Diversity of Toxic Components from the Venom of the Evolutionarily

Mar 21, 2007 - envenomation, Australian elapids have been the focus of much research attention including the neutralization of the clinical effects of...
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Diversity of Toxic Components from the Venom of the Evolutionarily Distinct Black Whip Snake, Demansia vestigiata Liam St Pierre,†,‡,⊥ Geoff W. Birrell,†,‡,⊥ Stephen T. Earl,†,‡ Tristan P. Wallis,† Jeffrey J. Gorman,† John de Jersey,§ Paul P. Masci,‡ and Martin F. Lavin*,†,‡ The Queensland Institute of Medical Research, Brisbane, Australia, School of Medicine, Central Clinical Division, The University of Queensland, Brisbane, Australia, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia Received March 21, 2007

Included among the more than 300 species of elapid snakes worldwide is the Australian genus Demansia, or whip snakes. Despite evidence to suggest adverse clinical outcomes from envenomation by these snakes, together with confusion on their true phylogenetic relationship to other Australian elapids, not a single toxin sequence has previously been reported from the venom of a Demansia species. We describe here a combined proteomic and transcriptomic approach characterizing the venom from the black whip snake, Demansia vestigiata. A total of 13 distinct toxin families were identified, including homologues of all of the major toxic components previously reported from the venom of other Australian elapids, such as factor X-like prothrombin activators, neurotoxins, phospholipases, cysteine rich secretory proteins, textilinin-like molecules, nerve growth factors, L-amino acid oxidases, vespryns, 5′ nucleotidases, metalloproteinases, and C-type lectins as well as a novel dipeptidyl peptidase family. Phylogenetic analysis of these sequences revealed an early evolutionary split of the black whip snake from all other characterized Australian snakes, with a low degree of sequence identity between D. vestigiata and the other snakes, across all toxin families. The results of this study have important implications not only for the further characterization of venom from whip snakes, but also for our understanding of the evolutionary relationship of Australian snake species. Keywords: Demansia vestigiata • Black Whip snake • toxin • venom • proteomics • cDNA cloning • snake venomics

Introduction Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Among the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family.1 Worldwide, the elapid family is composed of approximately 61 genera and 300 species and are primarily defined by their unique venom delivery system which entails two permanently erect front fangs at the end of a relatively immobile maxilla.2 Because of the complexity of their venom and significant pharmacological outcomes of envenomation, Australian elapids have been the focus of much research attention including the neutralization of the clinical effects of a bite, as well as the analysis of individual components responsible for these effects at both the structural and func* Address correspondence to Professor Martin Lavin, The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, P.O. Box Royal Brisbane Hospital, Herston, Brisbane, 4029, Australia. Tel: 617 3262 0341. Fax: 617 3362 0106, E-mail: [email protected]. † The Queensland Institute of Medical Research. ‡ School of Medicine, The University of Queensland. § School of Molecular and Microbial Sciences, The University of Queensland. ⊥ These authors contributed to the work equally. 10.1021/pr0701613 CCC: $37.00

 2007 American Chemical Society

tional levels.3,4 Despite this, little consideration has been given to the venom components from the pharmacologically significant Australian elapid genera, Demansia, or whip snakes. The black whip snake, Demansia vestigiata, and the closely related species, Demansia papuensis, are sympatric, oviparous, diurnal elapids that inhabit northern and northeastern Australia and parts of Papua New Guinea.5 It is believed that the black whip snake, which was first described by Macleay (1884), is the fastest moving land snake in Australia with a diet composed mainly of lizards and occasionally frogs.6,7 They may grow up to 1.8 m in length, with dark brown to black dorsal scales with a thin, whip-like tail.8 Previously, both species were classified under a single name, Demansia atra, until further distinguished by distinct differences in size, scalation, and color.9 This has led to some confusion in the literature since most of the small number of previous studies into this snake refer to a single species, D. atra, and do not distinguish between D. papuensis and D. vestigiata. Despite a considerable body of clinical information on envenomation by other Australian elapid snakes, there is no clearly defined clinical description of the outcome of bites from Demansia species.5,10 Traditionally, Australian elapid snakes have been classified according to a number of parameters including internal and external morphology, immunological distances, and ecological Journal of Proteome Research 2007, 6, 3093-3107

