Investigation of the estuarine stonefish (Synanceia horrida) venom composition

Journal of Proteomics 201 (2019) 12–26

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Investigation of the estuarine stonefish (Synanceia horrida) venom composition Rebekah Ziegmana, Eivind A.B. Undheimb, Gregory Bailliea, Alun Jonesa, Paul F. Alewooda, a b

T ⁎

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, 4072, Australia Centre for Advanced Imaging, The University of Queensland, St Lucia, Queensland 4072, Australia

A R T I C LE I N FO

A B S T R A C T

Keywords: Proteomics Transcriptomics Stonefish Synanceia horrida Venom Toxins

The Estuarine stonefish (Synanceia horrida) is recognised as one of the most venomous fish species in the world but the overall venom composition has yet to be investigated using in-depth transcriptomic and proteomic methods. To date, known venom components are restricted to a hyaluronidase and a large, pore-forming toxin known as Stonustoxin (SNTX). Transcriptomic sequencing of the venom gland resulted in over 170,000 contigs with only 0.4% that were homologous to putative venom proteins. Integration of the transcriptomic data with proteomic data from the S. horrida venom confirmed the hyaluronidase and SNTX to be present, together with several other protein families including major contributions from C-type lectins. Other protein families observed included peroxiredoxin and several minor protein families such as Golgi-associated plant pathogenesis related proteins, tissue pathway factor inhibitors, and Kazal-type serine protease inhibitors that, although not putative venom proteins, may contribute to the venom's adverse effects. Biological significance: Proteomic analysis of milked Synanceia horrida venom, paired with transcriptomic analysis of the venom gland tissue revealed for the first time the composition of one of the world's most dangerous fish venoms. The results demonstrate that the venom is relatively less complex compared to other well-studied venomous animals with a number of unique proteins not previously found in animal venoms.

1. Introduction Stonefish, consisting of five species of the genus Synanceia, are regarded as the most venomous of the nearly 3000 known venomous fish species [1,2]. They are widely distributed throughout the shallow, coastal waters of the Indo-Pacific region where their excellent camouflage renders them a significant hazard to humans. Exhibiting a highly sedentary lifestyle, stonefish will simply raise their venomous dorsal spines when they feel threatened, which can often result in the envenomation of unsuspecting humans while swimming or beachcombing [3,4]. Along with the 13 dorsal spines, stonefish venom apparatus also include two pelvic and three anal spines. Venom glands sit in paired anterolateral grooves on either side of each spine. The distal end of the gland is attenuated to form a duct-like structure in the spine grooves [5]. An integumentary sheath covers each venom spine with its associated glands. As there is no musculature associated with stonefish venom apparatus, mechanical pressure is required for envenomation to occur. Proximal compression of the integumentary sheath on the venom gland squeezes venom up through the spine grooves into the puncture

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wound caused by the sharp spines. The inability of stonefish to voluntarily inject their venom speaks to the defensive nature of the venom, which was designed to dissuade predators such as sharks, rays, eels, and sea snakes as opposed to predatory venom meant to subdue potential prey. The venom glands consist of large glandular cells surrounded by supporting cells that stabilise the venom gland [6]. Microscopy of the gland cells showed they lack Golgi apparatus and rough endoplasmic reticulum, which are associated with protein-secreting cells. Instead, the cells were filled with granules, denoting a holocrine-type secretion strategy [7], in which the plasma membrane ruptures, destroying the cell and releasing the contents. Although it generally been accepted that the venom toxins evolved from epidermal secretions [8], it has also been suggested that the lack of significant supporting cells and loose attachment of skin over the venom gland point to a non-epidermal origin for the venom glands [7]. Stonefish envenomations result in a range of symptoms with the most notable being extreme pain disproportionate to the size of the puncture wound in nearly all cases [9]. Prodigious edema of the affected area is also highly prevalent [10,11]. Other reported symptoms

Corresponding author. E-mail address: [email protected] (P.F. Alewood).

