Article pubs.acs.org/jpr
Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution Jure Skejic,*,†,∥ David L. Steer,‡ Nathan Dunstan,§ and Wayne C. Hodgson∥ †
Department of Biochemistry and Molecular Biology, BIO21 Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia ‡ Monash Biomedical Proteomics Facility, Monash University, 23 Innovation Walk, Clayton, Victoria 3800, Australia § Venom Supplies Pty Ltd., Stonewell Road, Tanunda, South Australia 5352, Australia ∥ Monash Venom Group, Department of Pharmacology, Monash University, 9 Ancora Imparo Way, Clayton, Victoria 3800, Australia S Supporting Information *
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 nervediaphragm preparation occurs sooner upon exposure to the venom (10 μg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic β-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. KEYWORDS: snake venom, proteome, toxicity, evolution, procoagulants, neurotoxins, Pseudonaja textilis, Oxyuranus scutellatus, label-free quantification, quantitative proteomics
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INTRODUCTION
The eastern Brown snake Pseudonaja textilis and coastal taipan Oxyuranus scutellatus are moderately to large-sized Australo-Papuan snakes, belonging to the Pseudonaja − Oxyuranus evolutionary group, which originated within the oxyuranine elapid radiation.8 These are medically important species in Australia and New Guinea, known to inflict a fatal bite in humans and domestic animals.9,10 Pseudonaja textilis feeds on rodents, lizards, and other small vertebrates, whereas rodents and other small mammals predominate in the diet of Oxyuranus scutellatus.11,12 Their prey is injected with venom, which contains toxic proteins that target the circulatory and neuromuscular physiological systems.13 The same physiological systems are affected in human envenomations, with victims
Regulatory mechanisms of gene transcription and mRNA translation have important effects on venom proteome composition. A study has shown that a mutation in the silencer region of a toxin gene can reduce the suppressive activity of the silencer and substantially increase the toxin gene expression.1 While variation in venom protein abundances between related snake lineages and populations has been extensively documented,2−4 few studies have addressed the role of venom protein expression in the modulation of snake venom toxicity.5,6 Furthermore, past studies have not directly quantified the expression of specific toxicity-inducing venom proteins using mass spectrometry-based quantitative proteomics, relying instead on percentage estimates obtained by general reverse-phase HPLC-based profiling of venom proteome and densitometry.2,7 © 2015 American Chemical Society
Received: August 14, 2015 Published: October 21, 2015 4896
DOI: 10.1021/acs.jproteome.5b00764 J. Proteome Res. 2015, 14, 4896−4906
Article
Journal of Proteome Research
venoms of two or more species. This is because unless all proteins comprising a venom proteome are identified, the relative percentages will change as new proteins become identified with additional mass spectrometry analyses. Furthermore, significant disadvantages of standard Coomassie staining and densitometry include low sensitivity, poor dynamic range, and protein-to-protein variability.30 With the advent of high-resolution mass spectrometry like Orbitrap,31−33 label-free quantification methods have emerged as a powerful tool for protein expression analysis. Spectral counting methods are associated with significant quantification errors and have largely been superseded by intensity-based methods.34,35 The area of an MS1 spectrum extracted ion chromatogram (XIC) is a measure of peptide abundance and can be used to infer relative and absolute abundances of proteins, as implemented in the Skyline MS1 filtering method.36 Ratios of MS1 XIC areas can be utilized for a comparison of relative abundances of peptides in different samples.37 The TOPn method is based on the discovery that the average mass spectrometry intensity response of the most intense peptides is directly proportional to the absolute abundance of the protein.34,38 This method was first introduced and experimentally validated with the three highest intensity peptides and is also known as the TOP3 method.38 A subsequent study found that the linear relationship between the MS intensity signal and protein concentration is maintained with a range of n values, including when one or two most intense peptides are used for quantification.39 Here the XIC approach was used for direct determination of the expression of snake venom proteins.
