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Toxins not neutralized by brown snake antivenom
Toxicology and Applied Pharmacology 213 (2006) 117 – 125 www.elsevier.com/locate/ytaap
Toxins not neutralized by brown snake antivenom Roopwant K. Judge a, Peter J. Henry b, Peter Mirtschin c, George Jelinek d, Jacqueline A. Wilce e,* a
Molecular Genetics and Evolution Group, Research School of Biological Sciences, Australian National University, Canberra 2601, ACT, Australia b School of Medicine and Pharmacology, University of Western Australia, Crawley 6009, Western Australia, Australia c Venom Supplies, Tanunda 5352, South Australia, Australia d School of Primary, Aboriginal and Rural Health Care, QEII Medical Centre, Western Australia, Australia e Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, VIC Victoria, Australia Received 13 July 2005; revised 29 August 2005; accepted 16 September 2005 Available online 26 October 2005
Abstract The Australian snakes of the genus Pseudonaja (dugite, gwardar and common brown) account for the majority of snake bite related deaths in Australia. Without antivenom treatment, the risk of mortality is significant. There is an accumulating body of evidence to suggest that the efficacy of the antivenom is limited. The current study investigates the protein constituents recognized by the antivenom using 2-DE, immuno-blot techniques and rat tracheal organ bath assays. The 2-DE profiles for all three snake venoms were similar, with major species visualized at 78 – 132 kDa, 32 – 45 kDa and 6 – 15 kDa. Proteins characterized by LC-MS/MS revealed a coagulant toxin (¨42 kDa) and coagulant peptide (¨6 kDa), as well as two PLA2 (¨14 kDa). Peptides isolated from ¨78 kDa and 15 – 32 kDa protein components showed no similarity to known protein sequences. Protein recognition by the antivenom occurred predominantly for the higher molecular weight components with little recognition of 6 – 32 kDa MW species. The ability of antivenom to neutralize venom activity was also investigated using rat tracheal organ bath assays. The venoms of Pseudonaja affinis affinis and Pseudonaja nuchalis incited a sustained, significant contraction of the trachea. These contractions were attributed to PLA2 enzymatic activity as pre-treatment with the PLA2 inhibitor 4-BPB attenuated the venom-induced contractions. The venom of Pseudonaja textilis incited tracheal contractility through a non-PLA2 enzymatic activity. Neither activity was attenuated by the antivenom treatment. These results represent the first proteomic investigation of the venoms from the snakes of the genus Pseudonaja, revealing a possible limitation of the brown snake antivenom in binding to the low MW protein components. D 2005 Elsevier Inc. All rights reserved. Keywords: Antivenom; Brown snake venom; Western blot; Organ bath; Phospholipase A2; Neurotoxin
Introduction The worldwide incidence of snake envenomation related death is estimated at over 100,000 each year, with the majority of these cases occurring in the Indo-Pacific region (Chippaux, 1998; WHO/SEARO, 1999). Within Australia, the occurrence of snake envenomation is high, with approximately 1000– 3000 cases reported on a yearly basis principally due to brown snake (Stewart, 2003; White, 1998; Cogger, 1975; Sutherland, 1992). In the absence of antivenom treatment, the Abbreviations: BCA, bicinchoninic acid; 2-DE, two-dimensional electrophoresis; 4-BPB, 4-bromophenacyl bromide; PLA2, phospholipase A2; IEF, isoelectric focusing; MQW, Milli Q water; PBS, phosphate-buffered saline. * Corresponding author. Fax: +61 3 9905 3726. E-mail address: [email protected] (J.A. Wilce). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.09.010
risk of mortality following brown snake envenomation is significant, especially in the case of attack by Pseudonaja affinis affinis (dugite), Pseudonaja textilis (common brown snake) or Pseudonaja nuchalis (gwardar). The venoms of these snakes contain a complex array of proteins that affect biological activities including blood coagulation and neuromuscular transmission (Sutherland and Tibballs, 2001). The characteristic clinical feature of brown snake envenomation is disseminated intravascular coagulation (DIC), ultimately resulting in incoagulable blood and hemorrhage, while the secondary symptoms of envenomation can include acute renal failure, neurotoxic paralysis and cardiac arrest (Williams et al., 1994; Jelinek and Breheny, 1990; Jelinek et al., 1991; White, 1987). The brown snake antivenom, produced by the Australian Commonwealth Serum Laboratories (CSL) and composed of
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horse immuno-globulins, is raised against venom from P. textilis only. While the antivenom has generally been successful in preventing death and treating victims of brown snake envenomation (Sutherland and Tibballs, 2001), there is increasing evidence to suggest the efficacy of the antivenom is not optimal. Firstly, the requirement for a substantially higher antivenom dosage (approximately 5 – 7 times the recommended dose) for victims of snakebite from the P. affinis affinis snake compared to snake bite from P. textilis snake has been reported (Jelinek and Breheny, 1990; Yeung et al., 2004). Additionally, several studies report a low affinity of the antiserum for key procoagulant proteins from P. textilis venom itself. Research carried out by Masci et al. (1998) demonstrated a low affinity for the prothrombin activator from P. textilis venom by the antivenom, while in vitro research using dog models suggested that an antivenom dose 10 – 25 times the recommended dose was required for preventing the procoagulant action of venoms from P. affinis affinis and P. textilis, respectively (Sprivulis et al., 1996; Tibballs et al., 1991). Furthermore, there have been reports of death following envenomation by P. textilis despite the speedy administration of large doses of antivenom (Henderson et al., 1993). In the current investigation, the principal aim was to characterize major groups of venom proteins recognized by the brown snake antiserum. Two-dimensional electrophoresis (2-DE) was employed to separate protein constituents from the crude venoms followed by immuno-blot analysis of the 2-DE gels using brown snake antivenom. In order to identify the main protein components present, LC-MS/MS was used to identify several representative protein species. Venom activity assays using nerve – muscle preparations from rat trachea were also employed in order to test the ability of the antivenom to neutralize the effects of brown snake venoms. This investigation represents the first comparison of the proteomes of venom constituents from P. affinis affinis, P. textilis and P. nuchalis. Additionally, the study characterizes the spectrum of protein species recognized and neutralized by the brown snake antivenom. The results may help to explain the limited efficacy of the antivenom and consequent requirement for higher antivenom doses for victims of dugite and gwardar envenomations. Such an improved understanding of the antivenom specificity could assist in the development of improved antivenoms with a more complete toxin neutralization profile. Methods Snake venom and antivenom Venoms from P. affinis affinis, P. textilis and P. nuchalis were provided by Peter Mirtschin in lyophilized form (Venom Supplies, Tanunda, South Australia). Brown snake antivenom was a gift from the Commonwealth Serum Laboratories (Parkville, Melbourne, Australia).
Two-dimensional electrophoresis (2-DE) Lyophilized venoms were prepared in rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethyl-ammonio]-1-
propane sulfonate), 1% carrier ampholytes and 40 mM dithiothreitol) at a protein concentration of 100 Ag/400 AL and stored at 20 -C until required. The protein content was quantitated using the BCA (bicinchoninic acid) assay (Smith et al., 1985; Pierce Chemicals, Illinois, USA). Immobiline strips (pH range 3 – 10; Amersham Biosciences, UK), placed in ceramic strip holders, were hydrated with 400 AL venom solution and a 1 mL covering of DryStrip fluid (Amersham Biosciences, UK). Prior to first dimension isoelectric focusing (IEF), the dehydrated strips were rehydrated to the original gel thickness of 0.5 mm. This step was performed for 20 h at room temperature. IEF was then carried out sequentially at 250 V for 1.5 h, 500 V for 2 h, 1000 V for 2.5 h and 8000 V for 12.5 h using an Ettan IPGphor II isoelectric focusing unit (Amersham Biosciences, UK). The strips were then washed in MQW and equilibration buffer (6 M urea, 10% SDS, 50 mM Tris – HCl, 20% (v/v) glycerol, 2.5% (w/v) acrylamide, 5 mM tri-n-butylphosphine) with the latter step performed for 25 min. Strips were embedded on top of reducing polyacrylamide gels with 0.1% (w/v) agarose and 0.01% (w/v) bromophenol blue. Reducing polyacrylamide gels (15% gel, 23 cm 20 cm) were prepared according to the methodology of Laemmli (1970). For immunoblot analysis, Kaleidoscope MW markers (Biorad, California, USA) were introduced into the gel matrix of the polyacrylamide gel used to separate venom proteins from P. affinis affinis. Second dimension SDS-PAGE was undertaken at 25 mA/gel (constant voltage at 200 V) for 5 h. Gels were stained with coomassie blue overnight and destained with water.