https://doi.org/10.1016/j.jprot.2019.04.002 Received 28 January 2019; Received in revised form 2 April 2019; Accepted 3 April 2019 Available online 04 April 2019 1874-3919/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

include muscular weakness, hypotension and disruption to cardiac activity [11–13]. Historically, deaths have also been attributed to stonefish envenomations, although the literature on these instances is scarce [14]. Despite the severity of envenomation symptoms, the composition of stonefish venom has been relatively understudied and its mechanisms of action are largely unclear. Known components of stonefish venoms include hyaluronidase [15], a 46 kDa protein from S. verrucosa venom with inotropic and chronotropic effects [16] named cardioleputin, and a 45 kDa lectin from S. verrucosa that causes agglutination of rabbit erythrocytes [17]. Large, dimeric pore forming toxins from both S. verrucosa and S. horrida have been identified, characterised and found to have high homology to each other [18–20]. A recent study on stonustoxin (SNTX), the pore-forming toxin from S. horrida, revealed it to be a member of the MACPF/CDC superfamily of perforin toxins which are known to be promiscuous against a range of cell types and could account for a number of the envenomation symptoms [21]. Interestingly, SNTX was found to lack the N-terminal signal sequence typically found in venom components through cDNA cloning, indicating the possibility of secretion by a non-traditional pathway [18]. In contrast, cDNA cloning of the S. horrida hyaluronidase showed that the enzyme was expressed as a precursor peptide, which included a 28 residue signal sequence to target it into the endoplasmic reticulum [15]. Recently, transcriptomic methods combined with proteomic data have been used to analyse the venoms from animals such as snakes, spiders, and cone snails [22]. However, at this point fewer than ten venomous fish species have undergone transcriptomic analysis, and only three of these integrated with proteomic data to evaluate venom composition [23–28]. Those with corresponding proteomic data include a ray species [26], one catfish species [24], and a fang blenny species [27]. While a number of putative venom proteins were identified in the venom gland transcriptomes and proteomes no known fish toxins were found, save for the well known pore-forming toxins related to stonustoxin that were identified in three scorpionfish transcriptomes [23]. Previously, the venom gland transcripts of the toadfish Thalassophryne nattereri were analysed using expressed sequence tags, although the only toxin proteins identified were natterins which had previously been discovered in the venom [29], and a C-type lectin (nattectin), which was identified in the venom after further study and characterised [30]. This study describes the analysis of the venom of the estuarine stonefish Synanceia horrida using integrated transcriptomic and proteomic methods (Fig. 1). Broadly speaking, it reveals that, although a wide range of putative venom toxin families are represented as homologous contigs in the venom gland transcriptome, relatively few of them are actually found in the secreted venom. Instead, the venom is largely dominated by C-type lectins. A number of shorter proteins with homology to segments of stonustoxin were also identified in the venom, along with stonustoxin itself. Other putative toxins identified in the venom include hyaluronidase and peroxiredoxin.

2.1. Venom gland and venom extraction Three specimens of S. horrida were obtained from Amity Point, North Stradbroke Island, QLD, AUS under the permit QS2014/MAN282 and in accordance with the University of Queensland Animal Ethics Committee approval (AEC approval number IMB/317/14/NHMRC). Venom was milked from the dorsal spines either by aspiration directly from the venom sac or by applying mechanical pressure onto the spines, forcing them to eject the venom into an eppendorf tube. The venom was then pooled, lyophilised, and stored at −20 °C. The venom gland tissue was collected 3–5 days after milking from two S. horrida specimens, as has previously been done for other animals [31–33] (the third stonefish was not deemed healthy enough for venom gland extraction after the crude venom had been milked). It was pooled and stored in RNAlater solution (Ambion) at −20 °C. 2.2. RNA extraction and Illumina sequencing Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Subsequently, mRNA was isolated using Dynabeads (Thermo Fisher) according to the manufacturer's protocol. A total of 452.0 ng mRNA (260/280 = 2.11) was obtained and submitted for sequencing. The mRNA was sequenced using a next-generation Illumina MiSeq Benchtop Sequencer at the Institute for Molecular Bioscience Sequencing Facility based on 2 × 150 bp strand-specific paired-end runs. Raw sequencing reads were then trimmed using Trimmomatic v0.35 [34] software to remove adapter sequences and low quality reads. The remaining reads were assembled into contigs and isocontigs using default parameters of Trinity software v 2.0.6 [35]. 2.3. RP-HPLC fractionation Crude venom was reconstituted in 0.5% trifluoroacetic acid (solvent A) and fractionated using an Everest C18 monomeric column (4.6 mm × 250 mm, 5 um, 300 Å) on a Shimadzu prominence HPLC system. UV absorbance was monitored at 214 nm and 280 nm over a linear gradient of 1.0% per minute from 100% solvent A to 60% 0.043% trifluoroacetic acid/90% acetonitrile (solvent B). Fractions were collected across this elution range and were lyophilised then reconstituted in solvent A and divided into four aliquots each of which were then lyophilised and stored at −20 °C. 2.4. Reduction/alkylation and trypsin digestion of fractions Two aliquots of each fraction were reconstituted in 45 μl of water and 5 μl of 1 M ammonium carbonate (pH 11). A reduction/alkylation solution consisting of 97.5% acetonitrile (ACN), 2% iodoethanol, and 0.5% triethylphosphine was mixed and 50 μl was added to each sample [36] Samples were then incubated at 37 °C for 2–3 h, at which point