experiencing hypotensive collapse, coagulopathy, and, in Oxyuranus bites, clinically significant neurotoxicity.14,15 Most South Australian populations of Pseudonaja textilis belong to the southeastern and coastal Queensland populations to the northeastern mitochondrial DNA clade.16 In the previous toxinological study of these regional P. textilis forms and Oxyuranus scutellatus, profound differences in the procoagulant activities and neurotoxicities of the venoms were found.13 At the equivalent concentration, the venoms of P. textilis initiated substantially faster onset of rat blood plasma coagulation than O. scutellatus venom. The differences in the onset of neuromuscular paralysis observed in the phrenic nervediaphragm preparation of the rat were dramatic. The venom of South Australian P. textilis paralyzed the rat diaphragm as much as 6.1 times faster than the venom of Queensland P. textilis. A particularly significant finding was that the contractile response of the avian skeletal muscle to exogenous acetylcholine was completely abolished upon exposure to South Australian P. textilis venom and largely preserved following exposure to Queensland P. textilis venom, indicating differences in the postsynaptic neurotoxicity of the venoms. Interestingly, the venom of South Australian P. textilis paralyzed the rat diaphragm substantially (2.8 times) faster than the venom of Oxyuranus scutellatus (at 10 μg/mL). Given significant differences in venom-induced neurotoxicity and coagulation pathology, the Pseudonaja/Oxyuranus group represents a good model for the study of venom toxicity evolution in snakes. Because of recent common ancestry, the qualitative protein composition of the venoms of these snakes is very similar. A step toward understanding the causes of the variation in the toxicities is quantification of venom protein expression. In this study, a subset of the venom proteome of Pseudonaja textilis and Oxyuranus scutellatus known to be involved in neurotoxicity, coagulopathy, and hypotension was analyzed with label-free quantitative proteomics. Venoms of brown snakes and taipans are complex mixtures of diverse proteins.17−20 Venom prothrombin activator is a large protein complex (>200 kDa), consisting of a serine protease enzyme (coagulation factor Xa-like) and a nonenzymatic cofactor (coagulation factor Va-like). This protein complex functions as a potent procoagulant, converting prothrombin to thrombin and rapidly clotting blood plasma.21,22 An intravenous injection of the purified venom prothrombin activator rapidly leads to coagulopathy and collapse of the circulatory system in rodents, resulting in immediate death. 13,23 The neurotoxins of Pseudonaja and Oxyuranus belong to PLA2 and three-finger toxin protein families. Both families of neurotoxins cause neuromuscular blockade, but the mechanism of activity differs. Presynaptic PLA2 β-neurotoxins act on the peripheral nerve terminals, whereas postsynaptic three-finger α-neurotoxins bind to the skeletal muscle nicotinic acetylcholine receptors, inhibiting neurotransmission.24−28 A common approach to quantitatively assay the composition of a snake venom proteome has been to run the sample on a reverse-phase HPLC and then to calculate the relative abundance of individual venom proteins as a percentage of the reverse-phase chromatographic peak profile. Because reverse-phase fractions often contain coeluting proteins, their relative abundance is estimated from SDS-Page bands with densitometry.7,29 While this combination of methods is useful to get an insight into venom protein family composition, it is not suitable for direct quantitative comparison of the expression of individual proteins between different samples, for example,
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EXPERIMENTAL SECTION
Venom Sampling
Venoms for this study were collected from adult individuals. Pseudonaja textilis specimens were captured in Barossa Valley, South Australia (n = 3) and in Mackay, Queensland (n = 2), and Oxyuranus scutellatus specimens (n = 3) are from Julatten, Queensland. The snakes were housed in the Venom Supplies (SA) facility, where venom extractions were carried out. Venoms from the individuals of each species were pooled and freeze-dried. Sample Preparation
The venom protein samples were weighed to ∼1 mg using a microbalance, and each sample was reconstituted to a concentration of 5 mg/mL in a solution of 10% acetonitrile, 20 mM ammonium bicarbonate. The equivalent of 100 μg of the protein sample was reduced in 2.5 mM DTT at 50 °C for 15 min, followed by alkylation with 10 mM iodoacetamide for 60 min in the dark at room temperature. Following alkylation a solution containing 1 μg trypsin (Promega, Madison, WI) in 20 mM ammonium bicarbonate was added and the samples incubated at 37 °C overnight. The final concentration of the protein digest mixture was 2 μg/μL. Shotgun LC−MS Protocol
For analysis by LC−MS, the samples were diluted to 0.2 μg/μL using a buffer of 2% acetonitrile and 0.1% formic acid and centrifuged at 16 000g for 5 min before transfer to an autosampler vial. The mass spectrometry instrument used for analysis was Q-Orbitrap, model Q Exactive, Thermo Scientific (Bremen, Germany), software: Thermo Excalibur 2.2. The LC instrument used was nano RS-HPLC, Ultimate 3000, Thermo 4897
DOI: 10.1021/acs.jproteome.5b00764 J. Proteome Res. 2015, 14, 4896−4906
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Journal of Proteome Research
presence of an extreme intensity outlier, the other two technical replicates were used for the mean intensity calculation. The Supporting Information contains Skyline-generated MS1 XIC peak areas of the distinct tryptic peptides used in label-free quantitative analyses (Supplementary Table S1) and individual protein identification and quantification data (Supplementary Table S2).