Immuno-blot analysis Following the second dimension separation of venom proteins from the brown snakes, 2-DE gels were incubated in 50 mL transfer buffer (5.81 g/L Tris, 2.93 g/L glycine, 0.36 g/L SDS (sodium dodecyl sulfate), 0.2% (v/v) methanol) for 15 min at room temperature. PVDF (polyvinylidene difluoride) membranes (Amersham, UK) were used for protein transfer, where the successful transfer of all Kaleidoscope MW markers was accomplished after 90 min at 12 V using the Hoefer semi-dry apparatus (Amersham, UK). To ensure all protein components were transferred onto the PVDF membranes under these conditions, a sample membrane was stained with ponceau S for 5 min, and destained for 10 min in water. Membranes were then treated with 80 mL blocking buffer 1 (3% (w/v) BSA (bovine serum albumin) in Trisbuffered saline tween-20 (TBST;10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween-20) for 1 h at room temperature. The membranes were then incubated overnight at 4 -C with brown snake antivenom (1/500-fold dilution) in 40 mL blocking buffer 2 (1% (w/v) BSA in TBST). Subsequently, membranes were washed three times for 5 min with 80 mL TBST at room temperature, after which detection of specific binding to the horse IgG antivenom was accomplished through the use of rabbit antihorse IgG conjugated to horseradish peroxidase obtained from the Sigma Chemical Co. (St. Louis, USA; 1/20,000-fold dilution in 40 mL blocking buffer 3; 0.2% (w/v) BSA in TBST). This incubation step was undertaken for 1 h at room temperature. Membranes were washed three times for 5 min with 80 mL TBST at room temperature. Peroxidase activity was visualized using the ECL detection system, where the autoradiographs (Hyperfilm ECL) were developed as described in the Amersham Biosciences manufacturer’s protocol (Amersham, UK).
LC-MS/MS Single protein spots from the venom of P. affinis affinis were excised from a representative gel stained with coomassie blue and submitted for tryptic digestion and LC-MS/MS to Proteomics International (Perth, Western Australia). Briefly, digested samples of 15 – 20 Ag were analyzed on a Qstar Pulsar MS/MS system (Applied Biosystems, California, USA) utilizing an inline Agilent 1100 capillary LC system incorporating a Zorbax C18 reverse phase column (California, USA). Mass spectra and collision MS/MS data were analyzed with Analyst QS and BioAnalyst software (Applied Biosystems, California, USA). To identify the venom peptides, searches were carried out using NCBI BLAST (Altschul et al., 1990) and the SWISSPROT library.
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Tension recording studies using rat isolated trachea Crude venom. Male Wistar SPF (specific pathogen-free) rats (8 – 10 weeks old) were anesthetized with Nembutal (200 mg/kg, intraperitoneal) and then sacrificed. The trachea was removed and sectioned into rings 2 mm in width. Tracheal sections were mounted onto stainless steel hooks attached to force displacement transducers (Grass FT03C; Grass Telefactor, Rhode Island, USA). A resting tension of 500 mg was applied. Trachea were immersed in Sigmacoate treated organ baths containing 1 mL Krebs bicarbonate solution with indomethacin (3 AM bath concentration in 0.9% NaCl) bubbled with carbogen (95% O2 and 5% CO2) at 37 -C. The cyclooxygenase inhibitor indomethacin was included in the buffer to avoid the generation of cyclooxygenase metabolites such as thromboxanes and prostoglandins. These can cause muscle contraction separately due to the action of 5-lipoxygenase metabolites that were shown in our previous study to be the likely mediators of PLA2 enzyme-induced muscle contraction (Judge et al., 2002). Changes in smooth muscle tension were recorded using a Grass Polygraph Model 7 B (Grass Telefactor, Rhode Island, USA). After equilibration (1 h), tracheal contractile function was tested by the addition of a supramaximal bolus dose of carbachol (30 AM). Following a 20 min rest and washout period, tissues were exposed to 10 AM cumulatively added carbachol, washed for a further 20 min and then exposed to the venoms of P. affinis affinis (100 Ag/mL), P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/mL) or the saline control (0.1 M PBS) for 5 min (n = 4). The muscle was then washed and rested for a further 30 min, and 10 AM carbachol applied cumulatively. Crude venom and brown snake antivenom. The response of tracheal segments to the venoms from the Pseudonaja sp. snakes in the presence of brown snake antivenom was assessed. A dose – response curve for the brown snake antivenom was conducted in order to gauge an appropriate dose that would not stimulate a tracheal response itself. Accordingly, 5 units/mL antivenom (where 1 unit is defined as the volume of antivenom that will neutralize 10 Ag venom) was incubated in the presence of each brown snake venom at room temperature for 15 min, after which the mixture was added to the organ baths (n = 3). Crude venom and 4-BPB. Venom-induced tracheal contractility in the presence of the PLA2 inhibitor 4-BPB was assessed. Tracheal preparations were incubated with 4-BPB (180 AM) for 15 min prior to venom addition (n = 3).
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Venom-induced contractions were expressed as a percentage of the response observed with 10 AM cumulative carbachol obtained at the start of experimentation.
Results Comparison of the Pseudonaja sp. proteomes It was of interest to compare the venom protein composition of P. affinis affinis, P. textilis and P. nuchalis as manifested on 2-DE gels. Fig. 1 depicts representative 2-DE gels of these snake venoms (n = 6 for P. affinis affinis; n = 3 for P. textilis and n = 4 for P. nuchalis). These gels demonstrate the separation of the venom protein constituents, where separation occurred by molecular weight (6 kDa to ¨132 kDa) in the vertical dimension and pI (pH 3 – 10) in the horizontal dimension. The pattern of protein distribution from each venom was reproducible, although replicates differed slightly in their intensity and dimensions and some streaking occurred. General similarities were noted among the three gels. In particular, major spots were visualized at 78 –132 kDa, 32– 45 kDa and 6 –15 kDa across all three gels. Several dominant spots were also visualized between 15 and 32 kDa. Interestingly, a few general differences were observed with regard to the quantity and intensity of spots. Protein constituents between 6 and 15 kDa were present in greater quantity in the venoms of P. affinis affinis and P. textilis than the venom of P. nuchalis, whereas constituents between 15 and 32 kDa were observed in relatively greater abundance in venom from P. nuchalis. Also, protein species ranging from 6– 15 kDa in venom from P. textilis were generally more acidic in nature than corresponding proteins from the venoms of P. affinis affinis and P. nuchalis. Recognition of venom proteins by brown snake antivenom
Statistical analysis All data are presented as the mean T SEM and group data initially compared using two-way analysis of variance. Post hoc analyses of group data were performed using an all pairwise multiple comparison procedure (Holm-Sidak method). P values less than 0.05 were considered significant.
Immuno-blots using brown snake antivenom of representative 2-DE gels depicting protein constituents from the venoms of P. affinis affinis, P. textilis and P. nuchalis are presented in Fig. 2. The profile of protein constituents
Fig. 1. Representative 2-DE gel images of the protein constituents present in the P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) proteomes. Spots from the P. affinis affinis 2-DE gel submitted for LC-MS/MS analysis are circled and labeled 1 – 7. The positions of the kaleidoscope MW markers introduced into the matrix of the P. affinis affinis 2-DE gel are indicated in panel A.
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Fig. 2. Representative 2-DE immuno blots of P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) venoms using brown snake antivenom to probe the venom/ antivenom protein interactions. Positions of the kaleidoscope MW markers as visualized on the immuno-blot of the P. affinis affinis 2-DE gel following transfer are indicated in panel A.
