Cloning, Synthesis and Functional Characterization of a Novel α-Conotoxin Lt1.3

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement “C1-C3, C2-C4” is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges “C1-C3, C2-C4” and “C1-C4, C2-C3” were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges “C1-C3, C2-C4” potently and selectively inhibited α3β2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges “C1-C4, C2-C3” showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3β2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.


Cloning of α-CTx Lt1.3
A novel α-CTx precursor was isolated from the genomic DNA of C. litteratus ( Figure 1). The mature toxin sequence was predicted as GCCSHPACSGANPYFC-NH 2 with a cysteine (C) pattern of CCX 4 CX 7 C (X represents any other amino acids). It belongs to cysteine framework I and the α4/7 CTx family, which is usually associated with an inhibitory activity for nAChRs. In accordance with conventional conotoxin nomenclature, the novel CTx was designated as Lt1.3 and its partial cDNA sequence of precursors has been submitted to GenBank and its genBank accession number is KF414121.

Peptide Synthesis and Disulfide Connectivity
Two major peaks were found in the one-step folding products of Lt1.3 linear peptide analyzed by HPLC ( Figure 2). According to the theoretical isomers of peptides with two disulfide bonds and the folding products, two Lt1.3 isomers (with disulfide connectivities "C 1 -C 3 , C 2 -C 4 " and "C 1 -C 4 , C 2 -C 3 ") were synthesized by the two-step oxidative folding strategy using the two different Acm-protected Lt1.3 linear peptides ( Figure 3). Co-elution assays were performed with either Lt1.3-I (Figure 2c) or Lt1.3-II (Figure 2d) plus the two products formed in the two-step oxidative folding reaction ( Figure 3). The results indicate that Lt1.3-II has a disulfide connectivity arrangement of "C 1 -C 3 , C 2 -C 4 ", and the Lt1.3-I has the arrangement of "C 1 -C 4 , C 2 -C 3 ". The Lt1.3 variants (Table 1) were also synthesized as described in the materials and methods section and assessed by analytical RP-HPLC. Their purities were >98% with the expected molecular weights.

Circular Dichroism Spectra of Lt1.3-II and Its Variants
The circular dichroism (CD) spectra of Lt1.3-II and its variants in 0.01 M phosphate buffer (50% TFE (2,2,2-trifluoroethanol)) showed some ellipticities around 208 nm and 220 nm ( Figure 4) although the minimum values of several Lt1.3 variants were lower than that of Lt1.3. However, Lt1.3-I did not show the helical CD spectrum. These results are consistent with the previous reports that α4/7-CTxs have a short helical structure if they have the disulfide bridges "C 1 -C 3 , C 2 -C 4 " and a conserved proline residue exists in loop2 [22]. Thus, this structural feature was used to confirm the disulfide bridges of Lt1.3 variants. It should be noted that the minimum ellipticities of Lt1.3 and its variant in 0.01 M phosphate buffer (pH = 7.20) were at 194 nm and 208 nm ( Figure S1), suggesting they may contain distorted α-helical structures.

Activities of Lt1.3 and Its Variants on nAChRs
Two-electrode voltage clamping was used to assess the effects of Lt1.3-II and Lt1.3-I on various neuronal nAChRs expressed by Xenopus oocytes. As shown in Figure 5, Lt1.3-II exhibited a strong inhibition on the rat neuronal subtype with an IC 50 of 44.8 nM, but not on the subtypes of α2β2, α2β4, α3β4, α4β2, α4β4, α7 and α9α10 ( Figure 5A, IC 50 > 10 µM). On the other hand, Lt1.3-I displayed no apparent inhibitory effects on nAChR subtypes (IC 50 > 10 µM) ( Figure S2). These results indicate that Lt1.3-II selectively inhibits the α3β2 subtype of nAChRs, and its disulfide connectivity arrangement "C 1 -C 3 , C 2 -C 4 " is very important for the potency. When Ser 9 was mutated to Ala, the inhibitory activity was similar. However, the replacement of Asn 11 , Asn 12 , Pro 13 , Tyr 14 , and Phe 15 with Ala resulted in a sharp decrease in inhibitory activity on α3β2, the IC 50 was decreased to 216 nM and <10 µM for Lt1.3[Y14A] and others ( Figure 5B), respectively.  Table 1. Data represent mean ± SEM.

