Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom

Abstract

ABSTRACT:Two presynaptic phospholipases A 2 (PLA 2 ), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (μBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10μg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA 2 .

Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

“…In the present work, a myotoxin phospholipase A 2 homologue from Bothrops moojeni venom, denominated BmooMtx, was purified by a combination of ion exchange and gel filtration chromatography (Figure 1 -A and B). The molecular weight observed at SDS-PAGE (16,500) was similar to those reported for MjTX-I (13,400), M-IV (14,000), BmTX-I (14,240) and BmooTX-I (15,000), from the same venom, a presynaptic phospholipase A 2 , neuwieditoxin-I (14,000), isolated from the venom of Bothrops neuwiedi pauloensis and an acidic phospholipase A 2 (14,000) from the venom of Crotalus durissus cascavella (35,36 (Figure 2 -A). These results show that the B. moojeni venom shares some common myotoxins that induce antibodies that crossreact with crude venoms of B. alternatus and B. leucurus.…”

Biochemical Characterization and Pharmacological Properties of New Basic PLA2BrTX-I Isolated fromBothrops roedingeri(Roedinger's Lancehead) Mertens, 1942, Snake Venom

“…The fact that the BrtX-I from Bothrops moojeni did not significantly affect the response to ACh and KCl, except when high doses were used, suggests that the venom presents a primordial presynaptic nature. Such neuromuscular blockade characteristics have been attributed to presynaptic-acting PLA 2 from snake [45, 46] as those of Crotalus durissus terrificus [47], Micrurus species [48, 49], and other Bothrops , Bothrops insularis [50], Bothrops pauloensis [47, 51], and Bothriopsis bilineata smargadina [52], which did not show any detectable effect on the nicotinic receptor and, in some cases, showed only a mild muscle alteration.…”

BmajPLA 2 -II, a basic Lys49-phospholipase A 2 homologue from Bothrops marajoensis snake venom with parasiticidal potential

“…Group II PLA 2 s can display a series of actions that result in toxic effects on victims such as myotoxicity [13], neurotoxicity [14], cytotoxicity [15,16] and genotoxicity [17][18][19]. Several studies focusing on the biological functions of PLA 2 s have discovered essential information of their implication in diseases such as rheumatoid arthritis, inflammation and bone erosion [20], cancer [21,22], and neurological disorders such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and epilepsy [23,24] There are also several studies investigating the antiparasitic effects of PLA 2 s against the parasites that cause leishmaniasis, Chahttp://dx.doi.org/10.1016/j.ijbiomac.2017.04.013 0141-8130/© 2017 Published by Elsevier B.V. gas disease and malaria.…”