Scientific (Bremen, Germany), software Thermo Excalibur 2.2 DCMS link. Tryptic digests were injected in a volume of 2 μL and concentrated on a 100 μm, 2 cm nanoviper pepmap100 trap column with loading buffer (2% acetonitrile, 0.1% formic acid) at a flow rate of 15 μL/min. The peptides then eluted and separated with a 50 cm Thermo RSLC pepmap100, 75 μm id, 100 Å pore size, reversed-phase nano column with a gradient starting at 97% buffer A (0.1% formic acid), between 4 and 5 min the concentration of buffer B (80% acetonitrile, 0.1% formic acid) increased to 10%, followed by a 30 min gradient to 40% B then to 95% B in 2 min, at a flow rate of 300 nL/min. The eluant was nebulized and ionized using the Thermo nano electrospray source with a distal-coated fused silica emitter (New Objective, Woburn, MA) with a capillary voltage of 1800 V. The MS data were acquired with the following parameters: resolution 70 000, AGC target 3e6, 120 ms Max IT. Peptides were selected for MSMS analysis in full MS/dd-MS2 (TopN) mode with the following parameter settings: TopN 10, resolution 17 500, MSMS AGC target 1e5, 60 ms Max IT, NCE 27, and 3 m/z isolation window. Underfill ratio was at 10% and dynamic exclusion was set to 15 s.
Pathophysiology Data
Pathophysiological data used in Figures 10 and 11 were obtained from the previous study.13 Rat diaphragm-phrenic nerve preparation experiment was conducted at 37 °C, using the venom concentration of 10 μg/mL (vehicle: water). Venom procoagulant activity was tested after administration of 0.5 μg of venom (in 50 μL saline) to 100 μL of citrated rat plasma (recalcified with 4 μmol CaCl2) at 37 °C. Contractile response of the avian skeletal muscle to exogenous acetylcholine (1 mM) was examined at 40 °C following exposure to 10 μg/mL venom. For a detailed description of the pathophysiological experiments, please refer to Skejic and Hodgson (2013) study.13
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Protein Identification Bioinformatics
Raw data from the LC−MS runs were processed with MaxQuant/Andromeda software,40 version 1.5.0.0, and searched against Squamata fasta subset of the UniProtKB database (119 890 sequences) using the following parameters: digestion enzyme: trypsin, missed cleavages: 2, fixed modifications: carbamidomethyl (C), variable modifications: oxidation (M), instrument: Orbitrap, first peptide search tolerance: 20 ppm, main peptide search tolerance: 6 ppm, fragment mass tolerance: 0.02 Da. Peptide spectrum matches were filtered with a high-confidence FDR of 0.01, and the protein FDR was set to 0.05. Protein identifications were based on unique tryptic peptides. Alignments of the toxin sequences from the Uniprot database were made with Clustal Omega, using the default settings for protein sequences.41 Matched tryptic peptides were mapped onto the venom protein sequence alignments to show the identified distinct and shared tryptic peptides and those used for XIC-based quantification of the protein family members.