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research articles and biochemical characteristics.11 Despite this, the phylogenetic relationship between Australian snakes has yet to be fully elucidated. DNA sequencing of cytochrome b and 16S rRNA mitochondrial genes from 19 of the 20 Australian elapid genera and morphological analysis of D. atra reveal a close relationship with the Papua New Guinean genus Aspidomorphus, or crowned snakes.2,12 This appears to be contradictory to a previous report on a closer relationship between Demansia and the Australian genera Pseudechis, Pseudonaja, and Oxyuranus based on immunological and hemipenial morphological means.13,14 Molecular sequence analysis has proven to be a powerful tool in that it not only resolves the order of divergence between species but also gives a measure of the timing of that divergence.15 Despite this, only five DNA sequences have been identified from D. atra and not a single toxin sequence from a Demansia species. The venom from a single Australian elapid may contain upward of 100 different proteins which are typically small in size, cysteine rich, and often exert a multitude of functions with specific molecular targets.16 No detailed analysis of the constituents of the venom from the black whip snake has previously been described. The venom from D. atra was reported to have an LD50 value of only 14.2 mg/kg as determined by subcutaneous injection in mice and was observed to be only weakly coagulant.1,17 A recent study has also indicated that the venom of D. papuensis demonstrates both myotoxic and weak neurotoxic effects.18 The venoms from other related Australian elapids produce symptoms including disorientation, paralysis, nausea, swelling, hemeotoxicity, myotoxicity, and respiratory failure in association with multiple coagulopathic effects.3 These symptoms predominantly arise from three of the major constituents within the venom: a factor Xa-like prothrombin activating enzyme, three-finger neurotoxins, and phospholipase A2 (PLA2) enzymes. However, with the advent of DNA cloning technology and more advanced proteomic methodologies, an increasing number of toxin families have been isolated and characterized from Australian snake venoms.19-21 A molecular analysis of the components from snake venoms is not only of interest for our understanding of the functionality of the toxin, but also on the origin and evolution of these proteins. The diversity of venom peptides are a direct result of their mode of evolution: toxin genes are subject to frequent duplication events within the snake genome, often followed by functional and structural diversification at an accelerated rate, as demonstrated by relatively conserved intronic sequences compared to exons.22,23 We describe here a detailed analysis of the venom from the Australian elapid, the black whip snake (D. vestigiata), incorporating a comprehensive proteomic survey in conjunction with cDNA cloning and identification of a number of unique toxin proteins. This study is the first account of any of the toxic components from the venom of this medically significant snake, and not only provides information on the phylogenetic and evolutionary relationship of this species to other elapid snakes, but also has important implications for the structural and functional relationship of toxins.

Experimental Section Venom Two-Dimensional Electrophoresis and Immunoblotting. Venom from a black whip snake (Demansia vestigiata) was obtained in lyophilized form and resuspended in 50% glycerol/50% PBS to a final protein concentration of 10 mg/ mL. Prior to first dimension isoelectric focusing (IEF), 30 µL 3094

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(300 µg total protein) of reconstituted venom was added to 170 µL of rehydration solution containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 10 mM DTT, and 0.2% (v/v) pH 3-10 Biolyte ampholytes (BioRad, Hercules, CA). The final volume of 200 µL was applied to a precast BioRad 11 cm 3-10 linear IPG strip. First dimension IEF was carried out in a BioRad Protean isoelectric focusing unit at 20 °C using a three-phase program: 250 V rapid gradient for 15 min, 250 V to 8000 V linear gradient for 3 h, and 8000 V rapid gradient to a total of 40 000 V/h. Prior to second dimension separation, the proteins on the IPG strip were reduced and alkylated and then applied to 12% Tris-HCl acrylamide gels (BioRad criterion, 13 cm × 10 cm) for electrophoresis at 200 V for 65 min. Gels were stained with a mass spectrometry compatible silver stain, based on a previously described method.24 Immunoblotting to detect the presence of γ-carboxyglutamic acid (Gla) residues and the heavy chain of factor X was performed as previously described.25 Detection of kunitz-type serine protease inhibitors (textilinin-like molecules) was also performed via immunoblotting. Antiserum to common brown snake (Pseudonaja textilis) recombinant factor Xa heavy chain and to native P. textilis textilinin-1 was raised in sheep against the purified proteins.26 Identification of Venom Proteins Using MALDI-TOF and TOF-TOF Mass Spectrometry and de Novo Peptide Sequencing. Protein spots from silver stained 2D PAGE were excised, washed in water, and destained as previously described.27 Trypsin was added and proteins were digested overnight at 37 °C prior to extraction. Extracted peptides were dried, resuspended in 50% ACN/0.1% TFA, mixed 1:1 with matrix (10 mg/ mL R-cyano-4-hydroxycinamic acid in 60% acetonitrile/25 mM ammonium bicarbonate), and spotted on a MALDI plate. Peptides were analyzed using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA) operated in positive ion reflectron mode. MS data were acquired using 2000 shots of a Nd:YAG laser at 355 nm with a 200 Hz repetition rate and fixed intensity. MS data were calibrated via a plate-wide external calibration using the 4700 Mass Standards Kit (Applied Biosystems). The top 50 most intense peptides detected for each spot in the MS mode were automatically selected for tandem MS (MS/MS) analysis using 3000 laser shots at a fixed intensity, approximately 20% greater than that used for MS. MS/MS data were calibrated against the MS/MS fragments of the m/z ) 1296.685 angiotensin I peptide in the standards. MALDI-TOF/TOF-MS/MS data from the 4700 Proteomics Analyzer were automatically analyzed using the GPS Explorer suite of software (Version 3.5 Build 321, Applied Biosystems). For each spot a combined MS and MS/MS analysis was performed in-house using a Mascot search engine (version 1.9) and the Celera Discovery System database (CDS Combined KBMS2.1.20030813 containing 1335729 sequences, dated May 5, 2006). MS peptide tolerance was 100 ppm, and MS/MS tolerance was 0.3 Da. All searches took into account carbamidomethylated cysteine and oxidized methionine. For the purposes of protein identification, no other post-translational modifications were considered. Criteria for positive protein identification were based on Mascot scores greater than the 95% confidence threshold, with candidates whose protein mass and pI correlated with the 2D PAGE spot automatically accepted. Those candidates with Mascot scores below the 95% confidence threshold but whose identity matched to a known snake venom protein were also included. Candidates still unknown were selected for de novo peptide sequencing, with MALDI-TOF/TOF-MS/MS data opened in Data Explorer ver-

