An N-Terminally Elongated Peptide From Conus rolani Defines a New Class of Ribbon α-Conotoxins Targeting Muscle nAChRs

Abstract

Conotoxins, peptides found in cone snail venoms, selectively target ion channels and receptors to incapacitate prey. α-Conotoxins are extensively investigated for their potent modulation of nicotinic acetylcholine receptors (nAChRs). This study describes the discovery and characterization of RoIA, a novel α-conotoxin from Conus rolani. RoIA belongs to the α4/4 conotoxin class and features an atypical N-terminal elongation of 18 amino acids. The biological activity of RoIA is assessed through both in vivo and in vitro assays using the three potential folding isoforms (globular, ribbon, and bead) of the full-length and truncated (lacking the N-terminal elongation) analogs. The full-length RoIA analog exhibits delayed but potent paralytic activity when administered to mice and fish; the ribbon isoform shows the highest potency. Notably, only the ribbon isoform of the truncated peptide is active on heterologously expressed muscle nAChRs, suggesting that the N-terminal elongation may be released in vivo or form interactions that are not recapitulated in vitro. This discovery challenges the prevailing understanding that native α-conotoxins adopt a globular conformation and illustrates that Conus can explore novel chemical spaces using an alternative disulfide bond connectivity. This research enhances our knowledge of the complex mechanisms by which toxins manifest their physiological effects.

Keywords: bioassay; disulfide connectivity; electrophysiology; nAChR; peptide stability; venom; α‐Conotoxin.

FIGURE 1

(A) Ro1.9 (RoIA) precursor sequence. The blue arrow indicates the boundary between the propeptide and the mature toxin region, while the gray arrow signifies the boundary between the mature toxin and the post‐peptide region. Cysteines are denoted in red, while the post‐peptide region is in gray. (B) The mature peptide sequence of Ro1.9. (C) Precursor sequences of Asprella clade α‐conotoxins related to Ro1.9. The signal sequence, where present, is indicated in blue, and the propeptide region is underlined. The signal sequence, propeptide, and mature toxin regions are each separated by a space, with gray highlighting indicating sequence homology in the mature peptide region.

FIGURE 2

Comparative alignment of Asprella clade a‐conotoxins. Nucleotide and amino acid sequence alignments of α4/4 conotoxins Ro1.9 (RoIA), Bh1.3, and Bh1.16 are displayed alongside C. rolani α4/7 conotoxins. The blue box indicates the signal peptide region, while the pink boxes highlight the predicted trypsin cleavage sites that facilitate the formation of the mature toxin. The gray box indicates the deleted region, which results in the absence of the canonical cleavage site in N‐terminally elongated α‐conotoxins. The red and orange arrowheads mark the propeptide and postpeptide cleavage sites in the elongated forms, respectively. The blue arrowhead highlights the propeptide cleavage site characteristic of classical α‐conotoxins. The mature toxin region is indicated in bold type, with cysteine residues highlighted in yellow. GenBank accession numbers—Ro1.9: PV156767, Bh1.16: PV156768, Ro1.1: PV164370, Ro1.3: PV164371, Ro1.4: PV164372.

FIGURE 3

Distinct disulfide connectivities of synthesized RoIA (A) and RoIA[18–30] (B) are denoted by color coding. # C‐terminal amidation.

FIGURE 4

Behavioral assay in mice. (A) scheme showing the location of the i.p. injection. NSS, normal saline solution. (B) Latency to crawling behavior and (C) latency to death of RoIA peptides upon injection of 10 nmol of the peptides. Peptide activities were compared using a two‐tailed t‐test (α = 0.05). ∞ = No activity observed. (D) Latency to crawling behavior and (E) latency to death of the RoIA peptides upon injection of 1 nmol of the peptides. All three isoforms, gRoIA (orange), rRoIA (green), and bRoIA (yellow), in the long RoIA (●) and short RoIA[18–30] (▲) versions were tested and compared to 1 nmol of α‐MI (pink), a highly selective α3/5 α conotoxin from C. magus. Peptide activities were compared using a one‐way ANOVA analysis. Groups were compared using Tukey's post hoc test. Data are represented as box plots. Within each box, the horizontal black line denotes median values; boxes extended from the 25th to the 75th percentile of each group's distribution values; vertical lines extended from the minimum to the maximum values. Individual values are shown as dots. Relevant statistical significances are highlighted in the figure. For further comparisons, please refer to the text. Data are shown as mean values ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = no significant.