recognized by the antivenom is similar across all three snake species. In general, the signals are stronger than those visualized in the coomassie stained gels, where several protein species were recognized by the antivenom but were not detected in the coomassie stained gels. A strong signal was observed for proteins of MW 32– 45 kDa, reflecting the dominance of proteins within this MW range to incite an immune response. Similarly, proteins ranging from 78 to 132 kDa were also strongly recognized by the antivenom. However, the absence of significant signal for toxins in the MW range of 6 to ¨32 kDa is a striking and consistent feature of the immuno-blots. The protein recognition that does occur in this molecular weight range occurs for quite differently positioned proteins among the venoms of the three Pseudonaja species. Identification of proteins from the venom of P. affinis affinis In order to identify sample protein constituents from the venoms of the Pseudonaja sp. snakes, gel spots were submitted for LC-MS/MS analysis (protein spots from one snake species were chosen to represent protein constituents from all three Pseudonaja sp. snakes). The similar pattern of protein species from the venoms of the Pseudonaja sp. snakes separated by 2-DE provided a sound rationale for this approach. Consequently, seven spots from a representative coomassie stained 2-DE gel of venom constituents from P. affinis affinis were excised and submitted for tryptic digest and peptide identification by mass spectrometry. The low MW components (MW < 32 kDa) were primarily targeted due to their relative dominance on the 2-DE gels. The location of the spots on the 2-DE pattern is depicted in Fig. 1A. All identified peptide sequences from these spots and the results of database searches using NCBI BLAST and SWISS-PROT are presented in Table 1. Only the highest peptide match is reported. Of the seven spots submitted for sequencing, only four contained sequences with similarity to proteins in the databank. Peptides from the three remaining spots appear to be unique. Two peptide sequences were identified from spot 2 (¨MW 42 kDa). One peptide possessed similarity to the Group D prothrombin
activator trocarin from the venom of the snake Tropidechis carinatus while the other peptide was similar to Factor X isolated from Ornithorhynchus anatinus (duck billed platypus). These proteins are involved in blood coagulation, where trocarin functions to convert prothrombin to thrombin (Joseph et al., 1999) while factor X is converted to factor Xa which in turn serves to activate thrombin (Poorafshar et al., 2000; Sutherland and Tibballs, 2001). While Factor X proteins have not been previously isolated from brown snake venoms, Factor X activators have been reported in venoms from the genus Pseudonaja as well as other Australian snake venoms (Marshall and Herrmann, 1983). It is not unexpected to discover such protein species since the molecular weights of characterized coagulant toxins from the venoms of Pseudonaja sp. range from 50 kDa to ¨250 kDa (Rao and Kini, 2002; Stocker et al., 1994). Spot 4 yielded three peptide sequences with significant identity to a PLA2 protein isolated from the venom of P. textilis designated Pt-PLA2, and a PLA2 protein isolated from the venom of the snake Gloydius halys pallas designated B-PLA2. Similarly, the peptide sequence obtained from spot 5 was identical to a segment of the PLA2 protein HTe obtained from the venom of the snake Notechis scutatus. Both peptide sequences from spot 6 were identical to segments from the plasmin inhibitor textilinin (isolated from the venom of P. textilis). The PLA2 enzymes, a primary class of proteins present in the venoms of Pseudonaja sp. and other Australian snakes (Sutherland and Tibballs, 2001), are characteristically 13 – 15 kDa in size (Kini, 1997). The pharmacological characteristics of PLA2 proteins are extremely diverse, with activities ranging from neurotoxicity, myotoxicity, antiplatelet effects, hemolysis, coagulant/anticoagulant effects and hemorrhage (Kini, 1997; Grieg Fry, 1998). Pt-PLA2 was found to induce procoagulation (Armugam et al., 2004), B-PLA2 hemolyzed erythrocytes (Pan et al., 1998) while HTe was found to induce hypotension, hemorrhage and neurotoxicity (Francis et al., 1995). Textilinin, isolated from the venom of P. textilis functions to neutralize plasmin activity, inhibiting the destructive action of this enzyme on fibrin polymers (Filippovich et al., 2002; Masci et al., 2000).
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Table 1 Sequence comparison of peptide fragments from Pseudonaja affinis affinis proteins 17 separated using 2DE
Identical (:) and conserved (.) amino acids are indicated.
Contractile response in rat tracheal tissue induced by the Pseudonaja venoms The proteomic investigation highlighted a deficiency in the interaction between the brown snake antivenom and protein constituents of the Pseudonaja sp. snakes of MW 6 kDa to <32 kDa. In snake venoms, such species predominantly include the PLA2 proteins, as confirmed by our analysis, and a-neurotoxins (Sutherland and Tibballs, 2001). To assess the interaction between the brown snake antivenom and PLA2 proteins from the Pseudonaja sp. snakes, a functional approach was undertaken using rat tracheal nerve/muscle preparations. The action of the venom of P. affinis affinis on tracheal contractility has been previously established. A strong single contractile response resulting from PLA2 protein activity was reported (Judge et al., 2002). In this study, 100 Ag/mL of the venom of P. affinis affinis was applied to tracheal preparations as a positive control (Fig. 3A), resulting in a contractile response 51 T 4% (n = 4) of the maximum carbacholinduced response (10 AM). Application of the venoms of P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/mL) to tracheal
preparations was also found to affect tracheal tone through the induction of muscle contraction (Figs. 3B and C, respectively). These responses were 52 T 7% (n = 4) and 43 T 7% (n = 4) of the maximum carbachol-induced response (10 AM). Peaks were observed approximately 3 min following venom administration, and were maintained during the observation period (5 min). Venom-induced contractions were reversed by washout, and did not impair the contractile function of the muscle tissue in response to carbachol following venom treatment. Brown snake antivenom does not inhibit venom-induced tracheal contractility To characterize the venom-induced response in the presence of the brown snake antivenom, the venoms of P. affinis affinis (100 Ag/mL), P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/ mL) were pre-incubated with 5 units/mL brown snake antivenom for 15 min at room temperature. All three venoms were found to induce a contractile response of tracheal muscle. The responses induced by the venoms of P. affinis affinis, P.
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Fig. 3. P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) venoms induce contraction of rat tracheal preparations. P. affinis affinis (100 Ag/mL venom protein), P. textilis (50 Ag/mL venom protein) and P. nuchalis (100 Ag/mL venom protein) venoms were added to tracheal segments (resting tension 500 mg), resulting in significant contractions (51 T 4%, 52 T 7% and 43 T 7% of the maximum contraction induced by 10 AM cumulative carbachol, respectively; n = 4).
textilis and P. nuchalis were 40 T 5% (n = 3), 48 T 9% (n = 3) and 44 T 5% (n = 3) of the maximum carbachol-induced response (10 AM), respectively (Fig. 4). The two-way ANOVA indicated that the level of contraction was independent of both the snake venom and the drug treatment. This suggests an
Fig. 4. Effect of brown snake antivenom on Pseudonaja sp. venom-induced contractions of rat trachea. In the presence of antivenom (AV), the venominduced contractions for P. affinis affinis, P. textilis and P. nuchalis venoms were 40 T 5%, 48 T 9% and 44 T 5% of the maximum carbachol-induced response (10 AM cumulative), respectively (P < 0.05; n = 3). This suggested that the antivenom did not mediate a significant effect on venom-induced contractions. Control venom responses were 51 T 4%, 52 T 7% and 43 T 7%, respectively (n = 4).
Fig. 5. Effect of the PLA2 inhibitor 4-BPB on Pseudonaja venom-induced contractions of rat tracheal preparations. 4-BPB inhibited contractions induced by the venom of P. affinis affinis and P. nuchalis (*P < 0.05), but not those induced by the venom of P. textilis. In the presence of the inhibitor, the venominduced contractions for P. textilis and P. nuchalis were 43 T 11% and 10 T 1% of the maximum carbachol-induced response, respectively (10 AM cumulative concentration) and no response was observed for P. affinis affinis venom treated preparations (n = 3). The control venom response was 51 T 4%, 52 T 7% and 43 T 7% for P. affinis affinis, P. textilis and P. nuchalis venoms, respectively (n = 4).
