Dr. Toni Petan

Department of Molecular and Biomedical Sciences
Jozef Stefan Institute
Jamova 39
SI-1000 Ljubljana

tel.: +386 1 477 33 06
fax: +386 1 477 39 84

email: toni.petan{at}ijs.si


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Neurotoxic and Enzymatic Action of Viper Venom Phospholipases A2.


(Toni Petan, PhD thesis summary)


Ammodytoxins (Atxs) are presynaptically neurotoxic secreted phospholipases A2 (sPLA2s) from venom of the long-nosed viper, Vipera ammodytes ammodytes. Ammodytin (Atn) I2 is a non-toxic sPLA2 from the same venom, while AtnL is a myotoxic sPLA2 homologue without enzymatic activity. Although the exact role of enzymatic activity in the process of presynaptic neurotoxicity of sPLA2s is still unknown, it has been shown that it is essential for full expression of the neurotoxic effect. In order to determine the role of different hydrophobic and aromatic residues on the interfacial binding surface (IBS) of Atxs, we prepared a number of recombinant mutant proteins of AtxA. By replacing several residues in the active site and calcium-binding loop of AtnL, we successfully prepared two enzymatically active mutants, which differed only in the substitution V31W. By carefully analysing the enzymatic characteristics of 16 wild-type and mutant snake venom sPLA2s of group IIA (Atxs, Atns and a weakly neurotoxic sPLA2 from the Russell's viper), we searched for possible differences in the enzymatic action of neurotoxic and non-neurotoxic sPLA2s.

          In this study, we show that Atxs are very efficient enzymes when acting on anionic as well as on zwitterionic aggregated phospholipid substrates. Anionic phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles are very good general substrates for Atxs. The specific enzymatic activities of our snake venom sPLA2s on vesicles composed of neutral phosphatidylcholine (PC) molecules were up to five orders of magnitude lower than that determined on anionic vesicles. The range of activities of the 16 spla2s on anionic PG and PS vesicles varied by up to 11- and 34-fold, respectively, while the activities on neutral PC vesicles showed a much higher variability and differed by up to 20,000-fold.

          The addition of anionic PS phospholipids to zwitterionic PC vesicles induced an increase in membrane-binding affinity leading to higher enzymatic activities of our enzymes. The membrane-binding affinities of sPLA2s determined on non-hydrolysable PS/PC vesicles correlated well with the enzymatic activities determined on hydrolysable vesicles of equal compositions. Certain aromatic IBS residues (Phe24, Phe124, Trp31 and Tyr119) have a very important role in productive binding of Atxs to both neutral and anionic membrane surfaces. However, the relative contribution of each single aromatic residue to membrane binding depends upon its location on the IBS, the orientation of its side-chain and the contribution of other IBS residues. Additionally, the presence of polar and basic residues at certain positions (24, 118 and 119) on the IBS of the snake venom sPLA2s can have a marked negative impact on membrane binding to PC-rich vesicles and consequently on their enzymatic activity. Phe24 plays an important role in both enzymatic activity and neurotoxicity of AtxA, but it has no influence on binding to two specific neuronal receptors, R25 and R180, and to calmodulin. The nature of the residue at position 31 can have a dramatic influence on enzymatic activity of Atxs, but it has a minor role in their neurotoxicity. In addition to enzymatic activity, the two AtnL mutants may have also acquired the ability to act as neurotoxins. Therefore, the marked difference in their toxicity could be a consequence of their 40-fold difference in enzymatic activity on zwitterionic membrane surfaces.

          Atxs can, surprisingly, reach full activity in the presence of low micromolar, and not necessarily milimolar as previously believed, concentrations of Ca2+ and they show relatively high stability in a highly reducing environment, comparable to that of the cytosol of mammalian cells. The relatively low calcium affinity of both non-toxic AtnI2 and F24N, a mutant of AtxA which displayed a 133-fold decrease in toxicity, indicate a possible role of calcium activation in the process of neurotoxicity. AtnI2 is unique among V. a. ammodytes sPLA2s in having low membrane binding affinity and low enzymatic activity on the PS-containing vesicles, as well as low affinity for Ca2+. The neutral IBS of AtnI2 and its lack of basic residues in comparison to highly basic Atxs may be crucial for its inability to form extensive non-specific electrostatic interactions with anionic membrane surfaces. Additionally, its low enzymatic activity on PS-containing membranes may reflect a weaker binding of the anionic head-group of PS in the active site of AtnI2.

          While Atxs are very efficient enzymes in comparison to the whole range of mammalian (non-toxic) sPLA2s, they also exhibit a very potent and specific neurotoxic action. Their high binding affinity and high enzymatic activity on different membrane surfaces, as well as their ability to act at low Ca2+ concentrations and in highly reducing conditions, is most probably of crucial importance in a specific, yet unidentified, step in the process of presynaptic neurotoxicity. If sPLA2s do enter the cytosol of nerve terminals during their neurotoxic action, they would be restrained in time (reducing environment) and space (local Ca2+ concentrations). In this case, besides the low affinity of AtnI2 for Ca2+, its low affinity for binding to PS-containing membrane surfaces could also play a critical role in its inability to act as a neurotoxin.


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