Ammodytoxins, Potent Presynaptic Neurotoxins, Are Also Highly Efficient Phospholipase A₂ Enzymes

Abstract

The enzymatic activity of ammodytoxins (Atxs), secreted phospholipases A(2) (sPLA(2)s) in snake venom, is essential for expression of their presynaptic neurotoxicity, but its exact role in the process is unknown. We have analyzed in detail the enzymatic properties of Atxs, their mutants, and homologues. The apparent rates of phospholipid hydrolysis by the sPLA(2)s tested vary by up to 4 orders of magnitude, and all enzymes display a strong preference for vesicles containing anionic phospholipids, phosphatidylglycerol or phosphatidylserine (PS), over those containing zwitterionic phosphatidylcholine (PC). Nevertheless, Atxs are quite efficient in hydrolyzing pure PC vesicles as well as PC-rich plasma membranes of intact HEK293 cells. The presence of anionic phospholipids in PC vesicles dramatically increases the interfacial binding affinity and catalytic activity of Atxs, but not of their nontoxic homologue ammodytin I(2), that displays unusually low binding affinity and enzymatic activity on PS-containing vesicles and HEK293 plasma membranes. Aromatic and hydrophobic residues on the interfacial binding surface of Atxs are important for productive binding to both zwitterionic and anionic vesicles, while basic and polar residues have a negative impact on binding to zwitterionic vesicles. When tightly bound to the membrane interface, Atxs can reach full enzymatic activity at low micromolar concentrations of Ca(2+). Although Atxs have evolved to function as potent neurotoxins that specifically target presynaptic nerve terminals, they display a high degree of phospholipolytic efficiency on various phospholipid membranes.

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

“…It is highly likely that the primary target, or acceptor, sites are proteins. This is because of the much lower affinity (mM-µM range) of sPLA 2 s for binding to the abundant zwitterionic phospholipid surfaces (i.e., cell membranes) Petan et al, 2005;Singer et al, 2002) and, following enzyme adsorption to the membrane surface, indiscriminate binding to and hydrolysis of phospholipid molecules at the catalytic site. Therefore, separate pharmacological sites on an sPLA 2 molecule recognizing different target binding sites should be the main structural determinants that differentiate their respective pharmacological actions, such as presynaptic or central neurotoxicity.…”

“…Indeed, there are numerous examples of sPLA 2 ß-ntxs with significantly different enzymatic properties, which are, however, not reflected in differences in toxicity; in fact even the most potent sPLA 2 ß-ntxs are weak enzymes (Petan et al, 2005;Pražnikar et al, 2008;Prijatelj et al, 2006bPrijatelj et al, , 2008Rosenberg, 1997). Nevertheless, sPLA 2 enzymatic activity is necessary for full expression of the ß-neurotoxic effect .…”

“…The enzymatic action of sPLA 2 s could lead to structural and functional destruction of cell membranes and organelles, like mitochondria or synaptic vesicles Rigoni et al, 2008), since the products of phospholipid hydrolysis are disruptive to many physiological processes by impairing the function of peripheral and integral membrane proteins and promoting membrane dysfunction by altering membrane asymmetry, curvature and fusogenicity Paoli et al, 2009;Rigoni et al, 2005). The apparent lack of correlation between in vitro enzymatic activity and lethal potency of Atxs or other neurotoxic sPLA 2 s Petan et al, 2005; can be explained by the strict localization of the sPLA 2 activity to particular target membrane(s) due to binding to highly specific extra-and intracellular protein acceptors (Paoli et al, 2009;Petan et al, 2005;. Our studies investigating the interfacial binding and kinetic properties of toxic sPLA 2 s and their mutants have provided important clues to understanding the role of enzymatic activity in the process of presynaptic neurotoxicity of sPLA 2 s. As described in detail below, despite their potent neurotoxic activity, Atxs are quite effective in hydrolysing pure phosphatidylcholine (PC) vesicles as well as PC-rich plasma membranes of mammalian cells, similarly to the most active mammalian group V and X sPLA 2 enzymes (Petan et al, 2005(Petan et al, , 2007.…”

“…The apparent lack of correlation between in vitro enzymatic activity and lethal potency of Atxs or other neurotoxic sPLA 2 s Petan et al, 2005; can be explained by the strict localization of the sPLA 2 activity to particular target membrane(s) due to binding to highly specific extra-and intracellular protein acceptors (Paoli et al, 2009;Petan et al, 2005;. Our studies investigating the interfacial binding and kinetic properties of toxic sPLA 2 s and their mutants have provided important clues to understanding the role of enzymatic activity in the process of presynaptic neurotoxicity of sPLA 2 s. As described in detail below, despite their potent neurotoxic activity, Atxs are quite effective in hydrolysing pure phosphatidylcholine (PC) vesicles as well as PC-rich plasma membranes of mammalian cells, similarly to the most active mammalian group V and X sPLA 2 enzymes (Petan et al, 2005(Petan et al, , 2007. We have also shown that, when tightly bound to the membrane surface, the Ca 2+ requirements of Atxs are in the micromolar range (Petan et al, 2005), opening up the possibility that such neurotoxins are also catalytically active in the subcellular compartments where Ca 2+ concentrations are low (Kovačič et al, 2009;Petan et al, 2005).…”

“…Our studies investigating the interfacial binding and kinetic properties of toxic sPLA 2 s and their mutants have provided important clues to understanding the role of enzymatic activity in the process of presynaptic neurotoxicity of sPLA 2 s. As described in detail below, despite their potent neurotoxic activity, Atxs are quite effective in hydrolysing pure phosphatidylcholine (PC) vesicles as well as PC-rich plasma membranes of mammalian cells, similarly to the most active mammalian group V and X sPLA 2 enzymes (Petan et al, 2005(Petan et al, , 2007. We have also shown that, when tightly bound to the membrane surface, the Ca 2+ requirements of Atxs are in the micromolar range (Petan et al, 2005), opening up the possibility that such neurotoxins are also catalytically active in the subcellular compartments where Ca 2+ concentrations are low (Kovačič et al, 2009;Petan et al, 2005). Moreover, Atxs are rapidly internalized in motoneuronal cells and are, surprisingly, translocated to the cytosol, where they specifically bind calmodulin (CaM) and 14-3-3 proteins, strongly opposing the dogma of the exclusively extracellular action of not only sPLA 2 -neurotoxins, but also of sPLA 2 s in general .…”