Chemical synthesis and characterization of two α4/7-conotoxins

Abstract

α-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two α-conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of α-conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat α7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle α1β1γδ receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

Figure 1

The cDNA sequences and predicted translation products of Mr1.1 (A) and Lp1.4 (B) The signal peptides and mature toxins are shaded. The codons of conserved Cys are shown in bold letter. The nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers DQ311077 and AY580325 for Mr1.1, DQ311056 and AY580324 for Lp1.4.

Figure 2

Oxidative folding of α4/7-conotoxins Mr1.1 and Lp1.4 (A) RP-HPLC oxidation profile of Mr1.1 using orthogonal protection on Cys residues. The folded product was loaded onto an analytical PepMap-C18 column in 90% solvent A and eluted using a linear gradient of 20–40% solvent B at a flow rate of 0.5 ml/min for 40 min. (B) The synthetic Lp1.4 prepared by site-directed disulfide formation. (C) The synthetic Lp1.4 prepared by the one-step oxidative folding procedure. (D) Co-elution profile of the two synthetic Lp1.4. Lp1.4 was separated on an analytical PepMap-C18 column and eluted using a flow rate of 1 ml/min using the following gradient: 0–5 min, solvent A; 5–7 min, 0–10% solvent B; 7–22 min, 15–25% solvent B. The molecular mass of the folded Lp1.4 in (B) and (C) was respectively analyzed by mass spectrometry to be identical (for Lp1.4 in B, observed value: 1755.4; for Lp1.4 in C, observed value: 1755.3), further confirming their identity.

Figure 3

Inhibitory effects of Mr1.1 and Lp1.4 on various mouse and rat nAChRs Each bar indicates the average percent response ± SD after toxin application to Xenopus oocytes expressing a variety of nAChRs. Peptides were tested three to five times against each receptor subtype.

Figure 4

Selectivity of Mr1.1 and Lp1.4 on various nAChRs Representative current traces for Mr1.1 and Lp1.4 at 1 µM on rat α7 and mouse α1β1γδ nAChR, respectively. Control traces are shown prior to peptide application. The arrow marks the first current trace elicited after a 10-min exposure to toxin. Subsequent current traces show peptide dissociation and washout. Two peaks of the ACh-induced response immediately after the 10-min incubation of Lp1.4 are obtained due to the displacement of salt bridges when the high-speed (5 ml/min) gravity-perfusion restored.

Figure 5

Acute effect of intraplantar injection of Mr1.1 on thermal paw withdrawal threshold in SD rat inflammation pain model Results are expressed as mean ± SEM (n = 8). Statistical analyses were performed using two-way repeated measures ANOVA. Asterisks represent significant PWL increase (P < 0.05) induced by injection of Mr1.1.