Fig. 1. Flow diagram of the transcriptome and proteome analysis used by this study to investigate S. horrida venom. 13

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using the ABSCIEX ProteinPilot 4.0 software. Those spectra with a confidence value under 95% were discarded. Spectral data was manually verified and subsequently used to identify the contigs present in the venom. Sequence alignments were performed using Clustal on Jalview software [37]. Homology modelling was performed on SWISS-MODEL [38] by manually selecting the most applicable template based on coverage and sequence similarity using the default parameters. To assemble the C-type lectin phylogenetic tree, first a database composed of Uniprot proteins from Euteleostomi species (349,784 sequences) was created and the stonefish C-type lectin sequences were BLASTed against the database. Those hits with E-values of e−15 and less were retained and all sequences (stonefish sequences, BLAST hits, and nattectin) were aligned using MAFFT v.7 [39]. This alignment was used to create a consensus tree using igTree [40]. Additionally, the MAFFT alignment was analysed using Bayesian inference on MrBayes 3.2 [41], which resulted in a tree that agreed with the consensus tree.

they were dried on a Christ RVC speedvac. One reduced and alkylated aliquot from each fraction was reconstituted in 100 mM ammonium carbonate (pH 8.5) with trypsin (1.0 mg/ml in 1.0 mM HCl) added at a protease to peptide ratio of between 1:20 to 1:100. Samples were incubated at room temperature for at least 1 h then microwaved for 4 min and lyophilised and stored at −20 °C. 2.5. 2D gel Lyophilised venom (~0.36 mg) was reconstituted in 125 μl of DeStreak Rehydration Solution (GE Life Sciences, USA). The sample was mixed and centrifuged to pellet insoluble material, then 1% Immobilized pH Gradient (IPG) buffer (pH 3–10 NL; GE Life Sciences) and 10 mM DTT were added to the supernatant before loading onto isoelectric focusing (IEF) strips (ReadyStrip, non-linear pH 3–10, 7 cm; Bio-Rad, USA) for 24 h of passive rehydration. Proteins were electrophoresed in an Ettan IPGphor3 IEF system (GE Life Sciences, USA) under the following conditions: 100 V for 1 h, 300 V for 200 Volt-hours (V-h), 300 to 1000 V for 300 V-h, 1000 to 5000 V for 4000 V-h, and 5000 V for 1250 V-h. The IPG strip was then equilibrated for 10 min in reducing equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 1.5% DTT) followed by a second incubation for 20 min in alkylating equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, 2% iodoacetamide). The IPG strip was then embedded on top of a 12.5% polyacrylamide gel (PowerPac Electrophoresis unit; Bio-Rad) and covered with 0.5% agarose. Second dimension electrophoresis was performed at 4 °C for 1 h at 150 V per gel. The resulting gel was stained overnight with 0.2% colloidal Coomassie brilliant blue G250 (34% methanol, 3% phosphoric acid, 170 g/l ammonium sulfate, 1 g/l Coomassie blue G250), then destained in 1% acetic acid/H2O. Visible spots were subsequently picked from the gel and digested overnight at 37 °C using sequencing-grade trypsin (Sigma, USA). Briefly, gel spots were washed with ultrapure water, destained (40 mM NH4CO3/50% acetonitrile (ACN)) and dehydrated (100% ACN). Gel spots were rehydrated in 10 μl of 20 μg/ml proteomics-grade trypsin (Sigma-Aldrich) and incubated overnight at 37 °C. Digests were eluted by washing the gel spots for 30 min with each of the following solutions: 50 μl 50% ACN/1% formic acid (FA), followed by 50 μl 70% ACN/1% FA. The samples were then dried by evaporation using a vacuum centrifuge and reconstituted in 20 μl of 1% FA prior to analysis by LC-MS/MS.

3. Results 3.1. Transcriptomic analysis of the venom gland The de novo sequencing of the S. horrida venom gland resulted in two sets of high quality raw reads with over 22 million reads each (Table 1). The Trinity assembly performed on the raw reads resulted in 179,687 contigs representing 100,647 different read clusters, with an average length of 1088 bp and a N50 value of 2197 bp. Using the “Get open reading frames (ORFs) or coding sequences (CDSs)” tool on the Galaxy platform [42], the coding DNA sequences (CDS) were extracted from the contigs, and were subsequently translated into their open reading frame (ORF) protein sequences. The ORFs were searched with BLASTp against SwissProt and Toxprot and the two results were manually verified against each other. Based on these results, 40.3% of the S. horrida venom gland contigs were identified as housekeeping proteins (proteins used in normal cellular function and not recognised as a putative toxin in the ToxProt database), while 59.3% did not have homology with any proteins available in the Swissprot database. Only the final 0.4% of contigs were found to be homologous (with an E-value of < 1.0 × 10−3) to known venom components and represented 806 sequences. The contigs with homology to known venom components were classified into 48 families and were grouped according to their putative activities. Only 16 families contained 10 or more stonefish contigs. The

2.6. Mass spectrometry Table 1 Summary statistics of the raw reads and assembled transcriptome for the S. horrida venom gland.

Samples (native, reduced/alkylated, trypsin digested, and gel spots) were reconstituted in 0.1% formic acid for LC-MS/MS mass spectrometry analysis on a Shimadzu Nexera uHPLC (Japan) coupled to a Triple TOF 5600 mass spectrometer (ABSCIEX, Toronto, Canada) equipped with a duo electrospray ion source. Samples were loaded onto a Zorbax 300SB-C18 column (1.8 um, 2.1 × 100 mm). Linear gradients of 1–40% solvent B (0.1% formic acid in 90% ACN, solvent A was 0.1% formic acid in water) over 20 min were used, followed by a rapid increase to 80% solvent B. The data was acquired and processed using Analyst TF 1.5.1 software (Framingham, MA, USA.)

Raw reads

2.7. Bioinformatics analysis Assembled contigs were analysed to extract their coding domains, which were then translated into their protein sequences. This reference database was submitted to BLASTp with the E-value upper limit set to 1E-3, in order to identify those contigs with homology to known proteins, and run against both the most recent UniProtKB-SwissProt and ToxProt databases. The results of these searches were manually compared and validated to verify contigs with homology to putative venom proteins. The reference database was matched to the LC-ESI-MS/MS spectra 14

R1

R2

# Reads Length interval Average length N50 %GC %N

22,119,825 35–151 bp 144.3 bp 151 bp 47.85 0.00

22,125,983 35–151 bp 144.3 bp 151 bp 48.12 0.00

# Contigs Length Interval Average Length N50 %GC %N

Fully assembled contigs 179,687 224–17,970 bp 1088 bp 2197 bp 45.2 0.0

# Sequences Length Interval Average Length N50

Open reading frames 2,119,035 30–5216 residues 66 residues 68 residues

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Fig. 2. Profile of putative venom proteins from the venom gland transcriptome of S. horrida showing toxin families with 10 or more representative contigs. Bars are colour-coded based on putative activity. Inset represents activity ratio of toxin families with < 10 representative contigs.

identity with Q801Z8, including the original signal sequence, but also included an additional 17 residues before the signal sequence (Fig. S3).

most abundant of these were the metallanoproteinases with 159 contigs (Fig. 2). C-type lectins and Stonustoxin contigs were also prominent with 131 and 93 corresponding contigs, respectively. The remaining 32 toxin families represented a range of physiological activities including small ion-channel targeting peptide toxins, phospholipases, enzymes, and proteins over 80 kDa (Table 2). The homologous putative venom proteins were from a range of animals including snakes, spiders, scorpions, bees, and several marine invertebrates. While few fish venom proteins have been identified and characterised, there were five contigs identified in the S. horrida venom gland transcriptome that were homologous to Natterins, a family of proteins with kininogenase activity from the venom of the toadfish Thalassophryne nattereri, as well as those contigs homologous to SNTX and the C-type lectin Nattectin from T. nattereri. A total of 93 stonefish contigs showed homology with stonustoxin subunits, with 33 total contig isomers representing 15 total read clusters corresponding to the alpha subunit and 60 contig isomers representing 26 read clusters corresponding to the beta subunit. Many of the corresponding ORFs were shorter sequences that aligned with fragments of the large Stonustoxin subunits, and several were larger than the known subunit sequences (Fig. S1). They ranged in length from 55 to 1272 residues with an average length of 232 residues. There were ORFs identified of the same length as the known subunits that were highly homologous with E-values of 0.0. Alignments of these ORFs with their corresponding SNTX subunit sequences showed an 87% sequence identity to the known alpha subunit and a 99% sequence identity to the beta subunit (Fig. S2). No signal sequence or pro region was identified for either subunit. Additionally, nine contigs from 5 read clusters with high homology to putative snake venom hyaluronidases were identified. While the hyaluronidase previously identified from S. horrida (Uniprot accession number Q801Z8) is currently unverified and therefore not included in the SwissProt database used, a separate BLAST search of Q801Z8 against the nine corresponding ORFs showed e-values ranging from 6 × 10−19 to 0.0. The most homologous ORF shared a 99% sequence