research articles

Black Whip Snake Venom Characterization

Table 1. Primer Sequences Used for the PCR Amplification of D. vestigiata Venom Gland cDNA Transcripts protein family

forward primer

reverse primer

vestiginin Dv SNTX Dv LNTX Dv NGF PLA2 Dv-1 to DV-7 Dv CRISP vestarin D Dv LAAO Dv 5′ nucleotidase Dv dipeptidyl peptidase Dv calglandulin

5′-ATG TCT TCT GGA GGT-3′ 5′-CGC AGG ATG AAA ACT CTG CTG C-3′ 5′-ATG AAA ACT CTG CTG CTG ACC-3′ 5′-TAA TGT CCA TGC TGT GCT AC-3′ 5′-TGC TTG CAG CTT CAC CAC TGA C-3′ 5′-GGA GTT ACA CTG GGG CTC-3′ 5′-ATG GCT CCT CAA CTA CTC CTC TG-3′ 5′-GAT GAA TGT CTT CTT TAT GTT ATA-3′ 5′-ATG ACA ACT TCT TGG AGT G-3′ 5′-CCC GCA ATG AAG ACT GTA GTG-3′ 5′-CGA GGA AAT GGC AGC AAC ACT AAC-3′

5′-TCA GGC AGC ACA GGT-3′ 5′-GCC ACT CGT AGA GCT AAT TGT TG-3′ 5′-GTC GAG ATG TCA AAG ACG CA-3′ 5′-TTG GAG CAA TCA ATG GTC-3′ 5′-TCC TCG CGC TGA AGC CTC TCA AA-3′ 5′-ACT GAA TGG GAG ATC AGC-3′ 5′-TTA GAG CGG ACC AGT GCT TGA CTC-3′ 5′-TTA AAG TTC ATT GTC ATT GCT CA-3′ 5′-TTA TTC TTC TTC TTC CTC-3′ 5′-AGG CAC TCC CAA CTT TAT GGC-3′ 5′-GTC TTA CTG AGT CAG TTT GAA GG-3′

sion 4.2 (Applied Biosystems) and deisotoped, and raw text peak lists exported. The peak lists were analyzed using the automatic de novo function of PEAKS Studio software version 2.4 (Bioinformatics Solutions Inc., Waterloo, Canada).28 Peptide sequences of six or more amino acids with 100% confidence call, using parent ion and fragment mass error tolerances of 0.1 Da, were collected and matched to the NCBI nonredundant protein database using the protein BLAST algorithm (version 2.2.14). RNA Isolation and cDNA Synthesis. A pair of venom glands were excised from a D. vestigiata specimen as identified by a qualified herpetologist and collected under Australian National Parks and Wildlife’s permit number W4\00261\01\SAA. Snap frozen glands were homogenized with a polytron, and RNA was isolated using the Tri Reagent method (Sigma-Aldrich, St. Louis, MO), precipitated in 2-propanol, washed with 70% ethanol, and resuspended in DEPC-treated water. First strand cDNA was synthesized from 1 µg of total RNA with an oligo(dT)12-18 primer via reverse transcription with 200 units of Superscript II RNase H- Reverse Transcriptase (Invitrogen, Mt. Waverly, Australia). The final reaction was ethanol precipitated, and cDNA samples were resuspended in sterile water and stored at -20 °C. Full-Length Toxin cDNA Identification. The full-length cDNA sequences of multiple toxin families were amplified via a combination of 3′RACE and PCR and subsequently cloned and sequenced. Briefly, PCR amplification was performed with 50 Fmol of each of the forward and reverse primers described in Table 1 for individual gene sequences, designed from toxin sequences previously characterized from other Australian elapid snakes. The reaction mix was made to a final volume of 25 µL containing approximately 200 µg of cDNA template, 1 unit of AmpliTaq Gold (Applied Biosystems) in 1× buffer, 2 mM MgCl2, and 200 µM dNTPs. All reactions were thermocycled and run with appropriate no-template controls. Resulting PCR products were analyzed on a 1% TAE agarose gel, excised, and purified with a HiYield Gel Extraction kit according to manufacturer’s instructions (Real Genomics, ChungHo, Taiwan) and then cloned via the pGEM-T vector system (Promega, Madison, WI). Additional vestiginin sequences were also identified via 3′RACE, using the forward primer (5′-GAG CTT CAT CAT GTC TTC TGG AGG TCT TCT TC-3′) with a SMART RACE cDNA Amplification kit according to manufacturer’s protocol (Clontech, Palo Alto, CA). Multiple clones were isolated for each toxin family and sequenced with an ABI Big Dye Terminator cycle sequence ready reaction kit (Perkin-Elmer, Norwalk, CT). Sequencing was performed in both the forward and reverse direction, results were assembled and analyzed with BioEdit software (Isis Pharmaceuticals Inc, Carlsbad, CA), and sequence comparisons were performed with other known protein families from related snake species.