Discussion
Currently, a dozen α4/7-CTXs have been found to target α3β2 and other nAChRs ( Table 2). They share similar amino acid residues in the loop1 region, especially the conserved first (Ser) and third amino acid (Pro) [23]. The difference in selectivity is mainly derived from the surface-exposed charge and polarity of the loop2 region [24]. For example, the polarity, shape, and size of residues of α-CTX PnIA at position 10 affect the potency and selectivity to α3β2 and α7 nAChRs, while hydrophobic residues at position 10 maintain potency at both subtypes, smaller hydrophobic residues increase selectivity to α3β2 nAChRs [23]. Lt1.3-II has a small residue Gly at this position, so it selectively targets the α3β2 (IC 50 = 44.8 nM) with high selectivity index (SI, >200) compared to other nAChR subtypes. The structure-activity relationship of Lt1.3 indicates that the substitution of Asn 11 , Asn 12 , Pro 13 , Tyr 14 , and Phe 15 by Ala results in the sharp decrease in the inhibitory activity to α3β2 but the substitution of Ser 9 ( Figure 5 Two major peaks (Lt1.3-I and Lt1.3-II) existed during the oxidative folding reaction of the linear Lt1.3 peptides. Further activity assay demonstrated that Lt1.3-II (disulfide connectivity of "C 1 -C 3 and C 2 -C 4 ") had a strong binding affinity to the α3β2 nAChRs, but Lt1.3-I, which has the disulfide connectivity of "C 1 -C 4 , C 2 -C 3 ", did not bind. This suggests that the globular and α-helical structure of Lt1.3-II is crucial for the potency to inhibit α3β2 nAChRs.
In conclusion, we found a novel α4/7 CTx Lt1.3 that targets specifically the α3β2 nAChR subtypes, and its isomer with the disulfide bridges "C 1 -C 4 , C 2 -C 3 " that specifically inhibits the GABA B R-coupled Cav2.2. This finding expands current knowledge of targets and selectivity of α-CTxs and provides a new structural motif to inhibit the GABA B R-coupled Cav2.2.

Cloning of Lt1.3 cDNA
Previous reports show that a long intron sequence exists between the exon I and exon II in the precursor gene sequence of α-conotoxins [21]. Exon I encodes the signal peptide and a part of the pro-region, while exon II encodes the other part of the pro-region, the mature peptide and 3 untranslated region . The sequences at the 5 and 3 end of introns are highly conserved and contains long dinucleotide (e.g., "GT", "CA") or trinucleotide ("CAT") repeats. Taking advantage of the conserved 3 end sequence of the intron, Lt1.3 was cloned from the genomic DNA of the venom ducts of Conus litteratus (C. litteratus) which was collected from the Cisha Island of the South China Sea, according to the kit protocol (TIANGEN catlog no: DP324, Beijing, China) [36]. The forward primer P1 (5 -GTGGTTCTGGGTCCAGCA-3 ) from the conserved 3 end sequence of intron identified in αA conotoxins and was paired with the reversed primer P2(5 -GTCGTGGTTCAGAUGGTC) from the conserved 3 -UTR sequences of A-family conotoxins described previously [14,36]. PCR amplification was carried out as follows: 94 • C, 4 min (1 cycle); 94 8 • C, 30 s; 55 8 • C, 30 s; 72 8 • C, 45 s (30 cycles); 72 8 • C, 10 min (1 cycle). The PCR products were analyzed by electrophoresis on agarose gel. The target band was excised from the gel and purified with Gel Extraction Mini Kit (Beyotime, Haimen, China). The purified PCR products were added to basyl A and ligated into the T-tailed plasmid vector pGEM-T for DNA sequencing. The predicted protein sequences were analyzed with software Seqtools (http://www.bio-soft.net/sms). The novel cDNA sequence Lt1.3 was identified following a comparison with the sequences available in the GenBank nucleotide sequence database and in the literature, and was deposited in the Genbank nucleotide sequence database.

