Abstract
The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.
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Acknowledgements
This work received funding from the Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) Grant number IN203118, and from SEP-CONACyT grant number 240616 awarded to GC. We acknowledge Dr. Alejandro Alagón and BSc. Felipe Olvera for providing elapid venoms and technical support, respectively. The patent filling advice from MBA Mario Trejo and M.Sc. Martin Patiño is also greatly acknowledged. We greatly appreciate the English grammar edition from Dr. Christopher David Wood. GDLR is a doctoral student from “Programa de Doctorado en Ciencias Bioquímicas” at the Universidad Nacional Autónoma de México (UNAM) and received a fellowship (No. 367094) from CONACYT.
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GD designed, expressed and evaluated, biologically and immunologically, the ScNtx, and wrote the manuscript; LCG constructed and cloned the ScNtx; XRR and ELV performed the pharmacological characterization; GC designed CD experiments, reviewed and wrote the manuscript.
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de la Rosa, G., Corrales-García, L.L., Rodriguez-Ruiz, X. et al. Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties. Amino Acids 50, 885–895 (2018). https://doi.org/10.1007/s00726-018-2556-0
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DOI: https://doi.org/10.1007/s00726-018-2556-0