Phylogenetic Analysis. Phylogenetic and molecular evolutionary analysis of venom gland-specific gene sequences was performed with MEGA version 2.1 software.29 Phylogenetic analysis was performed for each toxin family identified, with comparisons made against homologues previously identified from other snake venoms. In the case of the cysteine-rich secretory protein (CRISP) family a sequence from the chimpanzee, Pan troglodytes (XP 001148468), was selected as an outgroup for analysis, which was performed with a Minimum Evolution test of phylogeny via the Neighbor-Joining method with 1000 bootstrap replicates. Novel CRISP sequences identified from the venom gland of the black whip snake, D. vestigiata (DQ917520),rough-scaledsnake,Tropidechiscarinatus(DQ917546), and small eyed-snake, Rhinoplocephalus nigrescens (DQ917547) were included in the phylogenetic analysis along with a number of other previously published sequences from different snake and lizard families. Investigations into the phylogenetic relationships of the other toxin families isolated from the venom of D. vestigiata were also performed for comparison to the CRISP phylogenetic tree.

Results and Discussion Identification of Venom Proteins by 2D PAGE and Mass Spectrometry. When venom proteins from D. vestigiata were separated on 2D PAGE and subsequently silver stained, greater than 100 protein spots were detected over the pI range of 3-10 using PDQuest software (BioRad) (Figure 1). As observed for proteins from other elapid snakes, several horizontal trains of spots were evident, particularly for higher molecular sized proteins, suggesting that they are isoforms of a single toxin family or represent post-translationally modified forms of individual toxins.25 Thirty-one protein spots were sampled from the 2D gels covering a range of molecular sizes and isoelectric points as indicated (Figure 1) and analyzed by MALDI-TOF and MALDI TOF/TOF-MS/MS. A summary of the toxin families identified via 2D PAGE and mass spectrometry sequencing, along with some of their protein characteristics, is provided in Table 2, with a complete list of fragmented peptide sequences and their BLAST homology matches found in Table 3. The combined approach of initial Mascot database searches followed by de novo peptide sequencing and PEAKS software analysis successfully identified stretches of amino acids within individual peptides with 100% confidence, a necessary requirement for organisms where many proteins are not known or highly conserved. A total of 10 different protein families were identified including L-amino acid oxidase, CRISP, C-type lectins, factor Xa-like protease, PLA2, long chain neurotoxin, and kunitz-type protease inhibitors (vestiginin). Kunitz-type protease inhibitors were identified as two spots (Figure 1, spots 1 and 2) with molecular sizes of less than 11 kDa and pI Journal of Proteome Research • Vol. 6, No. 8, 2007 3095

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Figure 1. Silver stained 2D PAGE of D. vestigiata venom proteins identified by tandem mass spectrometry and de novo peptide sequencing. Numbered circles show protein spots excised and identified (Table 2). Lines to labels show multiple protein spots identified in the same toxin family. 5′Nuc. ) 5′ nucleotidase; CRISP ) cysteine rich secretory protein; FXa ) factor Xa; LAAO ) L-amino acid oxidase; LNTX ) long chain neurotoxin; Metallo. ) metalloprotease. Table 2. 2D PAGE Spots with Toxin Family Identities, Calculated and Observed Molecular Masses, and Isoelectric Points (Mr and pI)a 2D PAGE spot

1, 2 3 4 5-11 12-18 19 20-23 24 25-29 30 31

toxin family

vestiginin long chain neurotoxin vespryn PLA2 C-type lectin CRISP FXa-like (heavy chain) FXa-like (light chain) metalloprotease L-amino acid oxidase 5′ nucleotidase

obsd pI

calcd Mr (kDa)