Peptide Synthesis and Disulfide Connectivity Analysis
Lt1.3 and its variants were synthesized using the method described previously [18,37]. Briefly, Lt1.3 or its mutant was assembled and then cleaved from Rink resin. The released peptides (0.1 mg/mL) were oxidized in 0.1 M NH 4 HCO 3 at room temperature, pH 8.0-8.2. The folding products were then purified by semi-preparative reverse phase-high performance liquid chromatography (RP-HPLC). The final products were assessed by analytical RP-HPLC. The primary sequences of Lt1.3 and its variants were listed in Table 1.
The disulfide connectivity of one-step oxidative folding products of Lt1.3 was analyzed by comparing the folded peptide products with known disulfide connectivity [38]. Briefly, linear peptides containing an acetamidomethyl (Acm)-protecting group at the C 2 -C 4 or C 1 -C 4 position were synthesized and then folded by incubation in 0.1 M NH 4 HCO 3 (pH 8.0) at room temperature for 24-48 h. The folded products were further oxidized with an iodine mixture containing 30% CH 3 CN/2% TFA/68% H 2 O for 10 min to yield peptides with the -S-S-bridges of "C 1 -C 3 , C 2 -C 4 " or "C 1 -C 4 , C 2 -C 3 ". The mixture of this second oxidized product and the one-step folding product of Lt1.3 was analyzed by HPLC to determine the disulfide connectivity. The disulfide connectivity of Lt1.3 variants was determined according to its CD spectra. If they display typical α-helical circular dichroism (CD) spectra, the disulfide connectivity will be "C 1 -C 3 , C 2 -C 4 ".

Two-Electrode Voltage-Clamp Recording on Oocytes Expressing nAChRs
cDNA preparation, oocyte harvest and expression of nAChR subunits were performed as described previously [13,36]. Briefly, each Xenopus oocyte was injected with 30-40 ng of cRNA and incubated with ND96 solution containing 2.5 mM pyruvic acid sodium, 0.1 mg/mL BSA and antibiotics (10 U/mL penicillin, 10 µg/mL streptomycin) (Gibco by Life Technologies, Grand Island, NY, USA) at 18 • C. Electrophysiological experiments were performed at days 2-5 post-injection at room temperature (22 • C). The oocytes were gravity-perfused in a recording chamber (50 µL) with ND96 at a rate of 1.5 mL/min. The membrane potential was clamped at −70 mV and the ACh-gated currents were recorded with a two-electrode voltage-clamp amplifier Axoclamp 900A (Axon Instruments Inc., Union City, CA, USA). The perfusion medium was automatically switched among ND96, Ach (in ND96) and toxin (in ND96) using MPS-2 multichannel perfusion system (Inbio Life Science Instrument Co., Ltd., Wuhan, China). Ach, in 200 µM, 30 µM, and 100 µM amounts in ND96, was used to activate α7, α9α10, and other nAChRs subtypes, respectively. For the low-dose response, the oocyte was perfused with the toxin solution until equilibrated (5~10 min) and then activated by ACh. In high-dose experiments (1 µM or greater), 5.5 µL of a 10-fold concentrated toxin solution was directly pipetted into static bath 5 min prior to the exposure of ACh pulses.
The dose-dependent response data were fit to the equation: response (%) = 100/[1 + ([toxin]/IC 50 ) n ], where n is the Hill coefficient and IC 50 is the inhibitor concentration giving half-maximal response, by non-linear regression analysis using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

HEK293T Cell Electrophysiology
HEK293T cells were transiently co-transfected with human GABAB1 and GABAB2 subunits (2 µg each, obtained from David J. Adams, University of Wollongong, Wollongong, Australia) and 0.2 µg mCherry fluorescent protein using Lipofectamine 2000 (Invitrogen, Van Allen Way, Carlsbad, CA, USA) according to the manufacturer's protocol. After 24 h, the cells were then transiently co-transfected with rat Cav2.2 channels (α1B, β3, and α2δ1 subunits with 1 µg each) and 0.2 µg of the enhanced green fluorescent protein using Lipofectamine 2000 as well. Two to three days after transfection, cells were seeded on glass coverslips pretreated with poly-L-lysine and incubated at 37 • C in 5% CO 2 for at least 6 h before recording.