ineffective neutralization of protein constituents responsible for tracheal muscle contractility by the antivenom. Contractile response in the presence of the PLA2 inhibitor 4-BPB The contractile response of tracheal nerve/muscle preparations following administration of the venom of P. affinis affinis has previously been attributed to PLA2 enzymatic activity, where treatment with the PLA2 inhibitor 4-BPB was found to completely neutralize the effects of the venom (Judge et al., 2002). In the current study, the effects of the venoms of P. textilis and P. nuchalis PLA2 proteins on tracheal contractility in the presence of 4-BPB were assessed and are depicted in Fig. 5. In a control experiment, the inhibitory effect of 4-BPB on contractility due to the venom of P. affinis affinis was confirmed (n = 3; two-way ANOVA, P < 0.05). 4-BPB also exhibited this inhibitory property on contractions induced by the venom of P. nuchalis, which were attenuated to 10 T 1% of the maximum carbachol-induced response (n = 3; two-way ANOVA, P < 0.05). However, application of venom of P. textilis to 4-BPB pre-treated tracheal preparations resulted in a strong contraction of tracheal muscle that was 43 T 11% of the maximum carbachol-induced response (n = 3). This suggested that PLA2 enzymatic activity from the venom of P. textilis was not a significant mediator of tracheal contractility. Discussion The current study investigates the complement of principal protein species within brown snake venoms that are effectively recognized and neutralized by the brown snake antivenom. It was of interest to examine the interaction of the antivenom with proteins from the three
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main Pseudonaja species as the commercial antivenom is raised solely against venom from P. textilis. The deficiency of information in this area is striking given the increasing number of reports indicating that high antivenom doses are required for treating victims of envenomation by the P. affinis affinis snake. 2-DE was utilized to separate and identify principal protein species. Comparison of the venom proteomes from the three Pseudonaja sp. snakes revealed a similarity in the spread of protein constituents with respect to MW and pI. The observation of high-MW (>78 kDa), mid-MW (32 –45) and low-MW (<32 kDa) protein species was consistent with a previously reported 1-DE analysis of P. affinis affinis venom proteins (Judge et al., 2002). The spread of protein components is also similar to the range of venom proteins from the Guatemalan snake Bothrops asper (Saravia et al., 2001). 2-DE analysis revealed primary spots present at 14, 20 –30 and 67– 94 kDa. However, there were no proteins visible above 94 kDa or below 14 kDa, in contrast to spots observed at these molecular weights on the protein gels of the Pseudonaja sp. 2-DE analysis of the venom proteomes of Naja naja atra and Gloydius halys also revealed a spread of components predominantly distributed from 97 – 30 kDa and 66– 20 kDa, respectively (Li et al., 2004). Interestingly, 1-DE analysis of 11 elapid venoms from the Micrurus snakes revealed the predominance of the 14 kDa species. Far less dominant were faint bands at 20 –30 kDa, 43 kDa, 67 kDa and 94 kDa (Prieto da Silva et al., 2001). Immuno-blotting using the commercial antivenom showed a similar pattern of recognition of proteins from the venoms of all three Pseudonaja species. This suggests that the venom proteins possess sufficient similarity across the species to warrant the use of antivenom, which is raised solely against the venom of P. textilis. Surprisingly, antivenom recognition occurred predominantly against the high-MW and mid-MW proteins. There was very little recognition of proteins between 6 kDa and ¨32 kDa by the Pseudonaja species. LC-MS/MS analysis of protein spots from the 2-DE gel of venom from P. affinis affinis revealed that the high-MW and mid-MW species are likely to include procoagulant proteins. This is consistent with the previously reported presence of toxins from brown snake venoms that interfere with blood homeostasis. Peptides derived from lowMW protein spots showed sequence similarity to several types of PLA2 enzymes as well as a plasmin inhibitor (textilinin) from P. textilis. While the presence of both short chain and long chain neurotoxins in the lower half of the 2DE gels was not verified by LC-MS/MS analysis, the existence of these peptides in the venoms of P. affinis affinis (Judge et al., 2002) and P. textilis (Gong et al., 1999) has been established. Thus, the antivenom may also be ineffective against the neurotoxin components of the venoms of the Pseudonaja sp. snakes that are among the principal protein constituents of Australian snake venoms (Sutherland and Tibballs, 2001). The absence of antivenom recognition of low-MW proteins from the venom of the Pseudonaja sp. prompted
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the use of a functional assay to examine contractile activity of the venom (potentially incited by either PLA2 enzymes or neurotoxin activity) in the presence of antivenom. The results suggest that the contractile function of rat trachea induced by all three venoms from the Pseudonaja sp. is not significantly attenuated by the antivenom. Further experiments utilizing the PLA2 inhibitor 4-BPB verified that the induced response by the venoms of P. affinis affinis and P. nuchalis was due to the enzymatic activity of the PLA2 proteins. In contrast, the contractile response to the venom of P. textilis was not incited by PLA2 enzymatic activity alone. Reports detailing the neurotoxic properties of the venom of P. textilis suggest the observed contractile activity may be mediated by the action of a pre-synaptic neurotoxin such as textilotoxin, a PLA2 protein with neurotoxic activity. Textilotoxin is the most neurotoxic snake venom protein isolated to date (Pearson et al., 1991; Pearson et al., 1993; Tyler et al., 1987) and its induction of contraction via neurotransmitter release is well documented (Harris, 1991; Nicholson et al., 1992). In our experiments, the antivenom did not recognize the low-MW protein components of the venoms of the Pseudonaja sp. In addition, the antivenom did not neutralize PLA2 enzymatic or putative neurotoxic activities that may be attributed to the low-MW species. However, the rat tracheal assay may not accurately reflect the human in vivo situation, and it is possible that that these toxins may escape neutralization by the antivenom in envenomed humans. In addition, other low-MW toxins that were not visibly active against the rat tracheal preparations may similarly remain unneutralized. The failure of snake antivenoms to neutralize specific venom components has been previously observed. Oshima-Franco et al. (1999) document a deficiency in the neutralizing capacity of Crotalus durissus terrificus antivenom for the venom, as well as the neurotoxic PLA2 protein crotoxin. Interestingly, a specific preparation comprising of anticrotoxin antibodies afforded optimal protection against the effects of both the venom and crotoxin to combat neuromuscular blockade. Similarly, Fry et al. (2001) reported a deficiency in the effectiveness of death adder antivenom in neutralizing the neurotoxic effects of several Australian death adder species. Application of these venoms to chick biventer cervicis preparations inhibited the twitch response of the muscle. However, a 10 min pre-incubation with 1 unit/mL antivenom failed to protect against twitch inhibition, whereas application of a higher dose of the antiserum (5 units/mL) was successful in reversing the inhibition of twitch response. Thus, while the commercial antivenom in its current form may provide adequate protection against the main clinical feature of envenomation in humans (such as disseminated intravascular coagulation), our study suggests that neutralization of lower molecular weight venom proteins may be limited. These include PLA2 protein and neurotoxin species which could underlie some of the secondary symptoms of envenomation in humans and the
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requirement for very high doses of antivenom in many cases. Further studies of the importance of these and other protein toxin species in the clinical features of envenomed humans would be of great interest. The development of an antivenom that recognizes and neutralizes the major PLA2 protein and neurotoxin species may yield greater success in treating brown snake envenomation victims using lower doses of the antiserum. Acknowledgments This research was supported by a Western Australian Raine Medical Research Foundation Grant (JAW) and an Australian Research Council Fellowship (JAW). References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403 – 410. Armugam, A., Gong, N., Li, X., Siew, P.Y., Chai, S.C., Nair, R., Jeyaseelan, K., 2004. Group IB phospholipase A2 from Pseudonaja textilis. Arch. Biochem. Biophys. 421, 10 – 20. Chippaux, J.P., 1998. Snake-bites: appraisal of the global situation. Bull. World Health Organ. 76, 515 – 524. Cogger, H., 1975. Reptiles and Amphibians of Australia. Reed Pty Ltd., London, pp. 7 – 25. Filippovich, I., Sorokina, N., Masci, P.P., de Jersey, J., Whitaker, A.N., Winzor, D.J., Gaffney, P.J., Lavin, M.F., 2002. A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties. Br. J. Haematol. 119, 376 – 384. Francis, B., Coffield, J.A., Simpson, L.L., Kaiser, I.I., 1995. New toxins from the venom of the common tiger snake (Notechis scutatus scutatus). Arch. Biochem. Biophys. 318, 481 – 488. Fry, B.G., Wickramaratna, J.C., Jones, A., Alewood, P.F., Hodgson, W.C., 2001. Species and regional variations in the effectiveness of antivenom against the in vitro neurotoxicity of death adder (Acanthophis) venoms. Toxicol. Appl. Pharmacol. 175, 140 – 148. Gong, N., Armugam, A., Jeyaseelan, K., 1999. Postsynaptic short-chain neurotoxins from Pseudonaja textilis. cDNA cloning, expression and protein characterization. Eur. J. Biochem. 265, 982 – 989. Grieg Fry, B., 1998. Structure – function properties of venom components from Australian elapids. Toxicon 37, 11 – 32. Harris, J.B., 1991. Snake Venoms: International Encyclopedia of Pharmacology and Therapeutics Section 134. Pergamon Press, New York, pp. 91 – 129. Henderson, A., Baldwin, L.N., May, C., 1993. Fatal brown snake (Pseudonaja textilis) envenomation despite the use of antivenom. Med. J. Aust. 158, 709 – 710. Jelinek, G.A., Breheny, F.X., 1990. Ten years of snakebite at Fremantle Hospital. Med. J. Aust. 153, 658 – 661. Jelinek, G.A., Hamilton, T., Hirsch, R.L., 1991. Admissions for suspected snake bite to the Perth adult teaching hospitals, 1979 to 1988. Med. J. Aust. 155, 761 – 764. Joseph, J.S., Chung, M.C., Jeyaseelan, K., Kini, R.M., 1999. Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa. Blood 94, 621 – 631. Judge, R.K., Henry, P.J., d’Aprile, A.C., Lynch, D., Jelinek, G.A., Wilce, M.C., Wilce, J.A., 2002. Identification of PLA(2) and alpha-neurotoxin proteins in the venom of Pseudonaja affinis (dugite). Toxicol. Appl. Pharmacol. 181, 184 – 191. Kini, R.M., 1997. Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism. John Wiley and Sons, Chichester, UK, pp. 1 – 28. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Li, S., Wang, J., Zhang, X., Ren, Y., Wang, N., Zhao, K., Chen, X., Zhao, C.,
Li, X., Shao, J., Yin, J., West, M.B., Xu, N., Liu, S., 2004. Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys. Biochem. J. 384, 119 – 127. Marshall, L.R., Herrmann, R.P., 1983. Coagulant and anticoagulant actions of Australian snake venoms. Thromb. Haemost. 50, 707 – 711. Masci, P.P., Mirtschin, P.J., Nias, T.N., Turnbull, R.K., Kuchel, T.R., Whitaker, A.N., 1998. Brown snakes (Pseudonaja genus): venom yields, prothrombin activator neutralization and implications affecting antivenom usage. Anaesth. Intensive Care 26, 276 – 281. Masci, P.P., Whitaker, A.N., Sparrow, L.G., de Jersey, J., Winzor, D.J., Watters, D.J., Lavin, M.F., Gaffney, P.J., 2000. Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model. Blood Coagul. Fibrinolysis 4, 385 – 393. Nicholson, G.M., Spence, I., Lloyd, D.R., Wilson, H., Harrison, B.M., Tyler, M., Howden, M.E.H., 1992. Presynaptic actions of textilotoxin, a neurotoxin from the Australian common brown snake (Pseudonaja textilis). 10th World Congress on Animal, Plant and Microbial Toxins, Singapore. , pp. 540 – 548. Oshima-Franco, Y., Hyslop, S., Prado-Franceschi, J., Cruz-Hofling, M.A., Rodrigues-Simioni, L., 1999. Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin. Toxicon 37, 1341 – 1357. Pan, H., Liu, X.-L., Ou-Yang, L.-L., Yang, G.-Z., Zhou, Y.-C., Li, Z.-P., Wu, X.-F., 1998. Diversity of cDNA’s encoding phospholipase A2 from Agkistrodon halys pallas venom and it’s expression in E. coli. Toxicon 36, 1155 – 1163. Pearson, J.A., Tyler, M.I., Retson, K.V., Howden, M.E., 1991. Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis): 2. The amino acid sequence and toxicity studies of subunit D. Biochim. Biophys. Acta 1077, 147 – 150. Pearson, J.A., Tyler, M.I., Retson, K.V., Howden, M.E., 1993. Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis): 3. The complete amino-acid sequences of all the subunits. Biochim. Biophys. Acta 1161, 223 – 229. Poorafshar, M., Aveskogh, M., Munday, B., Hellman, L., 2000. Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus. Immunogenetics 52, 19 – 28. Prieto da Silva, A.R., Yamagushi, I.K., Morais, J.F., Higashi, H.G., Raw, I., Ho, P.L., Oliveira, J.S., 2001. Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snake venoms. Toxicon 39, 949 – 953. Rao, V.S., Kini, R.M., 2002. Pseutarin C, a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa – Va complex. Thromb. Haemost. 88, 611 – 619. Saravia, P., Rojas, E., Escalante, T., Arce, V., Chaves, E., Velasquez, R., Lomonte, B., Rojas, G., Gutierrez, J.M., 2001. The venom of Bothrops asper from Guatemala: toxic activities and neutralization by antivenoms. Toxicon 39, 401 – 405. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76 – 85. Sprivulis, P., Jelinek, G.A., Marshall, L.M., 1996. Efficacy and potency of antivenoms in neutralizing the procoagulant effects of Australian snake venoms in dog and human plasma. Anaesth. Intensive Care 24, 379 – 381. Stewart, C.J., 2003. Snake bite in Australia: first aid and envenomation management. Accid. Emerg. Nurs. 11, 106 – 111. Stocker, K., Hauer, H., Muller, C., Triplett, D.A., 1994. Isolation and characterization of textarin, a prothrombin activator from eastern brown snake (Pseudonaja textilis) venom. Toxicon 32, 1227 – 1236. Sutherland, S.K., 1992. Deaths from snakebite in Australia. Med. J. Aust. 157, 740 – 746.
R.K. Judge et al. / Toxicology and Applied Pharmacology 213 (2006) 117 – 125 Sutherland, S.K., Tibballs, J., 2001. Australian Animal Toxins. Oxford Univ. Press, London, pp. 30 – 50. Tibballs, J., Sutherland, S.K., Kerr, S., 1991. Studies on Australian snake venoms: Part II. The haematological effects of brown snake (Pseudonaja) species in the dog. Anaesth. Intensive Care 19, 338 – 342. Tyler, M.I., Barnett, D., Nicholson, P., Spence, I., Howden, M.E., 1987. Studies on the subunit structure of textilotoxin, a potent neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis). Biochim. Biophys. Acta 915, 210 – 216. White, J., 1987. Elapid Snakes: Aspects of Envenomation. Queensland Museum Press, Brisbane, Australia, pp. 8 – 17.
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White, J., 1998. Envenoming and antivenom use in Australia. Toxicon 36, 1483 – 1492. WHO/SEARO, 1999. WHO/SEARO guidelines for the clinical management of snake bites in the Southeast Asian region. Southeast Asian J. Trop. Med. Public Health 30, 1 – 85. Williams, V., White, J., Mirtschin, P.J., 1994. Comparative study on the procoagulant from the venom of Australian brown snakes (Elapidae; Pseudonaja spp.). Toxicon 32, 453 – 459. Yeung, J.M., Little, M., Murray, L., Jelinek, G.A., Daly, F.F.S., 2004. Antivenom dosing in 35 patients with severe brown snake (Pseudonaja) envenoming in Western Australia over 10 years. Med. J. Aust. 181, 703 – 706.
Toxins not neutralized by brown snake antivenom Roopwant K. Judge a, Peter J. Henry b, Peter Mirtschin c, George Jelinek d, Jacqueline A. Wilce e,* a
Molecular Genetics and Evolution Group, Research School of Biological Sciences, Australian National University, Canberra 2601, ACT, Australia b School of Medicine and Pharmacology, University of Western Australia, Crawley 6009, Western Australia, Australia c Venom Supplies, Tanunda 5352, South Australia, Australia d School of Primary, Aboriginal and Rural Health Care, QEII Medical Centre, Western Australia, Australia e Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, VIC Victoria, Australia Received 13 July 2005; revised 29 August 2005; accepted 16 September 2005 Available online 26 October 2005
Abstract The Australian snakes of the genus Pseudonaja (dugite, gwardar and common brown) account for the majority of snake bite related deaths in Australia. Without antivenom treatment, the risk of mortality is significant. There is an accumulating body of evidence to suggest that the efficacy of the antivenom is limited. The current study investigates the protein constituents recognized by the antivenom using 2-DE, immuno-blot techniques and rat tracheal organ bath assays. The 2-DE profiles for all three snake venoms were similar, with major species visualized at 78 – 132 kDa, 32 – 45 kDa and 6 – 15 kDa. Proteins characterized by LC-MS/MS revealed a coagulant toxin (¨42 kDa) and coagulant peptide (¨6 kDa), as well as two PLA2 (¨14 kDa). Peptides isolated from ¨78 kDa and 15 – 32 kDa protein components showed no similarity to known protein sequences. Protein recognition by the antivenom occurred predominantly for the higher molecular weight components with little recognition of 6 – 32 kDa MW species. The ability of antivenom to neutralize venom activity was also investigated using rat tracheal organ bath assays. The venoms of Pseudonaja affinis affinis and Pseudonaja nuchalis incited a sustained, significant contraction of the trachea. These contractions were attributed to PLA2 enzymatic activity as pre-treatment with the PLA2 inhibitor 4-BPB attenuated the venom-induced contractions. The venom of Pseudonaja textilis incited tracheal contractility through a non-PLA2 enzymatic activity. Neither activity was attenuated by the antivenom treatment. These results represent the first proteomic investigation of the venoms from the snakes of the genus Pseudonaja, revealing a possible limitation of the brown snake antivenom in binding to the low MW protein components. D 2005 Elsevier Inc. All rights reserved. Keywords: Antivenom; Brown snake venom; Western blot; Organ bath; Phospholipase A2; Neurotoxin
Introduction The worldwide incidence of snake envenomation related death is estimated at over 100,000 each year, with the majority of these cases occurring in the Indo-Pacific region (Chippaux, 1998; WHO/SEARO, 1999). Within Australia, the occurrence of snake envenomation is high, with approximately 1000– 3000 cases reported on a yearly basis principally due to brown snake (Stewart, 2003; White, 1998; Cogger, 1975; Sutherland, 1992). In the absence of antivenom treatment, the Abbreviations: BCA, bicinchoninic acid; 2-DE, two-dimensional electrophoresis; 4-BPB, 4-bromophenacyl bromide; PLA2, phospholipase A2; IEF, isoelectric focusing; MQW, Milli Q water; PBS, phosphate-buffered saline. * Corresponding author. Fax: +61 3 9905 3726. E-mail address: [email protected] (J.A. Wilce). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2005.09.010
risk of mortality following brown snake envenomation is significant, especially in the case of attack by Pseudonaja affinis affinis (dugite), Pseudonaja textilis (common brown snake) or Pseudonaja nuchalis (gwardar). The venoms of these snakes contain a complex array of proteins that affect biological activities including blood coagulation and neuromuscular transmission (Sutherland and Tibballs, 2001). The characteristic clinical feature of brown snake envenomation is disseminated intravascular coagulation (DIC), ultimately resulting in incoagulable blood and hemorrhage, while the secondary symptoms of envenomation can include acute renal failure, neurotoxic paralysis and cardiac arrest (Williams et al., 1994; Jelinek and Breheny, 1990; Jelinek et al., 1991; White, 1987). The brown snake antivenom, produced by the Australian Commonwealth Serum Laboratories (CSL) and composed of
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horse immuno-globulins, is raised against venom from P. textilis only. While the antivenom has generally been successful in preventing death and treating victims of brown snake envenomation (Sutherland and Tibballs, 2001), there is increasing evidence to suggest the efficacy of the antivenom is not optimal. Firstly, the requirement for a substantially higher antivenom dosage (approximately 5 – 7 times the recommended dose) for victims of snakebite from the P. affinis affinis snake compared to snake bite from P. textilis snake has been reported (Jelinek and Breheny, 1990; Yeung et al., 2004). Additionally, several studies report a low affinity of the antiserum for key procoagulant proteins from P. textilis venom itself. Research carried out by Masci et al. (1998) demonstrated a low affinity for the prothrombin activator from P. textilis venom by the antivenom, while in vitro research using dog models suggested that an antivenom dose 10 – 25 times the recommended dose was required for preventing the procoagulant action of venoms from P. affinis affinis and P. textilis, respectively (Sprivulis et al., 1996; Tibballs et al., 1991). Furthermore, there have been reports of death following envenomation by P. textilis despite the speedy administration of large doses of antivenom (Henderson et al., 1993). In the current investigation, the principal aim was to characterize major groups of venom proteins recognized by the brown snake antiserum. Two-dimensional electrophoresis (2-DE) was employed to separate protein constituents from the crude venoms followed by immuno-blot analysis of the 2-DE gels using brown snake antivenom. In order to identify the main protein components present, LC-MS/MS was used to identify several representative protein species. Venom activity assays using nerve – muscle preparations from rat trachea were also employed in order to test the ability of the antivenom to neutralize the effects of brown snake venoms. This investigation represents the first comparison of the proteomes of venom constituents from P. affinis affinis, P. textilis and P. nuchalis. Additionally, the study characterizes the spectrum of protein species recognized and neutralized by the brown snake antivenom. The results may help to explain the limited efficacy of the antivenom and consequent requirement for higher antivenom doses for victims of dugite and gwardar envenomations. Such an improved understanding of the antivenom specificity could assist in the development of improved antivenoms with a more complete toxin neutralization profile. Methods Snake venom and antivenom Venoms from P. affinis affinis, P. textilis and P. nuchalis were provided by Peter Mirtschin in lyophilized form (Venom Supplies, Tanunda, South Australia). Brown snake antivenom was a gift from the Commonwealth Serum Laboratories (Parkville, Melbourne, Australia).
Two-dimensional electrophoresis (2-DE) Lyophilized venoms were prepared in rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS (3-[(3-Cholamidopropyl)dimethyl-ammonio]-1-
propane sulfonate), 1% carrier ampholytes and 40 mM dithiothreitol) at a protein concentration of 100 Ag/400 AL and stored at 20 -C until required. The protein content was quantitated using the BCA (bicinchoninic acid) assay (Smith et al., 1985; Pierce Chemicals, Illinois, USA). Immobiline strips (pH range 3 – 10; Amersham Biosciences, UK), placed in ceramic strip holders, were hydrated with 400 AL venom solution and a 1 mL covering of DryStrip fluid (Amersham Biosciences, UK). Prior to first dimension isoelectric focusing (IEF), the dehydrated strips were rehydrated to the original gel thickness of 0.5 mm. This step was performed for 20 h at room temperature. IEF was then carried out sequentially at 250 V for 1.5 h, 500 V for 2 h, 1000 V for 2.5 h and 8000 V for 12.5 h using an Ettan IPGphor II isoelectric focusing unit (Amersham Biosciences, UK). The strips were then washed in MQW and equilibration buffer (6 M urea, 10% SDS, 50 mM Tris – HCl, 20% (v/v) glycerol, 2.5% (w/v) acrylamide, 5 mM tri-n-butylphosphine) with the latter step performed for 25 min. Strips were embedded on top of reducing polyacrylamide gels with 0.1% (w/v) agarose and 0.01% (w/v) bromophenol blue. Reducing polyacrylamide gels (15% gel, 23 cm 20 cm) were prepared according to the methodology of Laemmli (1970). For immunoblot analysis, Kaleidoscope MW markers (Biorad, California, USA) were introduced into the gel matrix of the polyacrylamide gel used to separate venom proteins from P. affinis affinis. Second dimension SDS-PAGE was undertaken at 25 mA/gel (constant voltage at 200 V) for 5 h. Gels were stained with coomassie blue overnight and destained with water.
Immuno-blot analysis Following the second dimension separation of venom proteins from the brown snakes, 2-DE gels were incubated in 50 mL transfer buffer (5.81 g/L Tris, 2.93 g/L glycine, 0.36 g/L SDS (sodium dodecyl sulfate), 0.2% (v/v) methanol) for 15 min at room temperature. PVDF (polyvinylidene difluoride) membranes (Amersham, UK) were used for protein transfer, where the successful transfer of all Kaleidoscope MW markers was accomplished after 90 min at 12 V using the Hoefer semi-dry apparatus (Amersham, UK). To ensure all protein components were transferred onto the PVDF membranes under these conditions, a sample membrane was stained with ponceau S for 5 min, and destained for 10 min in water. Membranes were then treated with 80 mL blocking buffer 1 (3% (w/v) BSA (bovine serum albumin) in Trisbuffered saline tween-20 (TBST;10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween-20) for 1 h at room temperature. The membranes were then incubated overnight at 4 -C with brown snake antivenom (1/500-fold dilution) in 40 mL blocking buffer 2 (1% (w/v) BSA in TBST). Subsequently, membranes were washed three times for 5 min with 80 mL TBST at room temperature, after which detection of specific binding to the horse IgG antivenom was accomplished through the use of rabbit antihorse IgG conjugated to horseradish peroxidase obtained from the Sigma Chemical Co. (St. Louis, USA; 1/20,000-fold dilution in 40 mL blocking buffer 3; 0.2% (w/v) BSA in TBST). This incubation step was undertaken for 1 h at room temperature. Membranes were washed three times for 5 min with 80 mL TBST at room temperature. Peroxidase activity was visualized using the ECL detection system, where the autoradiographs (Hyperfilm ECL) were developed as described in the Amersham Biosciences manufacturer’s protocol (Amersham, UK).
LC-MS/MS Single protein spots from the venom of P. affinis affinis were excised from a representative gel stained with coomassie blue and submitted for tryptic digestion and LC-MS/MS to Proteomics International (Perth, Western Australia). Briefly, digested samples of 15 – 20 Ag were analyzed on a Qstar Pulsar MS/MS system (Applied Biosystems, California, USA) utilizing an inline Agilent 1100 capillary LC system incorporating a Zorbax C18 reverse phase column (California, USA). Mass spectra and collision MS/MS data were analyzed with Analyst QS and BioAnalyst software (Applied Biosystems, California, USA). To identify the venom peptides, searches were carried out using NCBI BLAST (Altschul et al., 1990) and the SWISSPROT library.
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Tension recording studies using rat isolated trachea Crude venom. Male Wistar SPF (specific pathogen-free) rats (8 – 10 weeks old) were anesthetized with Nembutal (200 mg/kg, intraperitoneal) and then sacrificed. The trachea was removed and sectioned into rings 2 mm in width. Tracheal sections were mounted onto stainless steel hooks attached to force displacement transducers (Grass FT03C; Grass Telefactor, Rhode Island, USA). A resting tension of 500 mg was applied. Trachea were immersed in Sigmacoate treated organ baths containing 1 mL Krebs bicarbonate solution with indomethacin (3 AM bath concentration in 0.9% NaCl) bubbled with carbogen (95% O2 and 5% CO2) at 37 -C. The cyclooxygenase inhibitor indomethacin was included in the buffer to avoid the generation of cyclooxygenase metabolites such as thromboxanes and prostoglandins. These can cause muscle contraction separately due to the action of 5-lipoxygenase metabolites that were shown in our previous study to be the likely mediators of PLA2 enzyme-induced muscle contraction (Judge et al., 2002). Changes in smooth muscle tension were recorded using a Grass Polygraph Model 7 B (Grass Telefactor, Rhode Island, USA). After equilibration (1 h), tracheal contractile function was tested by the addition of a supramaximal bolus dose of carbachol (30 AM). Following a 20 min rest and washout period, tissues were exposed to 10 AM cumulatively added carbachol, washed for a further 20 min and then exposed to the venoms of P. affinis affinis (100 Ag/mL), P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/mL) or the saline control (0.1 M PBS) for 5 min (n = 4). The muscle was then washed and rested for a further 30 min, and 10 AM carbachol applied cumulatively. Crude venom and brown snake antivenom. The response of tracheal segments to the venoms from the Pseudonaja sp. snakes in the presence of brown snake antivenom was assessed. A dose – response curve for the brown snake antivenom was conducted in order to gauge an appropriate dose that would not stimulate a tracheal response itself. Accordingly, 5 units/mL antivenom (where 1 unit is defined as the volume of antivenom that will neutralize 10 Ag venom) was incubated in the presence of each brown snake venom at room temperature for 15 min, after which the mixture was added to the organ baths (n = 3). Crude venom and 4-BPB. Venom-induced tracheal contractility in the presence of the PLA2 inhibitor 4-BPB was assessed. Tracheal preparations were incubated with 4-BPB (180 AM) for 15 min prior to venom addition (n = 3).
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Venom-induced contractions were expressed as a percentage of the response observed with 10 AM cumulative carbachol obtained at the start of experimentation.
Results Comparison of the Pseudonaja sp. proteomes It was of interest to compare the venom protein composition of P. affinis affinis, P. textilis and P. nuchalis as manifested on 2-DE gels. Fig. 1 depicts representative 2-DE gels of these snake venoms (n = 6 for P. affinis affinis; n = 3 for P. textilis and n = 4 for P. nuchalis). These gels demonstrate the separation of the venom protein constituents, where separation occurred by molecular weight (6 kDa to ¨132 kDa) in the vertical dimension and pI (pH 3 – 10) in the horizontal dimension. The pattern of protein distribution from each venom was reproducible, although replicates differed slightly in their intensity and dimensions and some streaking occurred. General similarities were noted among the three gels. In particular, major spots were visualized at 78 –132 kDa, 32– 45 kDa and 6 –15 kDa across all three gels. Several dominant spots were also visualized between 15 and 32 kDa. Interestingly, a few general differences were observed with regard to the quantity and intensity of spots. Protein constituents between 6 and 15 kDa were present in greater quantity in the venoms of P. affinis affinis and P. textilis than the venom of P. nuchalis, whereas constituents between 15 and 32 kDa were observed in relatively greater abundance in venom from P. nuchalis. Also, protein species ranging from 6– 15 kDa in venom from P. textilis were generally more acidic in nature than corresponding proteins from the venoms of P. affinis affinis and P. nuchalis. Recognition of venom proteins by brown snake antivenom
Statistical analysis All data are presented as the mean T SEM and group data initially compared using two-way analysis of variance. Post hoc analyses of group data were performed using an all pairwise multiple comparison procedure (Holm-Sidak method). P values less than 0.05 were considered significant.
Immuno-blots using brown snake antivenom of representative 2-DE gels depicting protein constituents from the venoms of P. affinis affinis, P. textilis and P. nuchalis are presented in Fig. 2. The profile of protein constituents
Fig. 1. Representative 2-DE gel images of the protein constituents present in the P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) proteomes. Spots from the P. affinis affinis 2-DE gel submitted for LC-MS/MS analysis are circled and labeled 1 – 7. The positions of the kaleidoscope MW markers introduced into the matrix of the P. affinis affinis 2-DE gel are indicated in panel A.
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Fig. 2. Representative 2-DE immuno blots of P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) venoms using brown snake antivenom to probe the venom/ antivenom protein interactions. Positions of the kaleidoscope MW markers as visualized on the immuno-blot of the P. affinis affinis 2-DE gel following transfer are indicated in panel A.
recognized by the antivenom is similar across all three snake species. In general, the signals are stronger than those visualized in the coomassie stained gels, where several protein species were recognized by the antivenom but were not detected in the coomassie stained gels. A strong signal was observed for proteins of MW 32– 45 kDa, reflecting the dominance of proteins within this MW range to incite an immune response. Similarly, proteins ranging from 78 to 132 kDa were also strongly recognized by the antivenom. However, the absence of significant signal for toxins in the MW range of 6 to ¨32 kDa is a striking and consistent feature of the immuno-blots. The protein recognition that does occur in this molecular weight range occurs for quite differently positioned proteins among the venoms of the three Pseudonaja species. Identification of proteins from the venom of P. affinis affinis In order to identify sample protein constituents from the venoms of the Pseudonaja sp. snakes, gel spots were submitted for LC-MS/MS analysis (protein spots from one snake species were chosen to represent protein constituents from all three Pseudonaja sp. snakes). The similar pattern of protein species from the venoms of the Pseudonaja sp. snakes separated by 2-DE provided a sound rationale for this approach. Consequently, seven spots from a representative coomassie stained 2-DE gel of venom constituents from P. affinis affinis were excised and submitted for tryptic digest and peptide identification by mass spectrometry. The low MW components (MW < 32 kDa) were primarily targeted due to their relative dominance on the 2-DE gels. The location of the spots on the 2-DE pattern is depicted in Fig. 1A. All identified peptide sequences from these spots and the results of database searches using NCBI BLAST and SWISS-PROT are presented in Table 1. Only the highest peptide match is reported. Of the seven spots submitted for sequencing, only four contained sequences with similarity to proteins in the databank. Peptides from the three remaining spots appear to be unique. Two peptide sequences were identified from spot 2 (¨MW 42 kDa). One peptide possessed similarity to the Group D prothrombin
activator trocarin from the venom of the snake Tropidechis carinatus while the other peptide was similar to Factor X isolated from Ornithorhynchus anatinus (duck billed platypus). These proteins are involved in blood coagulation, where trocarin functions to convert prothrombin to thrombin (Joseph et al., 1999) while factor X is converted to factor Xa which in turn serves to activate thrombin (Poorafshar et al., 2000; Sutherland and Tibballs, 2001). While Factor X proteins have not been previously isolated from brown snake venoms, Factor X activators have been reported in venoms from the genus Pseudonaja as well as other Australian snake venoms (Marshall and Herrmann, 1983). It is not unexpected to discover such protein species since the molecular weights of characterized coagulant toxins from the venoms of Pseudonaja sp. range from 50 kDa to ¨250 kDa (Rao and Kini, 2002; Stocker et al., 1994). Spot 4 yielded three peptide sequences with significant identity to a PLA2 protein isolated from the venom of P. textilis designated Pt-PLA2, and a PLA2 protein isolated from the venom of the snake Gloydius halys pallas designated B-PLA2. Similarly, the peptide sequence obtained from spot 5 was identical to a segment of the PLA2 protein HTe obtained from the venom of the snake Notechis scutatus. Both peptide sequences from spot 6 were identical to segments from the plasmin inhibitor textilinin (isolated from the venom of P. textilis). The PLA2 enzymes, a primary class of proteins present in the venoms of Pseudonaja sp. and other Australian snakes (Sutherland and Tibballs, 2001), are characteristically 13 – 15 kDa in size (Kini, 1997). The pharmacological characteristics of PLA2 proteins are extremely diverse, with activities ranging from neurotoxicity, myotoxicity, antiplatelet effects, hemolysis, coagulant/anticoagulant effects and hemorrhage (Kini, 1997; Grieg Fry, 1998). Pt-PLA2 was found to induce procoagulation (Armugam et al., 2004), B-PLA2 hemolyzed erythrocytes (Pan et al., 1998) while HTe was found to induce hypotension, hemorrhage and neurotoxicity (Francis et al., 1995). Textilinin, isolated from the venom of P. textilis functions to neutralize plasmin activity, inhibiting the destructive action of this enzyme on fibrin polymers (Filippovich et al., 2002; Masci et al., 2000).
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Table 1 Sequence comparison of peptide fragments from Pseudonaja affinis affinis proteins 17 separated using 2DE
Identical (:) and conserved (.) amino acids are indicated.
Contractile response in rat tracheal tissue induced by the Pseudonaja venoms The proteomic investigation highlighted a deficiency in the interaction between the brown snake antivenom and protein constituents of the Pseudonaja sp. snakes of MW 6 kDa to <32 kDa. In snake venoms, such species predominantly include the PLA2 proteins, as confirmed by our analysis, and a-neurotoxins (Sutherland and Tibballs, 2001). To assess the interaction between the brown snake antivenom and PLA2 proteins from the Pseudonaja sp. snakes, a functional approach was undertaken using rat tracheal nerve/muscle preparations. The action of the venom of P. affinis affinis on tracheal contractility has been previously established. A strong single contractile response resulting from PLA2 protein activity was reported (Judge et al., 2002). In this study, 100 Ag/mL of the venom of P. affinis affinis was applied to tracheal preparations as a positive control (Fig. 3A), resulting in a contractile response 51 T 4% (n = 4) of the maximum carbacholinduced response (10 AM). Application of the venoms of P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/mL) to tracheal
preparations was also found to affect tracheal tone through the induction of muscle contraction (Figs. 3B and C, respectively). These responses were 52 T 7% (n = 4) and 43 T 7% (n = 4) of the maximum carbachol-induced response (10 AM). Peaks were observed approximately 3 min following venom administration, and were maintained during the observation period (5 min). Venom-induced contractions were reversed by washout, and did not impair the contractile function of the muscle tissue in response to carbachol following venom treatment. Brown snake antivenom does not inhibit venom-induced tracheal contractility To characterize the venom-induced response in the presence of the brown snake antivenom, the venoms of P. affinis affinis (100 Ag/mL), P. textilis (50 Ag/mL) and P. nuchalis (100 Ag/ mL) were pre-incubated with 5 units/mL brown snake antivenom for 15 min at room temperature. All three venoms were found to induce a contractile response of tracheal muscle. The responses induced by the venoms of P. affinis affinis, P.
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Fig. 3. P. affinis affinis (A), P. textilis (B) and P. nuchalis (C) venoms induce contraction of rat tracheal preparations. P. affinis affinis (100 Ag/mL venom protein), P. textilis (50 Ag/mL venom protein) and P. nuchalis (100 Ag/mL venom protein) venoms were added to tracheal segments (resting tension 500 mg), resulting in significant contractions (51 T 4%, 52 T 7% and 43 T 7% of the maximum contraction induced by 10 AM cumulative carbachol, respectively; n = 4).
textilis and P. nuchalis were 40 T 5% (n = 3), 48 T 9% (n = 3) and 44 T 5% (n = 3) of the maximum carbachol-induced response (10 AM), respectively (Fig. 4). The two-way ANOVA indicated that the level of contraction was independent of both the snake venom and the drug treatment. This suggests an
Fig. 4. Effect of brown snake antivenom on Pseudonaja sp. venom-induced contractions of rat trachea. In the presence of antivenom (AV), the venominduced contractions for P. affinis affinis, P. textilis and P. nuchalis venoms were 40 T 5%, 48 T 9% and 44 T 5% of the maximum carbachol-induced response (10 AM cumulative), respectively (P < 0.05; n = 3). This suggested that the antivenom did not mediate a significant effect on venom-induced contractions. Control venom responses were 51 T 4%, 52 T 7% and 43 T 7%, respectively (n = 4).
Fig. 5. Effect of the PLA2 inhibitor 4-BPB on Pseudonaja venom-induced contractions of rat tracheal preparations. 4-BPB inhibited contractions induced by the venom of P. affinis affinis and P. nuchalis (*P < 0.05), but not those induced by the venom of P. textilis. In the presence of the inhibitor, the venominduced contractions for P. textilis and P. nuchalis were 43 T 11% and 10 T 1% of the maximum carbachol-induced response, respectively (10 AM cumulative concentration) and no response was observed for P. affinis affinis venom treated preparations (n = 3). The control venom response was 51 T 4%, 52 T 7% and 43 T 7% for P. affinis affinis, P. textilis and P. nuchalis venoms, respectively (n = 4).
ineffective neutralization of protein constituents responsible for tracheal muscle contractility by the antivenom. Contractile response in the presence of the PLA2 inhibitor 4-BPB The contractile response of tracheal nerve/muscle preparations following administration of the venom of P. affinis affinis has previously been attributed to PLA2 enzymatic activity, where treatment with the PLA2 inhibitor 4-BPB was found to completely neutralize the effects of the venom (Judge et al., 2002). In the current study, the effects of the venoms of P. textilis and P. nuchalis PLA2 proteins on tracheal contractility in the presence of 4-BPB were assessed and are depicted in Fig. 5. In a control experiment, the inhibitory effect of 4-BPB on contractility due to the venom of P. affinis affinis was confirmed (n = 3; two-way ANOVA, P < 0.05). 4-BPB also exhibited this inhibitory property on contractions induced by the venom of P. nuchalis, which were attenuated to 10 T 1% of the maximum carbachol-induced response (n = 3; two-way ANOVA, P < 0.05). However, application of venom of P. textilis to 4-BPB pre-treated tracheal preparations resulted in a strong contraction of tracheal muscle that was 43 T 11% of the maximum carbachol-induced response (n = 3). This suggested that PLA2 enzymatic activity from the venom of P. textilis was not a significant mediator of tracheal contractility. Discussion The current study investigates the complement of principal protein species within brown snake venoms that are effectively recognized and neutralized by the brown snake antivenom. It was of interest to examine the interaction of the antivenom with proteins from the three
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main Pseudonaja species as the commercial antivenom is raised solely against venom from P. textilis. The deficiency of information in this area is striking given the increasing number of reports indicating that high antivenom doses are required for treating victims of envenomation by the P. affinis affinis snake. 2-DE was utilized to separate and identify principal protein species. Comparison of the venom proteomes from the three Pseudonaja sp. snakes revealed a similarity in the spread of protein constituents with respect to MW and pI. The observation of high-MW (>78 kDa), mid-MW (32 –45) and low-MW (<32 kDa) protein species was consistent with a previously reported 1-DE analysis of P. affinis affinis venom proteins (Judge et al., 2002). The spread of protein components is also similar to the range of venom proteins from the Guatemalan snake Bothrops asper (Saravia et al., 2001). 2-DE analysis revealed primary spots present at 14, 20 –30 and 67– 94 kDa. However, there were no proteins visible above 94 kDa or below 14 kDa, in contrast to spots observed at these molecular weights on the protein gels of the Pseudonaja sp. 2-DE analysis of the venom proteomes of Naja naja atra and Gloydius halys also revealed a spread of components predominantly distributed from 97 – 30 kDa and 66– 20 kDa, respectively (Li et al., 2004). Interestingly, 1-DE analysis of 11 elapid venoms from the Micrurus snakes revealed the predominance of the 14 kDa species. Far less dominant were faint bands at 20 –30 kDa, 43 kDa, 67 kDa and 94 kDa (Prieto da Silva et al., 2001). Immuno-blotting using the commercial antivenom showed a similar pattern of recognition of proteins from the venoms of all three Pseudonaja species. This suggests that the venom proteins possess sufficient similarity across the species to warrant the use of antivenom, which is raised solely against the venom of P. textilis. Surprisingly, antivenom recognition occurred predominantly against the high-MW and mid-MW proteins. There was very little recognition of proteins between 6 kDa and ¨32 kDa by the Pseudonaja species. LC-MS/MS analysis of protein spots from the 2-DE gel of venom from P. affinis affinis revealed that the high-MW and mid-MW species are likely to include procoagulant proteins. This is consistent with the previously reported presence of toxins from brown snake venoms that interfere with blood homeostasis. Peptides derived from lowMW protein spots showed sequence similarity to several types of PLA2 enzymes as well as a plasmin inhibitor (textilinin) from P. textilis. While the presence of both short chain and long chain neurotoxins in the lower half of the 2DE gels was not verified by LC-MS/MS analysis, the existence of these peptides in the venoms of P. affinis affinis (Judge et al., 2002) and P. textilis (Gong et al., 1999) has been established. Thus, the antivenom may also be ineffective against the neurotoxin components of the venoms of the Pseudonaja sp. snakes that are among the principal protein constituents of Australian snake venoms (Sutherland and Tibballs, 2001). The absence of antivenom recognition of low-MW proteins from the venom of the Pseudonaja sp. prompted
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the use of a functional assay to examine contractile activity of the venom (potentially incited by either PLA2 enzymes or neurotoxin activity) in the presence of antivenom. The results suggest that the contractile function of rat trachea induced by all three venoms from the Pseudonaja sp. is not significantly attenuated by the antivenom. Further experiments utilizing the PLA2 inhibitor 4-BPB verified that the induced response by the venoms of P. affinis affinis and P. nuchalis was due to the enzymatic activity of the PLA2 proteins. In contrast, the contractile response to the venom of P. textilis was not incited by PLA2 enzymatic activity alone. Reports detailing the neurotoxic properties of the venom of P. textilis suggest the observed contractile activity may be mediated by the action of a pre-synaptic neurotoxin such as textilotoxin, a PLA2 protein with neurotoxic activity. Textilotoxin is the most neurotoxic snake venom protein isolated to date (Pearson et al., 1991; Pearson et al., 1993; Tyler et al., 1987) and its induction of contraction via neurotransmitter release is well documented (Harris, 1991; Nicholson et al., 1992). In our experiments, the antivenom did not recognize the low-MW protein components of the venoms of the Pseudonaja sp. In addition, the antivenom did not neutralize PLA2 enzymatic or putative neurotoxic activities that may be attributed to the low-MW species. However, the rat tracheal assay may not accurately reflect the human in vivo situation, and it is possible that that these toxins may escape neutralization by the antivenom in envenomed humans. In addition, other low-MW toxins that were not visibly active against the rat tracheal preparations may similarly remain unneutralized. The failure of snake antivenoms to neutralize specific venom components has been previously observed. Oshima-Franco et al. (1999) document a deficiency in the neutralizing capacity of Crotalus durissus terrificus antivenom for the venom, as well as the neurotoxic PLA2 protein crotoxin. Interestingly, a specific preparation comprising of anticrotoxin antibodies afforded optimal protection against the effects of both the venom and crotoxin to combat neuromuscular blockade. Similarly, Fry et al. (2001) reported a deficiency in the effectiveness of death adder antivenom in neutralizing the neurotoxic effects of several Australian death adder species. Application of these venoms to chick biventer cervicis preparations inhibited the twitch response of the muscle. However, a 10 min pre-incubation with 1 unit/mL antivenom failed to protect against twitch inhibition, whereas application of a higher dose of the antiserum (5 units/mL) was successful in reversing the inhibition of twitch response. Thus, while the commercial antivenom in its current form may provide adequate protection against the main clinical feature of envenomation in humans (such as disseminated intravascular coagulation), our study suggests that neutralization of lower molecular weight venom proteins may be limited. These include PLA2 protein and neurotoxin species which could underlie some of the secondary symptoms of envenomation in humans and the
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