7 10

6.5 and 7 5.5

7 7

12 14 to 17 12 to 30 25 30 24 35 to 72 60 60

10 5 to 8 5.5 to 8 10 8 to 9 5.5 5 to 8 7 8.5

obsd Mr (kDa)

12 14 16 26 25 11 66 59 65

calcd pI

8.3 5.7 10.1 5.2 to 8.7 8.2 9.4 8.3 5.0 5.5 7.2 5.6

a Note: Observed values represent the protein spots identified from the 2D PAGE (Figure 1). Calculated values were derived from the deduced D. vestigiata cDNA sequence or the closest homologue in NCBI database if D. vestigiata sequence was not available.

approximately 7 and 7.5. Antiserum raised against the kunitztype protease inhibitor from P. textilis, Textilinin-1, demonstrated multiple immunoreactive spots on 2D PAGE (Figure 2A) in addition to the two identified by mass spectrometry, suggesting additional isoforms may be present in the venom. Another low molecular sized protein identified was a vespryn at approximately 12 kDa and pI 10 (Figure 1, spot 4). Vespryns contain a B30.2/SPRY domain and are related by sequence homology to human butyrophilin and B30.2 proteins.30 They have been identified in the venom of the king cobra (Ophiophagus hannah), the monocled cobra (Naja kaouthia) and the Brazilian viper (Lachesis muta) and found to have hypolocomotion and hyperalgesia activities in a mouse model, suggesting a role in prey immobilization.31 Other proteins identified in the low molecular size range include a long chain neurotoxin 3096

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(Figure 1, spot 3), PLA2s (Figure 1, spots 5 to 11) with pI ranging from approximately 4.5 to 8, and C-type lectins (Figure 1, spots 12 to 18). While the PLA2s had a molecular size range of 14 kDa to 17 kDa, the C-type lectins ranged from 14 kDa to 22 kDa, suggesting a greater range of post-translational modification. A protein isoform of the CRISP family of proteins (Figure 1, spot 19) was identified with a molecular size of 25 kDa and pI 10 which is consistent with the size and charge of reported Australian elapid venom CRISP proteins.4 A train of spots was identified as the factor Xa-like heavy chain (Figure 1, spots 20 to 23) at approximately 26 kDa to 33 kDa and from pI 8.5 to 9.5. The light chain of this molecule was also identified at approximately 25 kDa and pI 5 (Figure 1, spot 24); however, the intensity of silver staining of the light chain was much lower. This is a similar staining pattern to the factor Xa-like protease in the Australian common brown snake, P. textilis.25 2D PAGE immunoblots of D. vestigiata venom were probed with antiserum raised against the heavy chain of P. textilis factor Xa-like protease (Figure 2B) and an antibody specific for γ-carboxyglutamic acid (Gla) shown to exist within the light chain of venom factor Xa (Figure 2C).32 The heavy chain immunoblot is in good agreement with the mass spectrometric identification of this molecule; however, the Gla immunoblot revealed a band of immunoreactivity that extends across the blot. This suggests that the light chain is resistant to silver staining since the corresponding region of the silver stained gel showed no staining (Figure 1). Metalloproteases were identified in a number of different protein spots on the 2D gel (Figure 1, spots 25 to 29) with molecular sizes ranging from 35 kDa to 72 kDa. The tryptic peptides used to identify the metalloproteases matched most closely to a metalloprotease, mocarhagin-1, from the Mozambique spitting cobra, Naja mocambique mocambique (Genbank AAM51550). Purified mocarhagin has been demonstrated to

phospholipase A(2), Notechis scutatus

phospholipase A(2), Notechis scutatus

phospholipase A(2), Notechis scutatus

phospholipase A(2), Notechis scutatus

phospholipase A(2), Dv PLA2-1, 2, and 3 C-type lectin-like protein 1

C-type lectin-like protein 1

C-type lectin-like protein-agkisacutacin A chain C-lectin-like protein C-type lectin-like protein 1, Bungarus multicinctus C-type lectin-like protein 1, Bungarus multicinctus

C-type lectin-like protein 1, Bungarus fasciatus

C-type lectin-like protein 1, Bungarus multicinctus

C-type lectin, Trimeresurus stejnegeri Dv CRISP

Dv vestarin (factor Xa-like heavy chain) Dv vestarin (factor Xa-like heavy chain) Dv vestarin (factor Xa-like heavy chain)

Dv vestarin (factor Xa-like heavy chain) Dv vestarin (factor Xa-like light chain)

metalloprotease, mocarhagin Naja mossambica

metalloprotease, mocarha gin Naja mossambica

metalloprotease - mocarhagin Naja mossambica

metalloprotease RATADAM15 precursor metalloproteinase, Bothrops asper metalloprotease cobrin, Naja naja metalloprotease - mocarhagin Naja mossambica L-amino acid oxidase precursor, Notechis scutatus 5-nucleotidase precursor (Ecto-5-nucleotidase)