FIGURE 5

Behavioral assay and LD₅₀ of RoIA in fish. (A) scheme showing the location of the i.p. injection. (B) latency to death of the RoIA peptides upon injection of 1 nmol/g of the peptides. Data are shown as mean values ± SD. All three isoforms, gRoIA (orange), rRoIA (green), and bRoIA (yellow), in the full‐length RoIA (●) and truncated RoIA[18–30] (▲) analogs were tested and compared using a one‐way ANOVA test. Groups were compared using Tukey's post hoc test. Data are represented as box plots. Within each box, the horizontal black line denotes median values; boxes extended from the 25th to the 75th percentile of each group's distribution values; vertical lines extended from the minimum to the maximum values. Individual values are shown as dots. Relevant statistical significances are highlighted in the figure. For the full comparisons, please refer to the text. ***p < 0.001; ****p < 0.0001. (C) Dose–response curve; cumulative frequency distribution of rRoIA and rRoIA[18–30] lethality in zebrafish. The lines represent the expected values (full line = rRoIA; dashed line = rRoIA[18–30]), and the symbols (● = rRoIA; ▲ = rRoIA[18–30]) represent the observed values. (D) Regression lines of the PROBIT values and the log (Dose). The lines represent the expected values (full line = rRoIA, y = 3.49x + 8.25; dashed line = rRoIA[18–30], y = 2.702x + 9.386), and the symbols (● = rRoIA; ▲ = rRoIA[18–30]) represent the observed values. The LD₅₀ corresponds to the dose value when y = 5.

FIGURE 6

Electrophysiological activity of RoIA analogs on mouse and fish muscle nAChR subtypes. Activity of RoIA and RoIA[18–30] at 100 nM as tested on (A) adult (α1β1δε) and (B) fetal (α1β1δγ subunits) mouse, and (C) adult (α1β1δε) and (D) fetal (α1β1δγ subunits) zebrafish muscle nAChRs expressed in Xenopus oocytes. Each data point is the average of the responses obtained from at least three oocytes. All three isoforms, gRoIA (orange), rRoIA (green), and bRoIA (yellow), in the long RoIA (●) and short RoIA[18–30] (▲) versions were tested and compared using a one‐way ANOVA test. Data are shown as mean ± SD. Groups were compared using Tukey's post hoc test. rRoIA[18–30] was significantly different from the other analogs. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 7

Serum stability assay. Stability of the individual isomers: ribbon (r), globular (g), and bead (b) of RoIA and RoIA[18–30] in mouse serum at 37°C sampled at 1 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h and 24 h. Data are shown as mean values relative to the peak intensity of a reference peptide ± SD, n = 3.

FIGURE 8

Behavioral assay of ribbon and globular isoforms of PIB. (A) PIB sequence showing the globular (gPIB) and ribbon (rPIB) cysteine connectivity. (B) HPLC traces of PIB random folding in the presence of glutathione (black) and molecular oxygen (air, black), rPIB (green), and gPIB (orange) collected on a C4 Vydac analytical column with a gradient ranging from 10% to 40% of buffer B90, with a flow rate of 1 mL/min. (C) Latency to crawling behavior and (D) latency to death in mice. Since 1 nmol of rPIB showed no significant effects in mice, a 10 nmol dose was used to compare rPIB and gPIB. Only two out of four mice died after i.p. injection of gPIB. Latency to death in fish (E) of rPIB was tested at 1 nmol/g. For C–D data are represented as box plots. Within each box, the horizontal black line denotes median values; boxes extended from the 25th to the 75th percentile of each group's distribution values; vertical lines extended from the minimum to the maximum values. Individual values are shown as dots. (F) Electrophysiological activity of gPIB and rPIB isoforms at 300 nM on mouse muscle nAChR (α1β1δε) expressed in Xenopus oocytes. The activity of the two isoforms was compared using a t‐test analysis. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.