Crotoxin (CTX) is the main toxic compound in the snake venom of the South American rattlesnake, Crotalus durissus terrificus. Crotoxin is a heterodimeric beta-neurotoxin, composed of an acidic, non-toxic and non-enzymatic subunit (CA), and a basic, weakly toxic, phospholipase A2 protein (CB). This neurotoxin causes paralysis by both pre- and postsynaptic blocking of acetylcholine signalling.
History
Crotoxin was identified in 1938 by researchers of the Department of Chemistry of the Instituto Butantan in São Paulo. The compound was first purified from the venom of Crotalus d. terrificus. These researchers found that 60 per cent of the venom consisted of a neurotoxic substance, later referred to as crotoxin.
Crotoxin was the first proteinic toxin to be crystallized (Protein crystallization). The first publication of this discovery already proved that the toxin worked with two elements, a toxic and a coagulating principle. Later was discovered that the crotoxin protein is not homogeneous, but consists of 2 subunits. The toxic effect of crotoxin is determined mainly by the phospholipase A2 action of CB. The CA subunit is non-enzymatic and non-toxic, but has blood coagulating functions (Coagulation), now known as crotapotin. Since 1966 until today, investigations into pharmacological applications for crotoxin are conducted.
Structure and reactivity
The structure of crotoxin is composed by the components CA and CB in a 1:1 molecular ratio.܁CA is a nontoxic and non-enzymatic acidic protein while CB is the toxic component, a phospholipase A2 protein. Both components form a noncovalent heterodimeric complex (Protein dimer). It was found that isoforms (Protein isoform) of CA and CB can form at least 16 distinct CTX complexes.
The CA subunit
The CA protein is formed by three disulfide-bonded polypeptide chains: α, β and γ. Alpha-helices (Alpha helix) with loops at the terminal positions are formed by the α and β chains. The γ chain forms a disordered loop. Component CA is present in the heterodimeric complex to prevent the binding of the phospholipase A2 to nonspecific binding sites.
The CB subunit
The CB subunit is a phospholipase A2 protein. The C-terminal (C-terminus) of the CB subunit is important for the interaction between both subunits as it interacts with an alpha helix of CA. The CA subunit thereby blocks a part of the enzyme surface of phospholipase A2, resulting in an impossibility to be activated. This means the phospholipase A2 cannot adsorb onto a lipid/water interface from the cell membrane. Residues on the CB subunit which are involved in the enzyme surface and blocked by the CA subunit are F24 and F119, which are phenylalanine amino acids. It however was found that these residues are not part of the active site.
The interface between CA and CB is formed by three tryptophan amino acids which play an important role in the stability of the crotoxin complex.
Reactivity
The different isoforms of both subunits CA and CB can form crotoxin complexes which can be subdivided into two classes: moderately toxic with a high phospholipase A2 activity or more toxic with a lower enzymatic activity. The isoforms thereby also play a role in the stability of the crotoxin complex. Less toxic complexes are less stable while the more toxic complexes are more stable. The more toxic crotoxin complexes therefore dissociate more slowly than the less toxic ones. The relation between toxicity and enzym activity is a result of the synergistic manner (Synergy) of action of both subunits. For this the CA subunit enhances the toxicity of the CB subunit while it reduces its enzyme activity and anticoagulant activity.
Mechanism of action
The primary mechanistic action of crotoxin is twofold, a presynaptic and a postsynaptic action.
Presynaptic
The presynaptic acetylcholine secretion inhibition is caused by the phospholipase A2 activity of the CB subunit. The exact mechanism of presynaptic acetylcholine inhibition by Phospholipase A2 (PLA2) is not known. While the presynaptic effect is caused by the CB subunit, it is enhanced by the presence of the CA subunit.
Hypotheses
There are some hypotheses and models that try to explain the pharmacological effects of phospholipase A2 (PLA2) activity.