7

8

9

10

11 12

13

14

16

17

18 19

20 21 22

23 24

25

26

27

28

Q9QYV0 ABB76282.1 AAF00693.1 AAM515501 DQ917521 spt/P21588

AAM51550.1

AAM51550.1

AAM 51550.1

DQ917519 DQ917518

DQ917519 DQ917519 DQ917519

AAD17252.1 DQ917520

Q90WI6

Q90WI8

AAF26286.2 AAB49518.1 AAK43586.1 Q90WI6

trm|Q90WI6

DQ917534, 5 and 6 trm|Q90WI6

S65624

S65624

S65624

S65624

S65624

S65624

DQ917524 and EF025514 DQ917524 and EF025514 DV917515 and DV917516 sp|Q27J48

accession no. (NCBI or Swiss Prot)

58.0/5.0 58.0/5.0 72.0/5.0 72.0/5.0 65.0/7.0 65.0/8.5

55.0/7.0

55.0/8.0

35.0/5.5

32.0/9.0 24.0/5.5

25.0/8.0 32.0/8.0 32.0/8.5

32/7.0 25.0/10.0

20.0/6.0

18.0/6.0

16.0/7.5 16.0/7.5 16.0/7.5 17.0/6.0

15.0/7.5

15.0/7.0 15.0/8.0

15.0/7.0

15.0/6.5

15.0/6.0

15.0/6.0

15.0/5.5

88.0/5.4 45.9/5.6 67.6/6.0 68.2/6.3 58.9/7.2 64.4/6.5

68.2/6.3

68.2/6.3

68.2/6.3

29.1/8.4 16.0/4.7

29.1/8.4 29.1/8.4 29.1/8.4

18.6/5.9 26.7/9.2

19.2/7.5

19.2/7.5

17.1/5.5 16.2/5.1 15.8/8.5 19.2/7.5

19.2/8.3

13.3/8.5 19.2/8.3

15.0/5.5

15.0/5.5

15.0/5.5

15.0/5.5

15.0/ 5.5

15.0/ 5.5

7.0/6.1 7.0/6.1 7.4/6.8 11.9/9.9

R at position 48 of the precursor protein) were isolated from the venom gland of D. vestigiata. Similarly, a single short chain neurotoxin, Dv SNTX-1, was also cloned, demonstrating 71% identity to a SNTX from the horned sea snake, Acalyptophis peroni (Genbank AAV33393) (Table 4 and Supporting Information Figure 1B and 1C). Classification of these proteins as long and short chain neurotoxins is based on the length of the mature protein as well as sequence relatedness to other known neurotoxins from BLAST homology searches. Despite conservation of hydrophobic residues within the propeptide sequence, as well as a number of cysteine residues in the mature protein, the long and short chain neurotoxins shared little identity. It is likely that homologues of these neurotoxins contribute to the weak postsynaptic neurotoxic effects reported from the venom of the closely related D. papuensis.18 Homologues to Other Known Toxin Families. A number of other cDNA transcripts were identified from D. vestigiata corresponding to an array of toxins and venom gland specific genes. These included three isoforms of a 242 amino acid venom nerve growth factor (NGF) transcript, differing by a single amino acid from each other (Table 4 and Supporting Information Figure 1D). The presence of NGF in the venom of Australian elapids has only been reported recently,55 with those

Black Whip Snake Venom Characterization

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Figure 6. Alignment of dipeptidyl peptidase sequences identified from the venom gland cDNA of Australian elapid snakes. Comparisons are made to two isoforms previously reported from the venom of Gloydius blomhoffi brevicaudus (Gbb) DPP4a and DPP4b, with Genbank accession numbers shown at the end of each sequence.

from D. vestigiata being significantly divergent in their sequence identity. Screening of the D. vestigiata venom gland cDNA also identified a single L-amino acid oxidase (LAAO) clone, whose putative functions include antibacterial and platelet aggregating activities, that demonstrated 83% identity

to a clone reported from the venom of the coastal taipan (Oxyuranus scutellatus) (Supporting Information Figure 1E).4 We also report for the first time in an Australian elapid, the presence of a 5′ nucleotidase clone (Table 4 and Supporting Information Figure 1F). 5′ Nucleotidases are postulated to Journal of Proteome Research • Vol. 6, No. 8, 2007 3103

