Functional expression in Escherichia coli of the disulfide-rich sea anemone peptide APETx2, a potent blocker of acid-sensing ion channel 3

Abstract

Acid-sensing ion channels (ASICs) are proton-gated sodium channels present in the central and peripheral nervous system of chordates. ASIC3 is highly expressed in sensory neurons and plays an important role in inflammatory and ischemic pain. Thus, specific inhibitors of ASIC3 have the potential to be developed as novel analgesics. APETx2, isolated from the sea anemone Anthopleura elegantissima, is the most potent and selective inhibitor of ASIC3-containing channels. However, the mechanism of action of APETx2 and the molecular basis for its interaction with ASIC3 is not known. In order to assist in characterizing the ASIC3-APETx2 interaction, we developed an efficient and cost-effective Escherichia coli periplasmic expression system for the production of APETx2. NMR studies on uniformly (13)C/(15)N-labelled APETx2 produced in E. coli showed that the recombinant peptide adopts the native conformation. Recombinant APETx2 is equipotent with synthetic APETx2 at inhibiting ASIC3 channels expressed in Xenopus oocytes. Using this system we mutated Phe15 to Ala, which caused a profound loss of APETx2's activity on ASIC3. These findings suggest that this expression system can be used to produce mutant versions of APETx2 in order to facilitate structure-activity relationship studies.

Keywords: APETx2; ASIC3; E. coli; NMR; heterologous expression.

Figure 1

(A) Schematic of construct encoding a His₆-MBP: APETx2 fusion protein for production of rAPETx2 in E. coli. (B) SDS-PAGE gel showing expression and purification of rAPETx2. Lane 1: molecular mass standards (the size of selected standards is shown on left of gel); Lanes 2 and 3: E. coli cells before and after induction with IPTG; Lanes 4 and 5: insoluble and soluble fractions resulting from rupture of E. coli cells; Lanes 6 and 7: purified MBP fusion protein before and after cleavage with TEV protease. (C) RP-HPLC chromatogram showing the final purification of folded rAPETx2.

Figure 2

(A) RP-HPLC chromatogram showing co-elution of synthetic and recombinant APETx2. (B) MALDI-TOF mass spectrum showing m/z of pure rAPETx2. (C) MALDI-TOF mass spectrum showing m/z of pure rAPETx2-F15A.

Figure 3

Overlaid 2D ¹H-¹⁵N HSQC spectrum of uniformly ¹⁵N-labelled wild-type rAPETx2 (blue) and a F15A mutant (red). Residues whose chemical shifts are most affected by the mutation of Phe15 to Ala are highlighted with green circles while the peak corresponding to F15 in the wild-type toxin is shown in a red box. The horizontal line connects the two peaks from the sidechain amide group of Asn8, and “W14 sc” indicates the peak from the sidechain indole NH group of Trp14.

Figure 4

(A) Representative current traces comparing the concentration-dependent inhibition of rat ASIC3 (expressed in Xenopus oocytes) by synthetic (s) and recombinant (r) APETx2. (B) Concentration-effect curves for sAPETx2 and an rAPETx2–F15A mutant.

Figure 5

Three-dimensional solution structure of APETx2 (PDB accession code 1WXN) showing the relative positions of the N- and C-termini, and the predicted interaction surface including the confirmed pharmacophore residues R17 [30] and F15 (this study).