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Dr.
Toni Petan
Department
of Molecular and Biomedical Sciences
email:
toni.petan |
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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