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Figure 7. (A) Alignment of cysteine rich secretory protein (CRISP) sequences deduced from multiple cDNA clones from the venom glands of a number of elapid snakes. Australian snake sequences include all those above the line. Genbank accession numbers are provided at the end of each sequence. Conserved cysteine residues involved in disulfide bond formation are shaded gray, and the cleavage site between propeptide and mature protein is shown. Novel sequences include those from D. vestigiata, R. nigrescens, and T. carinatus. (B) Phylogenetic comparison of CRISP sequences identified from the venom of snakes and lizards. Australian elapid sequences are indicated by a hatched box. The early evolutionary split of D. vestigiata from other Australian snakes is evident and was confirmed by phylogenetic analysis of other venom gland specific genes. A chimpanzee (Pan troglodytes) CRISP sequence was selected as an outgroup for this analysis. 3104

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assist the envenomation process by generating purines from endogenous precursors within the prey which then contribute to the neurotoxic and hypotensive effects in bite victims.56 They are also known to affect hemeostasis by inhibiting platelet aggregation, with anillic acid and Con-A inhibition studies suggesting an interaction with one or more factors of the intrinsic pathway to inhibit coagulation.35 The existence of LAAO and 5′ nucleotidases in the venom of D. vestigiata was also confirmed by 2D PAGE and mass spectrometry. Identification of venom gland specific transcripts within the black whip snake was not limited to secreted toxins alone. A highly conserved calglandulin-like clone was also isolated from D. vestigiata (differing by a single N>D substitution at residue 119 compared to other elapid calglandulin-like proteins) and has been implicated in the processing and export of toxins from the venom gland and into the venom (Table 4 and Supporting Information Figure 1A).4,57 The high degree of conservation within calglandulin compared to secreted toxin families is not only a reflection of the important role calglandulin plays in the processing of toxins, but also an indication of the relatively increased rates of evolution observed in toxin gene sequences, as demonstrated by highly conserved intronic sequences compared to exons.22 Phylogenetic Distinctiveness of D. vestigiata. Despite numerous attempts for the taxonomic classification of Australian elapid snakes, the position of the Demansia species, or whip snakes, has yet to be clearly resolved.2,11,58 Investigations of the phylogenetic relationships of elapid snakes on the basis of immunological, karyological, and electrophoretic data all suggest that Demansia are distinct from all other Australo-Papuan snake species.59-61 Classification on the basis of cytochrome b and 16S ribosomal RNA sequencing, as well as on morphological grounds, also indicate that Demansia are distantly removed from other Australian elapids, although are more closely related to the New Guinea genus Aspidomorphus, made up of three cryptozoic species.2,12 Given their diverse distribution throughout the venoms of lizards and snakes, as well as the fact that they are present often as a single isoform in most of these species, the CRISP family serve as an excellent phylogenetic marker for examining the evolutionary relationship of venomous animals. Phylogenetic analysis of the black whip snake sequence was performed with a Minimum Evolution test of phylogeny via the Neighbor-Joining method, incorporating known CRISP gene sequences as well as those identified here. The results in Figure 7B demonstrate distinct clustering of the snake family and lizard sequences analyzed. Although the D. vestigiata sequence was most closely related to those of previously identified Australian elapid sequences (hatched box, Figure 7B), it was observed to separate from the other Australian snakes, suggesting an early evolutionary split from other terrestrial Australian elapids. Indeed, when examining the phylogenetic relationship of proteins from the other toxin families, including the factor X proteases, NGF, dipeptidyl peptidase, and LAAO in which only one or a few isoforms were present, the early evolutionary split of D. vestigiata is also evident (data not shown). The phylogenetic tree in Figure 7B also demonstrates clustering of the two Pseudechis species, as well as the two Oxyuranus species forming a clade with Pseudonaja textilis. This phylogenetic tree closely reflects that of the evolutionary relationship previously reported for Australian snakes.2,61,62 There are a total of seven known Demansia species since the split of D. atra into D. vestigiata and D. papuensis.9,63 Further molecular characterization is required to

