptide synthesis of Tx6.7
The mature peptide of conotoxin Tx6.7 was synthesized on a Rink amide resin using a standard Fmoc strategy according to previously reported methods [17]. The three pairs of cysteines were protected by triphenylmethyl (Trt), acetamidomethyl (Acm), or methoxybenzyl (Mob) separately and the three disulfide bonds were successively formed by oxidation of oxygen, iodine, or potassium ferricyanide. After oxidation, the mature peptide was purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and the molecular weight was confirmed by mass spectrometry analysis. After purification by HPLC, the purity of synthetic Tx6.7 was more than 98%.

Whole-cell patch clamp for DRG cells
Acutely separated DRG cells were isolated as previously described [18]. SD rats (30 days old) were purchased from the Guangzhou University of Chinese Medicine Experimental Animal Center (NO. SYXK (Yue) 2018-0182). All animal procedures were carried out according to the approved protocol (GDY2002208) of the Institutional Animal Care and Use Committee at Guangdong Medical University. The rats were euthanized, and the dorsal root ganglia tissue was removed quickly and cut into small pieces. The ganglia were treated with 0.1% collagenase and 0.05% trypsin. After centrifugation, the DRG cells were suspended in essential DMEM with 10% (v/v) fetal bovine serum and incubated in a 5% CO 2 fully humidified environment at 37 °C.

Whole-cell patch clamp for HEK293 and ND7/23 cells
HEK293 and ND7/23 cells, which were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) with STR Authentication, were cultured in DMEM with 10% fetal bovine serum. Plasmids of human Na V 1.3 (hNa V 1.3), human Na V 1.4 (hNa V 1.4), human Na V 1.7 (hNa V 1.7), human Ca V 1.2 (hCa V 1.2) and human Ca V 2.2 (hCa V 2.2) were separately transfected into HEK293 cells using Lipofectamine 3000 (Invitrogen, America), while plasmids of human hNav1.8 and hNavβ1 were cotransfected into ND7/23 cells.

MTT cytotoxicity assay
The conotoxin Tx6.7 was examined for cytotoxic activities against ND7/23 cell lines. Cytotoxicity assays were carried out in vitro using MTT staining according to the procedures [19]. The peptide concentrations used were 0, 0.01, 0.1, 1, 10, and 100 μM for each well, respectively. Three separate experiments were carried out, and six replicate wells were used to determine each point. After 48 h of incubation, the cells were stained with MTT and placed in a BIO-RAD model 680 microplate reader to determine the absorbance at 490 nm.

Analgesic activity bioassays
All animal procedures were carried out according to the approved protocol (GDY2002208) of the Institutional Animal Care and Use Committee at Guangdong Medical University. Female Kunming mice (weight 18-22 g) were purchased from the Guangzhou University of Chinese Medicine Experimental Animal Center (NO. SYXK(Yue)2018-0182). Ziconotide (ω-conotoxin MVIIA) was used as a positive control, while artificial cerebrospinal fluid (aCSF) was used as a negative control. The aCSF contained the following composition: 48 mM NaCl, 3 mM KCl, 1.4 mM CaCl 2 , 0.8 mM MgCl 2 , 1.5 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 , and 250 mM NaHEPES, pH = 7.35. Thirty Kunming mice were randomly divided into three groups. Each mouse was intrathecally injected with 20 pmol Tx6.7 or Ziconotide. The pain threshold was set as the time from when mice were placed on the 55 °C hot plate to when they licked their foot. The pain threshold was tested at 12 h before drug administration and 0.5 h, 1 h, 2 h, 3 h, and 4 h after drug administration.

Sequence identification of the O1-conotoxin Tx6.7
The conotoxin Tx6.7 was cloned from the genomic DNA of Conus textile using primers designed based on the conserved 3' end of the intron before the mature peptide region and the 3' UTR elements in the O1 superfamily (Fig. 1A). The cloned peptide sequence of Tx6.7 comprises 35 amino acid residues, including four amino acids at the end of the pro-region and 31 amino acids in the whole mature peptide (Fig. 1B). According to the alignment of typical conotoxins in the O1 superfamily (Fig. 1C), Tx6.7 has high sequence similarity with μO-MrVIA, MrVIB and MfVIA, and should have the same disulfide connectivity (I-IV, II-V and III-VI).

