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Mar Drugs
2017 Jun 09;156:. doi: 10.3390/md15060170.
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Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human α9α10 Nicotinic Acetylcholine Receptor.
Jiang S
,
Tae HS
,
Xu S
,
Shao X
,
Adams DJ
,
Wang C
.
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Conotoxins are a pool of disulfide-rich peptide neurotoxins produced by cone snails for predation and defense. They are a rich reservoir of novel ligands for ion channels, neurotransmitter receptors and transporters in the nervous system. In this study, we identified a novel conotoxin component, O-conotoxin GeXXVIIA, from the venom of Conus generalis. The native form of this component is a disulfide-linked homodimer of a 5-Cys-containing peptide. Surprisingly, our electrophysiological studies showed that, in comparison to the folded monomers, the linear peptide of this toxin had the highest inhibitory activity at the human α9α10 nicotinic acetylcholine receptor (nAChR), with an IC50 of 16.2 ± 1.4 nM. The activities of the N-terminal and C-terminal halves of the linear toxin are markedly reduced compared with the full-length toxin, suggesting that the intact sequence is required to potently inhibit the hα9α10 nAChR. α9α10 nAChRs are expressed not only in the nervous system, but also in a variety of non-neuronal cells, such as cochlear hair cells, keratinocytes, epithelial and immune cells. A potent inhibitor of human α9α10 nAChRs, such as GeXXVIIA, would facilitate unraveling the functions of this nAChR subtype. Furthermore, this unusual nAChR inhibitor may lead to the development of novel α9α10 nAChR-targeting drugs.
Figure 1. Purification and identification of O-conotoxin GeXXVIIA from Conus generalis. (a) Purification of crude venom extracted from C. generalis (shown in inset) on a ZORBAX C18 semi-preparative column. The asterisk indicates the fraction containing GeXXVIIA. The elution gradient is 0–50% Buffer B for 0–50 min with a flow rate of 0.5 mL/min. (b) Analytical scale purification of the GeXXVIIA-containing fraction from panel (a) on a C18 reverse-phase analytical column. The peak with a molecular mass of 9695 Da is that of GeXXVIIA. The elution gradient is 10–30% Buffer B for 0–10 min and 30–39% Buffer B for 10–37 min with a flow rate of 0.5 mL/min. (c) Purification of the reduced GeXXVIIA after being treated with DTT on a C18 reverse-phase analytical column. The elution gradient is 10–45% Buffer B for 0–35 min with a flow rate of 0.5 mL/min. (d) Purification of the GeXXVIIA peptide after being alkylated with NEM on a C18 reverse-phase analytical column. The elution gradient is the same as that of panel (c).
Figure 2. cDNA sequence of O-GeXXVIIA. (a) The cDNA sequence of GeXXVIIA and the cDNA-encoded precursor sequence. The coding region of cDNA is shown in capital letters. The signal peptide sequence is shadowed, and the mature peptide sequence is underlined. Between the signal sequence and mature toxin region is the pro-peptide region. * represents the stop codon. (b) Alignment of the precursor sequences of GeXXVIIA, Mik41 [19], and GeXIVA [20]. Mature toxin sequences are underlined with the Cys residues highlighted in bold.
Figure 3. Preparation and nAChR-inhibitory activities of the monomeric isomers of GeXXVIIA. (a) Separation of four GeXXVIIA monomeric isomers (m1, m2, m3, and m4) refolded in vitro on a C18 reverse-phase analytical column. The elution is isocratic at 22% Buffer B with a flow rate of 0.5 mL/min. The molecular masses of these four isomers are the same, 4807 Da, indicating that two disulfide bonds are formed in each isomer. (b) The inhibition of 5 μM GeXXVIIA-m1, -m2, -m3, and -m4 on ACh-evoked peak current amplitude mediated by hα3β2, hα3β4, hα7, hα4β4, hα4β2, hα9α10, and rodent (r) α1β1εδ nAChRs (n = 1 oocyte for each nAChR subtype).
Figure 4. Preparation and nAChR-inhibitory activities of the linear GeXXVIIA and its N- and C-terminal fragments. (a) Left panel: Preparation of the linear peptide of GeXXVIIA (GeXXVIIA-L) alkylated with IAA on a C18 reverse-phase analytical column. Its molecular mass is 5096 Da. The elution gradient is 15–37% Buffer B for 0–22 min with a flow rate of 0.5 mL/min. Right panel: Two fragments were obtained after the digestion of GeXXVIIA-L by Asp-N protease. The elution gradient is 5–38% acetonitrile for 0–33 min with a flow rate of 0.5 mL/min. The N-terminal (Nter) and C-terminal (Cter) fragments of GeXXVIIA-L have a molecular mass of 3070 Da and 1598 Da, respectively. Below are the sequences of GeXXVIIA-L, Nter and Cter peptides, with the IAA-blocked Cys residues shown in red. (b–d) Superimposed ACh-evoked currents mediated by rα1β1εδ and hα9α10 nAChRs in the absence (C, control) and the presence of 5 μM different GeXXVIIA peptides (P). Arrows (▼) indicate ACh application. The peptides are GeXXVIIA-L (b), GeXXVIIA-L-Nter (c), and GeXXVIIA-L-Cter (d).
Figure 5. Antagonist potencies of GeXXVIIA-L and GeXXVIIA-L-Nter at the rodent α1β1εδ and human α9α10 nAChRs. (a) Inhibition of ACh-evoked peak current amplitude mediated by rα1β1εδ and hα9α10 nAChRs by GeXXVIIA-m1, GeXXVIIA-L, GeXXVIIA-L-Nter, and GeXXVIIA-L-Cter at 5 μM (n = 1 oocyte) and 300 nM (n = 3–7 oocytes) peptide concentrations. Error bars indicate SEM. (b) Concentration-response curves obtained for GeXXVIIA-L and GeXXVIIA-L-Nter inhibition of rα1β1εδ and hα9α10 nAChRs expressed in Xenopus laevis oocytes. Full-length GeXXVIIA-L exhibits potent inhibitory activity at hα9α10 receptors with an IC50 of 16.2 ± 1.4 nM (n = 4–8 oocytes for each data point).
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