research articles fully elucidate the exact relationship of species within this genus, and this study represents an excellent platform for this type of analysis. The identification of members of the same toxin families from other Australian snakes for the first time (in particular from the small eyed snake, R. nigrescens, for which this study represents one of the first descriptions of toxin from this species) also provides further detail toward these phylogenetic relationships. Clinical Implications of the D. vestigiata Venom Proteome. The combined molecular and proteomic analysis described here identified a total of 13 distinct toxin families from the venom of the black whip snake, D. vestigiata. These results provide a good correlation between the presence of the transcript of toxin genes in the venom gland and their corresponding protein products in the venom. This study represents the first description of any toxin sequence from the venom gland of a Demansia species and provides significant insight into how these venom components might collectively act to contribute to the toxicity of the venom. The presence of a factor X-like protein, vestarin D, as well as PLA2s are consistent with coagulopathic effects, along with the vestiginins (which demonstrate significant homology to the kunitz-type serine protease inhibitors) and metalloprotease that may also interfere with the coagulation cascade at different levels. The long and short chain neurotoxins, PLA2s and CRISPs, through their capacity to cause blockage of specific ion channels, would also be expected to contribute to the neurotoxicity and/or myotoxicity associated with envenomation by Demansia species.5,18 To date, clinical outcomes resulting from envenomation by whip snakes still remain poorly defined. A survey by Currie (2004) identified a total of 22 confirmed bites by D. papuensis, D. olivacea, and D. atra in the Northern Territory over a 14 year period; however, only occasional, nonspecific systemic effects were observed in bite victims.10 The results in the present study indicate that the venom of the black whip snake contains all of the components common to other more “venomous” Australian snakes, and hence the potential for this venom to be toxic in humans needs further consideration. Given the degree of variation in sequences observed in the black whip snake compared to other Australian snakes, the toxic components from the venom of D. vestigiata may have diverged to the point that they are no longer as specific or potent in their mechanisms of action against human receptor targets, which may in part account for the failure to observe significant toxic effects within bitten humans. This is supported by evidence suggesting a range of activities for other toxins which arise from only subtle sequence variations, while maintaining a common protein backbone scaffold.64 Similarly, previous studies into the venom from the closely related yellowfaced whip snake (Demansia psammophis) indicate that there is a great amount of venom variation within a species as a result of divergence, which may also account for variable clinical outcomes observed.65 The findings of this study also have important implications for the administration of antivenom during the treatment of a bite by a Demansia species. Current protocol calls for the use of polyvalent antivenom, as previously the exact toxic effects of the venom and the specific components within it have not been clearly defined.5 Furthermore, previous LC/MS analysis of D. papuensis venom indicates the presence of toxins of molecular masses not present in the venom of the nearest antivenom match.66 Comparison of the black whip snake toxin sequences identified in this study to those from which the five Journal of Proteome Research • Vol. 6, No. 8, 2007 3105

research articles current antivenoms are raised (including Pseudonaja textilis, Notechis scutatus, Acanthophis anatarcticus, Pseudechis species, and Oxyuranus species) reveals no significant identity with any one species.67 This confirms the variability in the efficacy of these antivenoms previously reported during in vitro studies of the venom from the closely related D. papuensis and D. psammophis and highlights the potential need for the development of more specific and targeted antivenoms in the future.18,65

Concluding Remarks This study has provided for the first time, a comprehensive proteomic and transcriptomic description of the toxin sequences from the venom of a Demansia species of snake. Although phylogenetic analysis of these sequences indicates that the black whip snake is evolutionarily removed from other Australian elapid snakes, the venom of D. vestigiata contains all of the components commonly observed in these species. Furthermore, a number of other toxin families were identified including a vespryn molecule, C-type lectin, 5′ nucleotidase, metalloproteinase, and dipeptidyl peptidase whose presence within the venom of Australian elapids has only become recently apparent. The functional role within the venom for many of these toxins has yet to be fully elucidated. Hence, the results of this study have a number of important implications not only for the molecular phylogenetic classification of Australian snakes but also for the detection and treatment of bites by this species. The 13 toxin proteins described here from D. vestigiata not only give an overall indication of the activity of the venom from this particular snake species, but also serve as an excellent platform for further characterization of the diversity, abundance, and exact functional role of each toxin family within snake venoms.

Acknowledgment. We wish to thank QRxPharma and the Australian Research Council for financial support and acknowledge Joe Sambono for the provision of venom glands. Supporting Information Available: Supporting Information Figure 1 contains further details on the transcriptomic identification of additional D. vestigiata cDNA sequences. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Broad, A. J.; Sutherland, S. K.; Coulter, A. R. The lethality in mice of dangerous Australian and other snake venom. Toxicon 1979, 17, 661-664. (2) Keogh, J. S.; Shine, R.; Donnellan, S. Phylogenetic relationships of terrestrial Australo-Papuan elapid snakes (subfamily Hydrophiinae) based on cytochrome b and 16S rRNA sequences. Mol. Phylogenet. Evol. 1998, 10, 67-81. (3) Fry, B. G. Structure-function properties of venom components from Australian elapids. Toxicon 1999, 37, 11-32. (4) St Pierre, L.; Woods, R.; Earl, S.; Masci, P. P.; Lavin, M. F. Identification and analysis of venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and related species. Cell. Mol. Life Sci. 2005, 62, 2679-2693. (5) Sutherland, S. K.; Tibballs, J. Australian Animal Toxins. The Creatures, Their Toxins and Care of the Poisoned Patient, 2nd ed.; Oxford University Press: New York, 2001. (6) Shine, R. Australian Snakes. A Natural History; Reed New Holland: Sydney, 1998. (7) Worrell, E. Reptiles of Australia; Angus and Robertson: Sydney, 1970. (8) Mirtschin, M.; Davis, R. Snake of Australia. Dangerous and Harmless; Hill of Content Publishing: Melbourne, 1992.

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