ABSTRACT
This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 µg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic ß-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.
Subject(s)
Coagulants/chemistry , Elapid Venoms/chemistry , Elapidae/genetics , Neurotoxins/chemistry , Peptide Fragments/chemistry , Serine Endopeptidases/chemistry , Amino Acid Sequence , Animals , Australia , Birds , Coagulants/isolation & purification , Coagulants/metabolism , Coagulants/toxicity , Computational Biology/methods , Diaphragm/drug effects , Diaphragm/physiology , Elapid Venoms/genetics , Elapid Venoms/isolation & purification , Elapid Venoms/metabolism , Elapid Venoms/toxicity , Elapidae/classification , Evolution, Molecular , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Gene Expression , Molecular Sequence Data , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Neuromuscular Junction/drug effects , Neuromuscular Junction/physiology , Neurotoxins/genetics , Neurotoxins/isolation & purification , Neurotoxins/toxicity , Peptide Fragments/isolation & purification , Phrenic Nerve/drug effects , Phrenic Nerve/physiology , Rats , Sequence Alignment , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , Serine Endopeptidases/toxicity , Species Specificity , Trypsin/chemistryABSTRACT
This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD) venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality) and Queensland (Mackay locality) populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver.
