Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis

Abstract

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A 2 s, and preand postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, ␥-carboxylation, and glycosylation. Snakes from the Australian elapid genus Pseudonaja are fast moving and highly venomous and are responsible for most deaths by snake bite in Australia. Of the eight classified species of Pseudonaja, Pseudonaja textilis, the Common or Eastern Brown snake, has the most lethal venom, surpassed only by Oxyuranus microlepidotus, the Inland Taipan. The venom is strongly procoagulant and has little hemolytic or myolytic activity (1). Several researchers have examined the venom from P. textilis and have isolated numerous toxins.These include postsynaptic ␣-neurotoxins (2-4), the presynaptic neurotoxin textilotoxin (5-7), phospholipase A 2 s (PLA 2 s) 1 (6, 8); serine protease inhibitors (9, 10), and a prothrombin activator (11, 12). Fry (13) has written a comprehensive review on the properties of venom components from Australian elapids.Barnett et al.(2) isolated a postsynaptic ␣-neurotoxin from the venom and named it pseudonajatoxin A. This protein is 117 amino acids in length, which is considerably larger than other snake neurotoxins, and like other ␣-neurotoxins acts by binding to acetylcholine receptors. Pseudonajatoxin B by contrast (3) contains only 71 amino acids and has considerable homology with other postsynaptic long ␣-neurotoxins. Six other ␣-neurotoxins have been cloned and expressed from venom gland RNA (4). These have fewer amino acids (57 or 58 residues) and possess lower neurotoxic activities than the short-chain ␣-neurotoxins found in other snakes. Textilotoxin is the most potent neurotoxin yet isolated from the venom of a land snake. It represents 3% by weight of the crude venom and 70% of its total lethality (1). It is comprised of five PLA 2 subunits, two of which are identical, and acts by blocking the release of acetylcholine following the arrival of an action potential at a nerve terminal (14). Armugam et al. (8) isolated four other PLA 2 s from the venom and found them to be group 1B PLA 2 s. In that study, analysis of DNA from the venom gland showed that only two genes and two cDNAs were responsible for the four PLA 2 proteins produced. This is similar to the short-chain ␣-neurotoxins (4) where alternative splicing of the P....

Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

“…Lately, 2DE has been the preferred technique used by several researchers to decipher the complexity of venoms due to its capability of separating a mixture of proteins based on two parameters, pI and MW (15)(16)(17)(18)(19). Owing to the close proximity of molecular weights among toxins bearing different net charges, SDS-PAGE was not able to provide efficient differentiation (6).…”

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity

“…Retained phospholipid is unlikely to have in vivo significance because P textilis venom proteins include phospholipases, which seem likely to degrade any retained phospholipid and therefore inhibit corresponding activity. 52 More interesting is the activity of FV MTTS/Y on platelets and membranes lacking PS, a lipid that is normally critical for full FV function. These results suggest that Pt-FV may be responsible for the ability of pseutarin C to activate prothrombin on cell membranes that would normally not support significant coagulation activity, such as unstimulated platelets.…”

The Role of Conventional Two-Dimensional Electrophoresis (2DE) and Its Newer Applications in the Study of Snake Venoms

“…Specifically in the field of venom proteome study, the 2DE technique remains important when it comes to the study of (1) subpopulation of venom proteins and (2) post-translational modifications (PTM) in venom proteins. In two studies that were done in similar manner, Serrano et al (2005) and Birrell et al (2006) investigated the diversity of venom proteins in the viperid venoms (Bothrops jararaca and Crotalid atrox) and the Australian Brown Snake venom (Pseudonaja textilis) respectively. Taking advantage of the efficient interface of 2DE with other biochemical techniques, the two groups of researchers subjected the obtained 2DE profiles to immunoblot analysis with antisera raised against specific venom protein groups that were of study interests.…”

“…Protein spots that have undergone PTM appeared as train of spots on the 2DE profiles, owing to their differences in pI values that gave rise to their non-identical migration profiles (Birrell et al, 2006). Both groups employed specific fluorescent dyes, Pro-Q Emerald and Pro-Q Diamond, for the study of post-translational modifications (PTM) in venom proteins (Birrell et al, 2006;Serrano et al, 2005). Apart from specific staining methods, other modifications can also be applied on the conventional 2DE protocol for the study of PTM.…”

“…Serrano et al (2005) did immunoblot analysis with antisera raised against metalloproteinase's, serine protease and phospholipase A2. Birrell et al (2006), on the other hand, did immunoblot analysis with antisera raised against prothrombinase complex, heavy chain of the Factor Xa-like protease, GIa residues, textilinins and textilotoxins. The results were specific 2DE profiles of these protein groups, giving rise to a more thorough understanding of venom complexity and providing insights for investigators who want to focus on these subpopulations of proteins in future studies.…”