Peptide synthesis and identification of Tx6.7
The O1-conotoxin Tx6.7 was synthesized by solid-phase chemical synthesis according to its sequence (DCHERWDWCPASLLGVI YCCEGLICFIAFCI). Triphenylmethyl (Trt), acetamidomethyl (Acm), and methoxybenzyl (Mob) were used to protect the three pairs of cysteines separately, and oxidation of oxygen, iodine or potassium ferricyanide were used to form the three disulfide bonds (Fig. 2A). The oxidized crude peptide was purified by reverse-phase HPLC (Fig. 2B), and the molecular weight was determined by mass spectrometry (Fig. 2C). Mass of the final peptide was 3574.5 Da, which was identical with the expected molecular weight, suggesting that conotoxin peptide Tx6.7 with three pairs of disulfide bonds was synthesized successfully.

The analgesic activity of Tx6.7
The analgesic activity of Tx6.7 was evaluated by the mouse hot plate assay, which was tested at 0.5 h, 1 h, 2 h, 3 h, and 4 h after intrathecal injection (Fig. 5). Ziconotide (ω-conotoxin MVIIA) was used as the positive control. The pain threshold was increased by 20 pmol Tx6.7 from 0.5 h to 4 h. The analgesic effects of Tx6.7 reached a maximum at 2 h with the pain threshold increasing by 98.91%. However, at each time point, Ziconotide showed a better analgesic effect than Tx6.7.

Discussion
The traditional methods to discover a new conotoxin are peptide purification directly from the crude venom of Conus or gene amplification from the venom duct cDNA library. In this study, we successfully obtained a novel conotoxin using a new approach of genomic DNA cloning. The gene structures of nine conotoxin superfamilies were reported in previous work, and most of them have two introns and three exons [14]. According to the conserved sequences of the beginning and ending portions of the introns, we designed primers and cloned a new O1 superfamily conotoxin, Tx6.7, from the genomic DNA of Conus textile. Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an effective approach to obtain novel conotoxins. The drawback of this approach is that only exon3 (corresponding to the mature region) was obtained from this genomic PCR experiment (Fig. 1). To obtain the signal and pro-regions, another two pairs of primers should be designed to clone the corresponding exon1 and exon2 sequences from two additional rounds of PCR experiments. As one of the O1 superfamily conotoxins, Tx6.7 was compared with eight other ω/μO-conotoxins in the O1 superfamily (Fig. 1C) [9,18]. From the alignment result, Tx6.7 has high sequence similarity with these μO family conotoxins (MrVIA, MrVIB and MfVIA) which inhibit sodium currents, but has low sequence similarity with ω family conotoxins (CVIB, CVIB, MVIIC, MVIIA and CVIC) which inhibiting calcium currents. However, patch clamp experiments on DRG, HEK293 and ND7/23 cells showed that Tx6.7 had a low inhibitory effect on hNa V 1.8 currents but significantly inhibited DRG calcium currents, hCa V 1.2 and hCa V 2.2 currents. The low sodium channel activity may be due to the lack of some key residues (such as the three prolines in MrVIA, MrVIB and MfVIA) relating to the inhibition of sodium currents in the amino acid sequence of Tx6.7. Point mutation of amino acid residues may improve the inhibitory effect of Tx6.7 on Na V 1.8. Na V 1.8 is selectively expressed in sensory neurons and plays an important role in neuropathic pain [20,21]. Ca V 2.2 is involved in the neurotransmitter release of nociceptive pathways from afferent terminals [22]. The inhibitory effects on these two channels suggested that Tx6.7 may have a certain analgesic effect. Ca V 3.2 channels are the major isoform expressed in nonpeptidergic and peptidergic nociceptive neurons [23]. The patch clamp results showed that Tx6.7 inhibited 24.67 ± 0.91% of the Ca V 2.2 currents. The observed analgesic activity in the hot plate test may be a result of Ca V 3.2 inhibition, which would be verified in future work. Ca V 1.2 channels are the main subtypes in ventricular cardiac muscle and are also present in the nervous system, secretory tissues and smooth muscle cells [24]. Inhibition of Ca V 1.2 channels by specific blockers is a recognized pharmacodynamic approach for treating cardiac ischemia and hypertension [25], and Tx6.7 could be used as a candidate for this treatment. However, the action concentrations of Tx6.7 on Na V 1.8, Ca V 2.2 and Ca V 1.2 are at the micromolar level, so the activity of Tx6.7 should be further improved by alanine scanning or other modifications.

Conclusion
This study provided an unconventional approach to obtain novel conotoxin sequences from the genomic DNA of cone snails. The conotoxin Tx6.7 cloned from Conus textile could significantly inhibit rat DRG calcium currents. In addition, Tx6.7 inhibited 56.61 ± 3.20% of the hCa V 1.2 currents and 24.67 ± 0.91% of the hCa V 2.2 currents, indicating that it could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Availability of data and materials
All data generated or analyzed during this study are included in this article.