Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema

The three-finger toxins (3FTxs) represent an extremely diverse protein family in elapid venoms, where the short chain α-neurotoxins are the most relevant toxin group from the clinical point of view. Essentially, the 3FTxs variability and the low proportions of α-neurotoxins in the venoms of North American coral snakes make it difficult to obtain effective elapid antivenoms against the envenomation symptoms caused mainly by these α-neurotoxins. In this work, thirty 3FTx transcript sequences were obtained from the venom glands of four coral snake species from Mexico (M. diastema, M. laticollaris, M. browni and M. tener). The transcripts were mined using a forward oligonucleotide based on the highly conserved signal peptide from the 3FTxs, and four of these transcripts, named MlatA1, B.D, B.E and D.H, encoded for short-chain α-neurotoxins. Additionally, one isoform of the D.H α-neurotoxin transcript was identified in the venom of M. diastema. The mature α-neurotoxin coded in the D.H transcript was heterologously expressed, and it was found soluble (4.2 mg/l) in the cytoplasm of a bacterial system. The recombinant D.H (rD.H) had an IC50 value of 31.5 ± 4.4 nM on nicotinic acetylcholine receptors of the muscular type expressed in rhabdomyosarcoma cells (TE671). The rDH also had an LD50 of 0.15 mg/kg mice, and it was evaluated as a potential immunogen in New Zealand rabbits. The protective capacity of rabbit sera was tested against two native coral snake α-neurotoxins, and against rD.H. One of the anti-rD.H rabbit sera was able to neutralize the lethality of all three neurotoxins when tested in groups of CD1 mice. This work contributes to the increasing understanding of the high diversity of 3FTxs, and shows that recombinant coral snake α-neurotoxins are promising supplements for hyperimmunization protocols for coral snake antivenom production.