Phospholipase A2 family

General Phospholipases A2 (PLA2s) (EC 3.1.1.4) are small (about 14 kDa), stable, calcium-dependent, disulfide-rich enzymes. They degrade membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. They are ubiquitously found in nature ih both intracellular and extracellular forms.

In mammals, PLA2s play important roles in fertilization, cell proliferation, smooth muscle contraction, hypersensitization and chronic inflammatory diseases. They are also important in cellular functions such as signal transduction via biosynthesis of prostaglandins and leukotrienes, and membrane homeostasis including the maintenance of cellular phospholipid pools and membrane repair through deacylation/reacylation (Kini, 2003 ).

Venom PLA2s are produced by almost all venomous animals, with snake venom PLA2s (svPLA2s) being the most studied. svPLA2s, in addition to their possible role in the digestion of the prey, show a wide variety of pharmacological effects. They exhibit pre-/post-synaptic neurotoxicity, myonecrosis, cardiotoxicity, anticoagulant property, inhibition/activation of platelet aggregation, hemorrhage, hemolysis, hypotensive and edema-inducing activities (Kini, 2003 ). It is noteworthy that not all these effects are exhibited by all PLA2 enzymes. This wide range of pharmacological effects has not been explained at the sequence and three-dimensional structure level, since snake venom PLA2s are highly similar. In addition, there is no clear correlation between catalysis and pharmacological activity. Indeed, natural catalytically inactive (Lys49-PLA2s or PLA2 homologs ) and the chemically inactivated svPLA2 enzymes conserve some of their biological activities.

Nomenclature
svPLA2s are divided in two groups. Those of group I are in the same group as pancreatic PLA2s present in mammals and are found in venom of Elapidae, Colubridae and Hydrophiidae snakes, while group II PLA2s belong to the Viperidae and are similar to mammalian non-pancreatic, inflammatory PLA2s.


Activity: myotoxin PLA2s are important myotoxic components in snake and bee venoms, inducing a local and systemic skeletal muscle degeneration. Myotoxic PLA2s bind to acceptors in the plasma membrane, which might be lipids or proteins and which may differ in their affinity for the PLA2s. Upon binding, myotoxic PLA2s disrupt the integrity of the plasma membrane by catalytically dependent or independent mechanisms, provoking a pronounced calcium influx which, in turn, initiates a complex series of degenerative events. Cell culture models of cytotoxicity indicate that some myotoxic PLA2s affect differentiated myotubes in a rather selective fashion, whereas others display a broad cytolytic effect. This difference of selectivity may explain the difference between PLA2s that induce predominantly local myonecrosis and those inducing both local and systemic myotoxicity. The former bind not only to muscle cells, but also to other cell types, thereby precluding a systemic distribution of these PLA2s and their action on distant muscles. In contrast, PLA2s that bind muscle cells in a more selective way are not sequestered by non-specific interactions with other cells and, consequently, are systemically distributed and reach muscle cells in other locations (Gutierrez and Ownby, 2003, Gutierrez et al., 2008 ).
Activity: postsynaptic neurotoxin Only a few PLA2s have been described as being postsynaptic neurotoxins. Bitanarin is the first postsynaptic neurotoxin PLA2 that has been shown to competitively block acetylcholine receptors (Vulfius et al., 2011 ). Other postsynaptic neurotoxins were described in the 1970s, such as the heterodimers crotoxin and vipoxin. Crotoxin was initially shown to cause blockade principally via a postsynaptic action (Brazil, 1966 ). However, although subsequent studies confirmed this postsynaptic activity, several studies demonstrated that the principal site of action of crotoxin at neuromuscular junctions was presynaptic (Sampaio et al., 2010 ). Vipoxin is another example of a neurotoxin described in the 1970s to have postsynaptic activities (Tchorbanov et al., 1978, Petrova et al., 2012 ).
Activity: presynaptic neurotoxin Presynaptically acting PLA2 neurotoxins interfere specifically with the release of acetylcholine from motor neurons, and their PLA2 activity is essential for the irreversible blockade of neuromuscular transmission. It has been suggested that structurally different PLA2 neurotoxins from different snake venoms bind to different receptors on the presynaptic membrane and enter the nerve ending through different import systems. In the nerve cell, they may impair the cycling of synaptic vesicles by phospholipid hydrolysis and by binding to specific protein targets such as calmodulin and 14-3-3 proteins in the cytosol and R25 in mitochondria. Electron microscopy studies of neuromuscular junctions treated with different presynaptically neurotoxic PLA2s as well as their triphasic effect on acetylcholine release suggest that these neurotoxins promote synaptic vesicle exocytosis but inhibit their retrieval from the presynaptic membrane (Petan et al., 2005 ).
Ammodytoxin A is a well-studied presynaptic neurotoxic PLA2.
Activity: blood coagulation cascade inhibiting toxin Although anticoagulant properties of several PLA2 enzymes have been studied, detailed mechanisms of the anticoagulant activities are not well known. The most common mechanism of this effect is binding to the activated coagulation factor FXa, thus inhibiting the formation of the prothrombinase complex. Another less-studied mechanism consists of the inhibition of the complex composed of tissue factor and coagulation factor VII (Kini, 2005 ).
Activity: hemolysis Hemolysis achieved by PLA2s can be explained by a direct lysis in consequence of PLA2 activity (enzymatic hydrolysis of red blood cell membranes), an indirect lysis due to disruption of the cell membrane by hydrolysis products, and a simultaneous direct and indirect lysis (Stoykova, et al., 2013 ).
Activity: platelet aggregation activating toxin PLA2s can induce platelet aggregation by release of arachidonic acid, which is generally found at the sn-2 position of membrane phospholipids. It is important to note that not all PLA2s affect platelet aggregation, despite their common catalytic activity (Kini and Evans, 1997).
Nomenclature
According to the classification of Kini and Evans (1997), PLA2s that solely initiate platelet aggregation belong to class A, whereas PLA2s that exhibit biphasic effects, i.e. they initiate aggregation at low concentration or with short incubation times, but inhibit platelet aggregation at high concentrations or with long incubation times belong to class C. Class C is divided in two groups. Group C1 shows dose-dependent biphasic effects, whereas group C2 shows no dose-dependent biphasic effects.
Activity: platelet aggregation inhibiting toxin PLA2s can inhibit platelet aggregation by physical destruction of the integrity of the platelet membrane via hydrolysis of the membrane phospholipids, which could affect the functions of receptors that play important roles in platelet aggregation. It is important to note that not all PLA2s affect platelet aggregation, despite their common catalytic activity (Kini and Evans, 1997).
Nomenclature
According to the classification of Kini and Evans (1997), PLA2s that solely inhibit platelet aggregation belong to class B, whereas PLA2s that exhibit biphasic effects, i.e. they initiate aggregation at low concentration or with short incubation times, but inhibit aggregation at high concentration or with long incubation times belong to class C. Classes B and C are both divided in two groups. The anti-platelet effects of group B1 are dependent on the enzymatic activity, whereas group B2 inhibit platelet aggregation by non-enzymatic mechanism, i.e. independent of phospholipid hydrolysis. Group C1 shows dose-dependent biphasic effects, whereas biphasic effects of group C2 are not dose-dependent.