Neuromuscular and lethal effects of phospholipase A from Vipera ammodytes venom

Thouin, L.G.; Ritonja, Anka; Gubenšek, Franc; Russell, Findlay E.

doi:10.1016/0041-0101(82)90107-6

Cited by 48 publications

(40 citation statements)

References 8 publications

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“…Specific enzymatic activity of recombinant ammodytoxin A was about 300 U/ mg in the egg yolk assay and its intraperitoneal LDs0 about 0.02 mg/kg of white mice. Almost the same values have been reported for ammodytoxin A isolated from the venom [15].…”

Section: Resultssupporting

confidence: 72%

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli

et al. 1993

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A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia eoli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

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Section: Resultssupporting

confidence: 72%

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli

et al. 1993

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“…The most toxic AtxA is 17 times more toxic than AtxC, a natural AtxA(F124I\K128E) mutant, and 28 times more toxic than AtxB, a natural AtxA(Y115H\R118M\N119Y) mutant [10]. This was later confirmed by site-specific antibodies directed toward the C-terminal-region epitopes, which neutralized the lethality of Atxs [11,12], and by site-directed mutagenesis [13].…”

Section: Introductionmentioning

confidence: 87%

“…The toxicity of the six-site mutant was close to that of AtxB (LD &! 580 µg\kg), a naturally occurring Atx isoform with pI 10.0 [10]. The substitutions drastically lowered the pI of the AtxA molecule from 10.2 to 6.2, but only moderately decreased the toxicity.…”

Section: Figure 2 CD Spectra Of the Atxa(k127t) And Six-site Mutantsmentioning

confidence: 99%

Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity

Prijatelj

Čopič

Križaj

et al. 2000

Biochemical Journal

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The positive charge concentrated at the C-terminal region of ammodytoxin (Atx) A, which is involved in presynaptic toxicity, has been reversed. A six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E, where K108N = Lys108 → Asn etc.) was prepared, in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones. The mutant was approximately 30-fold less lethal, but still neurotoxic. Consistent with this, its binding affinity for the neuronal receptors decreased by only a factor of five. Additionally, a single-site mutant of AtxA was prepared, with substitution at only one position (K127T) out of six mutated in the six-site mutant. Its toxicity indicated that most, if not all, of the six mutated residues partially contribute to the decreased lethality of the multiple-site mutant.

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“…Ammodytoxin (Atx) A is the most potent of three presynaptically toxic PLA # s from the venom of the western sand viper (Vipera ammodytes ammodytes) [13]. Three Atx-binding proteins, R25 [14], R180 [15] and calmodulin [16], have been identified recently in porcine cerebral cortex, and may have a potential role in presynaptic toxicity.…”

Section: Introductionmentioning

confidence: 99%

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

Petan

Križaj

Gubenšek

et al. 2002

Biochemical Journal

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Ammodytoxins (Atxs) are group II phospholipases A2 (PLA2s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA2 toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe24, in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp24 mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40–80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA2 activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe24 also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn24 mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe24 in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.

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Neuromuscular and lethal effects of phospholipase A from Vipera ammodytes venom

Cited by 48 publications

References 8 publications

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli

Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

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Neuromuscular and lethal effects of phospholipase A from Vipera ammodytes venom

Cited by 48 publications

References 8 publications

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli

Charge reversal of ammodytoxin A, a phospholipase A2-toxin, does not abolish its neurotoxicity

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

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Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A₂, in Escherichia coli