The bound AChBP and toxin-AChBP complexes were eluted in 12 l of His elution buffer (300 mM imidazole, 50 mM sodium phosphate buffer pH 8.0, 300 mM NaCl, 0.01% Tween-20) and resuspended 1:1 in 2 SDS-PAGE gel loading dye under reducing or nonreducing conditions and loaded onto 15% SDS-PAGE gels. elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from and a humanized neuronal 7 version (7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while 7-AChBP alone did not protect against (Egyptian cobra) venom lethality spp.), kraits (spp.), mambas (spp.), and coral snakes (spp.). Elapid envenoming often causes postsynaptic neurotoxic effects by blocking neuromuscular transmission that can culminate in respiratory paralysis and death (Gutirrez et al., 2017). Three-finger toxins (3FTxs) play a key role, with many competitively binding to postsynaptic neuromuscular and neuronal nicotinic acetylcholine receptors (nAChRs) to inhibit the binding of acetylcholine (Chang, 1979). Importantly, ever since the discovery of -bungarotoxin, snake venom neurotoxins have been extensively employed for characterizing nAChRs and understanding the basis of neurotransmission. Therefore, a comprehensive knowledge of their targets and mechanisms of action remains important for a range of biological disciplines. Three-finger toxins are one of many snake venom toxin types that are encoded by a multilocus gene family (Casewell et al., 2013). The frequent GNF-6231 duplication of toxin-encoding genes coupled with bursts of accelerated evolution results in snake venom composition varying at every taxonomic level, including inter- and intraspecifically (Chippaux et al., 1991; Casewell et al., 2013; Casewell Rabbit polyclonal to SGSM3 et al., 2014). Moreover, elapid venom proteomes are usually dominated by 3FTxs, which typically consist of numerous isoforms that comprise over 60% of venom proteins (Tasoulis and Isbister, 2017). These are small disulfide-bond rich proteins with a globular core and three -stranded loops that extend from the core as fingers and can be classified GNF-6231 into short-chain, long-chain, weak and nonconventional neurotoxins, cardiotoxins, and others (Kessler et al., 2017). Short-chain 3FTxs are small proteins consisting of 60C62 amino acid residues that contain four disulfide bridges, while long-chain -neurotoxins (long-chain 3FTxs) have 66C74 amino acid residues and display an additional disulfide bond. The small size (7C12 kDa) of these potent neurotoxins poses a therapeutic challengethey are weakly immunogenic (Tan et al., 2016b; Wong et al., 2016), yet all snakebite therapeutics (antivenoms) are manufactured from IgG of venom-immunized animals. The antineurotoxic efficacy of current antivenoms is further compromised because only 10C15% of the resulting IgG binds venom proteins (Casewell et al., 2010), with the remainder generated in response to environmental antigens to which the animals are exposed. Furthermore, antivenom efficacy is highly snake species-specific, resulting in limited cross-reactivity to venom toxins not included in the immunizing mixture (Tan et al., 2016a; Oh et al., 2019)an inevitable result of venom variation (Williams et al., 2011). To circumvent this challenge, many antivenom manufacturers use mixtures of venoms as immunogens. However, this often results in polyspecific antivenoms requiring much higher therapeutic doses [e.g., 5C10 vials (50C100 ml) of 50C100 mg/ml antibodies] to effect cure, which, in turn, results GNF-6231 in high incidences of foreign protein-related adverse reactions (de Silva et al., GNF-6231 2016) and prohibitively expensive treatment costs for impoverished snakebite victims [e.g., 48C315 USD/vial in Africa (Harrison et al., 2017)]. Therefore, a therapeutic approach providing cross-generic neutralization of snake venom neurotoxins (i.e., irrespective of the snake species responsible for the bite) and, at low therapeutic doses, would GNF-6231 be highly valuable. Acetylcholine binding proteins (AChBPs) are mimics of the extracellular ligand-binding domain of nAChRs and have been used extensively in structural studies of complexes of the receptor with a wide range of nicotinic agonists (Brejc et al., 2001), partial agonists (Hibbs et al., 2009), antagonists (Brams et al., 2011), and allosteric modulators (Spurny et al., 2015). AChBPs form pentameric complexes in which five subunits radially assemble around a central vestibule. The pharmacological properties of AChBPs from the freshwater snail (Ls-AChBP) most closely resemble the human 7-nAChR (Smit et al.,.