RESULTS
Venom Prothrombin Activator (Factor X and V) Expression
Two related coagulation factor X-like proteins were found to be highly expressed and similarly abundant in the venom of Queensland (Mackay) Pseudonaja textilis: pseutarin C venom prothrombin activator catalytic subunit (Q56VR3) and coagulation factor X isoform 2 (Q1L658) (Figure 1). The
XIC Label-Free Quantitative Proteomic Analysis
Skyline (version 3.1.0.7382) spectral libraries were created from MaxQuant/Andromeda search results and analyzed with Skyline Bibliospec module. To produce extracted ion chromatograms (MS1 XIC), the Thermo.raw files acquired on Orbitrap Q Exactive were processed with Skyline DDA with MS1 filtering protocol.36 The resolution was set to 70 000 at m/z = 200. Peak integration was automatic but was also visually inspected and manually adjusted when necessary. Three isotopic precursors were extracted, but only the most abundant was used for quantification, as in the study Rardin et al.37 Protein abundance was inferred by the label-free TOPn method.38,39 Specifically, unique tryptic peptides were ranked from the most intense to least intense for each protein. On the basis of the unique peptide availability in the highest intensity region, the two most intense unique peptides (TOP2) were used for quantification of venom factor X and V proteins and the most intense unique peptide (TOP1) for quantification of individual β-neurotoxin chains and 3FTx α-neurotoxin variants. Statistical analyses were conducted in GraphPad Prism 6.01, utilizing standard Student’s t test and one-way ANOVA on log10 (X) transformed intensities. In very rare instances of the
Figure 1. Venom prothrombin activator expression. Label-free quantitative analysis revealed high expression of venom factor X enzymes in the venoms of Australian eastern brown snake Pseudonaja textilis. Queensland P. textilis shows higher expression of venom prothrombinases than South Australian P. textilis. A related species, the coastal taipan Oxyuranus scutellatus, produces a significantly lesser amount of these proteins. Error bars in this and subsequent graphs represent ± 1 s.e.m. 4898
DOI: 10.1021/acs.jproteome.5b00764 J. Proteome Res. 2015, 14, 4896−4906
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difference in the TOP2 abundance values between these proteins was not significant (Student’s t test, p = 0.5074). In addition to these two proteins, a variant containing the tryptic peptide DTHFITGIVSWGEGCAQTGK was found. This sequence is homologous to the much more abundant DTHFITGIVSWGEGCAR of the typical pseutarin C (Q56VR3). The first peptide is also found in the sequences of coagulation factor X isoform 1 (Q1L659) of Pseudonja textilis and oscutarin C (Q58L96) of O. scutellatus (Figure 4). The ratio of the MS1 XIC peak areas of the homologous peptides (DTHFITGIVSWGEGCAQTGK/DTHFITGIVSWGEGCAR) in Queensland P. textilis venom was 9.92 × 10−4, indicating very low expression of the DTHFITGIVSWGEGCAQTGKcontaining variant. The same homologous sequences were also detected in the venom sample from South Australian (Barossa) P. textilis population (Figure 4); however, in contrast with Queensland P. textilis venom, the DTHFITGIVSWGEGCAQTGK-containing venom factor X variant was greatly expressed in South Australian P. textilis venom (Figure 1). The XIC ratio of the homologous peptides (DTHFITGIVSWGEGCAQTGK/DTHFITGIVSWGEGCAR) in South Australian P. textilis venom was 1.38. Current analysis was unable to detect tryptic peptides unique to coagulation factor X isoform 2 (Q1L658) in the venom sample from the South Australian (Barossa) population. In the venom of O. scutellatus from Queensland (Julatten), only a single venom factor X protein was found, matching the typical oscutarin C in the Uniprot database (Q58L96) (Figure 4). The total summed XIC intensity (relative abundance) of venom factor X proteins was approximately 2 times higher in Queensland P. textilis than in South Australian P. textilis venom sample and 8 times fold higher in Queensland P. textilis than in
Figure 2. PLA2 β-neurotoxin expression. Textilotoxin is the only substantially expressed neurotoxin in Queensland (Mackay) P. textilis venom, resulting in predominant presynaptic neurotoxicity of the secretion. Presynaptic β-neurotoxins are substantially more abundant in Queensland than in South Australian P. textilis venom. These neurotoxins are highly expressed in Oxyuranus scutellatus venom.
