A comparative study of the performance of E. coli and K. phaffii for expressing α-cobratoxin.

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs.

Acetylcholine-Binding Protein Affinity Profiling of Neurotoxins in Snake Venoms with Parallel Toxin Identification.

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins.

Evolution of Three-Finger Toxin Genes in Neotropical Colubrine Snakes (Colubridae).

Snake venom research has historically focused on front-fanged species (Viperidae and Elapidae), limiting our knowledge of venom evolution in rear-fanged snakes across their ecologically diverse phylogeny. Three-finger toxins (3FTxs) are a known neurotoxic component in the venoms of some rear-fanged snakes (Colubridae: Colubrinae), but it is unclear how prevalent 3FTxs are both in expression within venom glands and more broadly among colubrine species. Here, we used a transcriptomic approach to characterize the venom expression profiles of four species of colubrine snakes from the Neotropics that were dominated by 3FTx expression (in the genera Chironius, Oxybelis, Rhinobothryum, and Spilotes). By reconstructing the gene trees of 3FTxs, we found evidence of putative novel heterodimers in the sequences of Chironius multiventris and Oxybelis aeneus, revealing an instance of parallel evolution of this structural change in 3FTxs among rear-fanged colubrine snakes. We also found positive selection at sites within structural loops or "fingers" of 3FTxs, indicating these areas may be key binding sites that interact with prey target molecules. Overall, our results highlight the importance of exploring the venoms of understudied species in reconstructing the full evolutionary history of toxins across the tree of life.

Domain loss enabled evolution of novel functions in the snake three-finger toxin gene superfamily.

Three-finger toxins (3FTXs) are a functionally diverse family of toxins, apparently unique to venoms of caenophidian snakes. Although the ancestral function of 3FTXs is antagonism of nicotinic acetylcholine receptors, redundancy conferred by the accumulation of duplicate genes has facilitated extensive neofunctionalization, such that derived members of the family interact with a range of targets. 3FTXs are members of the LY6/UPAR family, but their non-toxin ancestor remains unknown. Combining traditional phylogenetic approaches, manual synteny analysis, and machine learning techniques (including AlphaFold2 and ProtT5), we have reconstructed a detailed evolutionary history of 3FTXs. We identify their immediate ancestor as a non-secretory LY6, unique to squamate reptiles, and propose that changes in molecular ecology resulting from loss of a membrane-anchoring domain and changes in gene expression, paved the way for the evolution of one of the most important families of snake toxins.

Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin's one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

The structural and functional divergence of a neglected three-finger toxin subfamily in lethal elapids.

Bungarus multicinctus is a widely distributed and medically important elapid snake that produces lethal neurotoxic venom. To study and enhance existing antivenom, we explore the complete repertoire of its toxin genes based on de novo chromosome-level assembly and multi-tissue transcriptome data. Comparative genomic analyses suggest that the three-finger toxin family (3FTX) may evolve through the neofunctionalization of flanking LY6E. A long-neglected 3FTX subfamily (i.e., MKA-3FTX) is also investigated. Only one MKA-3FTX gene, which evolves a different protein conformation, is under positive selection and actively transcribed in the venom gland, functioning as a major toxin effector together with MKT-3FTX subfamily homologs. Furthermore, this lethal snake may acquire self-resistance to its ß-bungarotoxin via amino acid replacements on fast-evolving KCNA2. This study provides valuable resources for further evolutionary and structure-function studies of snake toxins, which are fundamental for the development of effective antivenoms and drug candidates.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

Development of an Inhibition Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detecting Cytotoxic Three-Finger Toxins (3FTxs) in African Spitting Cobra Venoms.

The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (p-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (p-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.

Development of a Broad-Spectrum Antiserum against Cobra Venoms Using Recombinant Three-Finger Toxins.

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins (P01391), short-chain α-neurotoxins (P60770), and cardiotoxin A3 (P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries.