Characterization of α‐Neurotoxin and Phospholipase A₂ Activities from Micrurus Venoms

Abstract

New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for a-neurotoxin and phospholipase A, activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A, activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven a-neurotoxins, five phospholipases A, and four toxin homologs. The amino acid sequence of the major a-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain a-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.Keywords: Micrurus; a-neurotoxin; phospholipase A, ; venom.The elapid representatives of the New World are coral snakes. They comprise a group of about 120 species and subspecies (genera Micrurus, Leptomicrurus and Micruroides) distributed from Southern United States to Argentina (Mc Dowell, 1987 ;Campbell and Lamar, 1989). Clinical observations following bites show mainly neurotoxic effects, and in severe cases of envenomation, death due to respiratory arrest (Weis and Mc Isaac, 1971;Ramsey et al., 1972;Vital Brazil et al., 1976Moussatche and Melendez, 1979). Experimental studies suggest an extended spectrum of activities in Micrurus venoms. Neurophysiological changes similar to those induced by a-neurotoxins have been observed, as well as presynaptic effects (Parmentier and Carpenter, 1976;Vital Brazil and Fontana, 1983, 1984;Goularte et al., 1995). Cardiotoxic, haemolytic and myotoxic activities have been reported also (GutiCrrez et al., 1980(GutiCrrez et al., , 1983(GutiCrrez et al., ,1986(GutiCrrez et al., ,1992Tan and Ponnudurai, 1992). The major obstacle to study Micrurus venoms is the small quantity of material that can be collected (BolanBs, 1972). Thus, the biochemistry and the pharmacology of components isolated from coral snake venoms are not well documented. After the first attempts to purify venom proteins (Ramsey et al., 1971), only two phospholi- et al., 1979;Arroyo et al., 1987). More recently, a comparative chromatography study on Brazilian coral snake venoms has been conducted (Da Silva et al., 1991). From an immunological point of view, Micrurus and other elapid venoms contain some crossreacting components (Minton, 1979;Alape-Girbn et al., 1...

MmTX1 and MmTX2 from coral snake venom potently modulate GABA A receptor activity

“…Next, the fraction at 58.70 min was transferred to a cation exchange 7.5 × 75 mm TSKSP-5 PW column (Beckman) and eluted after a 10-min step with a 0-30% linear gradient of 0.5 M ammonium acetate in 0.005 M ammonium acetate at pH 8.0 at a flow rate of 0.5 mL/min. Edman sequencing was carried out on a cysteine-reduced and s-carboxymethylated derivative (22). Toxin molecular masses were determined using electrospray ionization quadripole time of flight Ultima (Waters) with MaxEnt for spectra deconvolution.…”

“…While examining the venom of Costa Rican coral snakes (22), we came across a major fraction that displayed evidence of GABA A -related toxicity in mice. Within this fraction, we found micrurotoxin1 (MmTX1) and MmTX2, two equally potent peptides with a primary sequence belonging to the PATE-SLURP1-LYNX1-Ly-6/neurotoxin-like family (23)(24)(25).…”

Neutralization of two North American coral snake venoms with United States and Mexican antivenoms

“…1). Envenomation by these snakes can cause death due to the alpha neurotoxins that cause muscle paralysis and respiratory arrest due to postsynaptic, nondepolarization blockage at the neuromuscular junction by binding competitively to the acetylcholine receptor (Lee, 1970 and1972;Pettigrew and Glass, 1985;Vital-Brazil, 1987;Alape-Giron et al, 1996;Rosso et al, 1996;Francis et al, 1997 ). The current NACSA will no longer be available after October 2008; and therefore, it is important to determine the ability of other coral snake antivenoms to neutralize the toxic effects of the North American coral snake venoms.…”

Comparative study on the ability of IgG and F(ab')2 antivenoms to neutralize lethal and myotoxic effects induced by Micrurus nigrocinctus (coral snake) venom.

“…[10][11][12] Although poorly studied, Micrurus venoms induce typical neurotoxic effects, with blockade of the neuromuscular synapse by ␣-neurotoxins that bind cholinergic receptors at the motor endplate. [13][14][15] Micrurus ␣-neurotoxins are similar to those of other elapids. 14,15 They are low molecular weight proteins that rapidly spread throughout tissues and bind acetylcholine receptor (AchR) with very high affinity.…”

“…[13][14][15] Micrurus ␣-neurotoxins are similar to those of other elapids. 14,15 They are low molecular weight proteins that rapidly spread throughout tissues and bind acetylcholine receptor (AchR) with very high affinity. 16 Moreover, Micrurus venoms also contain myotoxic phospholipases A 2 .…”