Figure 3. Three-finger α-neurotoxin expression. Enormous regional differences in the expression of postsynaptic α-neurotoxins in the venoms of Pseudonaja textilis have been discovered. Long-chain (or type II) α-neurotoxins are particularly highly expressed proteins in the venom of South Australian P. textilis. Expression of α-neurotoxins is very low in the venom of Queensland P. textilis. 4899
DOI: 10.1021/acs.jproteome.5b00764 J. Proteome Res. 2015, 14, 4896−4906
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Journal of Proteome Research
Figure 5. Tryptic peptide map of β-neurotoxins (PLA2 protein family). Notation as in Figure 4.
Figure 4. Tryptic peptide map of venom prothrombin activator enzymes (venom factor X protein family). Identified tryptic peptides of venom factor X proteins were mapped onto the protein sequence alignments. Colors denote the venoms in which the tryptic peptides were identified: P. textilis QLD (red), P. textilis SA (blue), O. scutellatus QLD (green), P. textilis QLD and P. textilis SA (purple), P. textilis QLD and O. scutellatus QLD (orange), P. textilis SA and O. scutellatus QLD (turquoise), P. textilis QLD, P. textilis SA, and O. scutellatus QLD (brown). Unique peptides are in bold. The most intense distinct tryptic peptides used for TOPn XIC quantification of individual protein family members are underlined. An amino acid in italics indicates the beginning of a new sequence of the adjacent tryptic peptide. Figure 6. Tryptic peptide map of α-neurotoxins (3FTx protein family). Notation as in Figure 4.
Oxyuranus scutellatus venom sample. One-way ANOVA on the XIC abundances of the venom factor X proteins produced a significant result (p < 0.0001), with Holm-Sidak’s multiple comparison test significant for all three venoms at the significance level (α) of 0.05. Venom factor V and X proteins are subunits of the venom prothrombin activator complex. Expectedly, the abundance of venom factor V was found to follow the trend observed in venom factor X abundance. ANOVA on the TOPn abundances of venom factor V proteins was significant (p < 0.0001), with Holm-Sidak’s multiple comparison test significant for all three venoms (at α = 0.05).
PLA2 β-Neurotoxin Expression
Subunits of the presynaptic neurotoxin textilotoxin were substantially more expressed in Queensland P. textilis venom than in South Australian P. textilis venom (Figure 2). In addition to the typical textilotoxin, peptide sequences matching those present in the subunits of taipoxin and neurotoxic PLA2 OS2 (Q45Z47) were detected in P. textilis venoms (Figure 5), but their expression levels were very low (Table S1). PLA2 βneurotoxins were the most abundant in the venom of O. scutellatus (Julatten, Queensland). The presynaptic neurotoxin PLA2 OS2 (Q45Z47) is evolutionarily most closely related to the taipoxin α-chain, and along with the taipoxin chains, it was found to be highly expressed in O. scutellatus venom (Figure 2). 4900
DOI: 10.1021/acs.jproteome.5b00764 J. Proteome Res. 2015, 14, 4896−4906
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Journal of Proteome Research
Comparisons of the Identical Tryptic Peptide XICs between SA and QLD Pseudonaja textilis Venoms
In this analysis, MS1 extracted ion chromatogram peak areas of the most intense peptides, which were present in both South Australian and Queensland P. textilis venoms, were compared individually (Figure 7). The XIC ratio of each homologous peptide between Queensland and South Australian P. textilis venoms was calculated (for each technical replicate). The ratios were transformed with the logarithm to the base 10. Student’s t test on the ratios was performed, with the null hypothesis Ho (μ = 0 if the XIC peak area ratio is not significantly different from zero) and Ha (μ ≠ 0 if the peptide XIC peak area difference between the venoms is significant). The Student’s t tests were significant (p values
