Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes

Abstract

Phospholipases A inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A (PLAs) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment.

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum

“…erythromelas and B . neuwiedi [ 32 ]), the rattlesnake ( Crotalus durissus terrificus [ 33 ] and Crotalus durissus collineatus [ 34 ]), the bushmaster snake ( Lachesis muta muta [ 35 ]), the coral snake ( Micrurus lemniscatus [ 36 ]) and the habu snake ( Trimeresurus flavoviridis [ 37 ]); as well as in the plasma of some non-venomous snakes as the Akamata ( Dinodon semicarinatus [ 38 ]), the Japanese striped snake ( Elaphe quadrivirgata [ 39 , 40 ]) and the Japanese rat snake ( Elaphe climacophora [ 41 ]).…”

“…γPLI has 30–90 kDa, with 3–6 non-covalent subunits of 15–31 kDa, which are composed of highly conserved structural units of two tandem cysteine repeats, characteristic of the three-finger motifs [ 42 ]. Although several PLIs have been identified and their sequences have been described through molecular techniques (such as cDNA sequencing), studies showing the isolation and characterization of these molecules from snake and other animals serum or plasma are scarce [ 32 , 35 , 36 , 44 ].…”

Gene Expression and Sequencing Techniques of Transcriptome to Elucidate Novel Function for Phospholipase (S) Enzyme

“…PLA 2 inhibitors (PLIs) isolated from snake blood may present differences among different snake species PLIs [20]. These molecules are oligomeric, globular, acidic glycoproteins with molecular mass ranging between 75 and 180 kDa.…”

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

“…SbγPLIs are the most widely distributed inhibitors among elapid and viperid species from the Old and New World [12,13,14]. Concerning non-venomous snakes, as far as we know, until now sbγPLIs were solely purified from Asian species [4,5,6,7,8,9].…”