PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer

Abstract

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC₅₀ 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

Keywords: Phymanthus crucifer; defensin-like fold; glutamate receptor; ion channels; neutoxin; sea anemone.

Figure 1

(A) Gel filtration profile of P. crucifer aqueous extract. The soluble material contained in 5 grams of whole-body homogenate (350 mg/90 mL) was fractionated on Sephadex G-50 (5 × 93 cm) at 2 mL/min using 0.1 mol/L ammonium acetate. Fractions of 20 mL each were collected; those within the elution volumes of 820 mL to 1460 mL were paralyzing to all of the crabs, and were pooled; (B) Cation-exchange chromatographic profile of the crab-paralyzing pool of chromatographic fractions from Sephadex G-50, in Fractogel EMD SO₃⁻ 650 M (1.8 × 5 cm); (C) Anion-exchange chromatographic profile of the non-retained fraction from the cation exchanger, in Fractogel EMD DEAE 650 M (1.8 × 5 cm). Both separations (B,C) were done at a flow rate of 1 mL/min using a 400-mL gradient, from 0.01 mol/L to 1 mol/L ammonium acetate. Eighty fractions of 5 mL each were collected in every chromatographic separation. Dashed lines in the ion-exchange chromatographic profiles represent the gradient of ammonium acetate. Fractions exhibiting toxicity to crabs were named I, II, III, and IV. The pools of fractions that inhibited acid-sensing ion channels are shown in both gel-filtration and cation-exchange chromatographic profiles, according to previous results with the same P. crucifer homogenate, using identical conditions [6]. PhcrTx1, an acid-sensing ion channel toxin from P. crucifer [6], eluted inhibiting pools of chromatographic fractions in the ASICs, as shown in (A,B). As shown, the crab-paralyzing zone and the ASICs inhibition zone barely overlapped in the gel filtration profile (A); and completely separated from each other in the cation-exchange profile (B). PhcrTx1 is not present among the crab-paralyzing chromatographic fractions isolated from the ion-exchange chromatographic separations.

Figure 2

Reversed-phase chromatographic profiles of crab-paralyzing fractions from ion-exchange chromatography. (A,B) Reversed-phase chromatographic profiles of fractions I and II previously separated from cation-exchange chromatography, respectively; (C,D) Reversed-phase chromatographic profiles of fractions III and IV previously separated from anion-exchange chromatography, respectively. Conditions: Hypersil H5 ODS column (4.6 × 250 mm), flow rate 0.8 mL/min, linear gradient from 0 to 80% B in 80 min. Chromatographic fractions showing toxicity to crabs are indicated in the figure (1 to 16); (E,F) Reversed-phase chromatographic purification of fraction number 5. Conditions: Discovery RPC18 HPLC column (4.6 × 250 mm), flow rate of 1 mL/min, gradient from 10 to 20% B in 5 min, followed by 20 to 30% in 50 min. The pure toxin was named PhcrTx2. The dashed line in every chromatographic profile represents the gradient of acetonitrile. The asterisk (*) in (A–F) represent the point where PhcrTx1 elutes in the same chromatographic conditions, according to previous results [6].

Figure 3

Effect of PhcrTx2 on a glutamate-evoked current in isolated snail neurons. (A) Glutamate-evoked (at 1 mM of Glu) currents registered at different holding potentials (from −100 mV to +80 mV) show a current reversal at about 0 mV, indicating that it most probably is a non-selective cation permeant channel; (B) Concentration-response relationship of the inhibitory effect of PhcrTx2 on glutamate-gated currents. Data were fitted by a dose-response function with an IC₅₀ of 4.7 µM. Each point represents the mean ± SE from four to seven neurons; (C) Representative current traces elicited by glutamate (1 mM) under control condition, in the presence of 30 µM PhcrTx2, and after washout of the toxin.

Figure 4

MALDI-TOF mass spectrum of PhcrTx2. An intense signal of m/z 5297.8 from the monoprotonated peptide ion [M + H]⁺ was detected, corresponding to a molecular mass of 5296.8 Da.

Figure 5

Multiple sequence alignment (MAFF-LINSi) of PhcrTx2 and related sequences listed in order of descending score, according to pairwise sequence alignments using the Smith–Waterman algorithm (EMBOSS water tool). Dark blue columns represent the alignment of identical amino acid residues, whereas the lighter colors represent the alignment of less conserved positions. The secondary structure features from experimental 3D structures are represented above their corresponding sequences. The PhcrTx2-related sequences correspond to the sea anemone toxins Am-2 (UniProtKB P69930), BDS-2 (UniProtKB P59084), BDS-1(UniProtKB P11494), BcIV (UniProtKB P84919), U-AITX-Bg1a (UniProtKB G0W2H7), U-AITX-Bg1c (UniProtKB G0W2H9), U-AITX-Bg3d (UniProtKB G0W2I1), U-AITX-Bg1b (UniProtKB G0W2H8), U-AITX-Bg3c (UniProtKB G0W2I0), APETx3 (UniProtKB B3EWF9), APETx1 (UniProtKB P61541), and APETx2 (UniProtKB P61542). The upper side of the figure shows the secondary structure elements predicted for PhcrTx2 using RaptorX, JNET, PSSPRED, and PSIPRED. The lower side of the figure shows the representative disulfide bridge pattern of the family Defensin 4, which is Cys5-Cys42, Cys7-Cys34, and Cys24-Cys43 in the multiple sequence alignment. The same S-S arrangement is expected for PhcrTx2, corresponding to Cys4-Cys40, Cys6-Cys32, and Cys22-Cys41 in its sequence, which fits the pattern I-V, II-IV, III-VI found in β-defensins.

Figure 6

(A) The three-dimensional (3D) structural model of PhcrTx2 (obtained by RaptorX) includes three antiparallel β-strands (residues 14–18 DKWIF, 30–34 DRCFM, and 37–42 GSVCCY); (B) Prediction of the ligand-binding residues of PhcrTx2 (the structural model is rotated on the x-axis, with respect to the (A) by CLIP-4D, using structural and evolutionary information from the multiple sequence alignment; (C) Surface electrostatic potential representation of PhcrTx2. The surface is colored according to the electrostatic potential: negative regions (in red), positive regions (in blue), and neutral regions (in gray). The orientation of the surface electrostatic potentials is the same as that in the ribbon representation (A). We also provided a color intensity scale to better represent the electrostatic potential. This figure was prepared with the PyMOL Molecular Graphics System, Schrödinger, LLC.