Sequences, geographic variations and molecular phylogeny of venom phospholipases and threefinger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A₂

Abstract

Eight phospholipases A2 (PLAs) and four three‐finger‐toxins (3FTx) from the pooled venom of Bungarus fasciatus (Bf) were previously studied and sequenced, but their expression pattern in individual Bf venom and possible geographic variations remained to be investigated. We herein analyze the individual venom of two Bf specimens from Kolkata (designated as KBf) to address this question. Seven PLAs and five 3FTx were purified from the KBf venoms, and respective cDNAs were cloned from venom glands of one of the snakes. Comparison of their mass and N‐terminal sequence revealed that all the PLAs were conserved in both KBf venoms, but that two of their 3FTx isoforms were variable. When comparing the sequences of these KBf‐PLAs with those published, only one was found to be identical to that of Bf Vb‐2, and the other five were 94–98% identical to those of Bf II, III, Va, VI and XI‐2, respectively. Notably, the most abundant PLA isoforms of Bf and KBf venoms contain Pro31 substitution. They were found to have abnormally low kcat values but high affinity for Ca2+. Phylogenetic analysis based on the sequences of venom group IA PLAs showed a close relationship between Bungarus and Australian and marine Elapidae. As the five deduced sequences of KBf‐3FTx are only 62–82% identical to the corresponding Bf‐3FTx from the pooled venom, the 3FTx apparently have higher degree of individual and geographic variations than the PLAs. None of the KBf‐3FTx was found to be neurotoxic or very lethal; phylogenetic analyses of the 3FTx also revealed the unique evolution of Bf as compared with other kraits.

In-vitro Neurotoxicity of Two Malaysian Krait Species (Bungarus candidus and Bungarus fasciatus) Venoms: Neutralization by Monovalent and Polyvalent Antivenoms from Thailand

“…Repetitive washing with physiological salt solution did not reverse the neurotoxic effects of either venom, suggesting that the toxins inducing this effect were irreversible or pseudo-irreversible. It also supports the previous findings on the detection and isolation of potent presynaptic and postsynaptic neurotoxins in B. candidus and B. fasciatus venoms [7,15,29]. Pseudo-irreversible antagonism occurs when the agonist (e.g., acetylcholine released from the nerve terminal) and antagonist (e.g., post-synaptic neurotoxins in the venom) compete for the receptor (e.g., skeletal muscle nicotinic receptor) but the antagonist dissociates from the receptor so slowly ( i.e.…”

“…This may indicate that the composition of the Malaysian krait venoms are slightly different than the venoms used in the production of the antivenoms. Indeed, differences in the composition/activity of B. candidus and B. fasciatus venoms from different populations have been previously reported [7,32]. It is also possible that higher concentrations of antivenoms are required due to the lower immunogenicity of low molecular neurotoxins [22,33] which are responsible for the in vitro effects.…”

“…There are two subspecies of Bungarus flaviceps , Bungarus flaviceps flaviceps and Bungarus flaviceps baluensis [2]. A high number of krait envenoming cases have been reported in India, Sri Lanka, Thailand and Vietnam [3,4,5,6,7,8]. However, envenoming by kraits is relatively uncommon in Malaysia [9,10].…”

“…, presynaptic and postsynaptic neurotoxins [7,12,13]. Presynaptic neurotoxins disrupt the release of the neurotransmitter acetylcholine from the nerve terminal while postsynaptic neurotoxins inhibit the binding of acetylcholine to the nicotinic receptors on the skeletal muscle [12].…”

Development of high-throughput screening assays for profiling snake venom Phospholipase A2activity after high-resolution chromatographic fractionation

“…The presence of these isoforms, and their abundances, can thus vary greatly according to species, but also according to, for example, population, age and sex of a snake, and (prey) ecology. [33][34][35] The generic svPLA 2 tertiary structure, may contain up to seven disulfide bridges. This means that for an enzyme, svPLA 2 s are considered to be rather stable and are therefore relatively resistant to heat, organic solvents, and acidic conditions.…”