Identification of a sensory neuron Cav2.3 inhibitor within a new superfamily of macro-conotoxins

Abstract

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new gene superfamily. The crystal structure of recombinant Mu8.1 shows structural similarity with conotoxins of the con-ikot-ikot superfamily, with both toxins displaying a saposin-like fold. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.

A single-chain variable fragment selected against a conformational epitope of a recombinantly produced snake toxin using phage display

“…The CyDisCo co-expression system has been shown to accommodate highly complex disulfide-bonded proteins, including Fab antibody fragments [17] (five disulfide bonds), the vtPA [18] (a tissue plasminogen activator fragment with nine intra-molecular disulfide bonds), Resistin [18] (five intra-molecular disulfide bonds and an inter-molecular disulfide bond), and a SARS-CoV-2 spike protein receptor binding domain [19] (five disulfide bonds). In the present study, the modified csCyDisCo system was used, which includes an additional protein-disulfide isomerase expressed in the venom gland of the cone snail species Conus geographus and has been successfully used to produce conotoxins with up to five disulfide bonds [20]. It is shown that a toxin recombinantly expressed in the csCyDisCo system can be used to select binders to the native toxins in vitro, and that the antigen-antibody interactions rely on structural epitopes.…”