Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum.

Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ±â€¯2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus.

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.