3.2. Venom proteome analysis Crude S. horrida venom was separated using 2D gel electrophoresis to analyse the larger protein components. This resulted in a large number of spots with various sizes and charges (Fig. 3). While many spots were identified as containing housekeeping proteins such as actin, heat shock proteins, and ubiquitin, a number of spots were identified as having homology to four groups of putative venom proteins. The most prolific of these were a wide range of spots identified with homology to one or both of the stonustoxin subunits. Manual inspection of the peptide fragments identified by ProteinPilot as belonging to the SNTX subunits showed that they represented distinct regions of the protein, indicating a number of cleavage sites (data not shown). In addition to SNTX, many spots were identified as containing C-type lectins. The other putative venom proteins identified in the 2D gel spots were hyaluronidase and peroxiredoxin-6. There were 12 other enzymes and proteins identified in various 2D gel spots (Table 3) including one with no BLAST hits on known proteins against the SwissProt database but containing a C2 superfamily domain. The crude venom was fractionated using RP-HPLC and the fractions were reduced and alkylated prior to trypsin digestion. The chromatographic profile showed three major peaks eluting between 40 and 50% solvent B, as well as a number of smaller peaks (Fig. 4). The high quality LC-MS/MS data obtained from these fractions was compared to the transcriptome database using Protein Pilot v4 software and the results were manually verified. Only those peptide fragments with a confidence of 95% or higher were used. A number of the proteins identified from the RP-HPLC fractionation confirmed those found in the 2D gel including gastrotropin, golgi-associated plant pathogenesis related protein (GAPR), glutathione peroxidase, superoxide dismutase, thioredoxin, and the protein with no BLAST hits containing a C2 superfamily domain. Interestingly, the only 15

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Table 2 Putative venom protein families with < 10 representative homologous S. horrida contigs. Activity

Toxin family

Animal with BLASTp hit

Number of contigs

Max, min E-value

Ion channels

Turripeptide Actitoxin Toxin C117 Theraphotoxin Scolpotoxin Translationally Controlled Tumor Protein Homolog A. superbus Venom Factor Neprilysin Serine Carboxypeptidase Ctenitoxin Hyaluronidase Venom peptide isomerase heavy chain Cyclotransferase L-amino-acid oxidase O-methyltransferase Carboxylesterase Amylase Coagulation Factor Prothrombin Activator Toxin BcsIII Phospholipase A2 Phospholipase B Phospholipase D Peroxiredoxin Scorpion Venom Peptide Sagatoxin Natterin Insulin-like growth factor-binding protein CRISP/Allergen Plancitoxin C-type Natriuretic Peptide Stichitoxin

Sea Snail Anemone Scorpion Spider Anemone Spider, Snake Snake Spider Bee Spider Snake Spider Snake Snake Wasp Bee Scorpion Snake Snake Anemone Snake Snake Spider Snake Scorpion Anemone Toadfish Spider Spider Crown-of-thorns Starfish Platypus Anemone

9 4 2 1 1 9 1 9 9 8 9 7 7 6 6 2 1 5 3 1 7 3 1 9 9 7 5 3 3 3 1 1

2.00 × 10−4, 1.00 × 10−8 3.00 × 10−8, 4.00 × 10−19 2.00 × 10−4 8.00 × 10−34 3.00 × 10−4 2.00 × 10−6, 2.00 × 10−65 1.00 × 10−14 2.00 × 10−5, 9.00 × 10−137 5.00 × 10−9, 1.00 × 10−155 2.00 × 10−7, 1.00 × 10−26 3.00 × 10−16, 6.00 × 10−136 3.00 × 10−6, 6.00 × 10−47 5.00 × 10−9, 2.00 × 10−134 2.00 × 10−8, 4.00 × 10−63 6.00 × 10−17, 4.00 × 10−81 7.00 × 10−20, 4.00 × 10−22 5.00 × 10−11 8.00 × 10−4, 0 7.00 × 10−4, 6.00 × 10−17 6.00 × 10−4 4.00 × 10−4, 2.00 × 10−26 3.00 × 10−81, 0 9.00 × 10−25 4.00 × 10−18, 6.00 × 10−4 8.00 × 10−4, 6.00 × 10−10 7.00 × 10−16, 2.00 × 10−17 8.00 × 10−9, 3.00 × 10−113 1.00 × 10−41, 4.00 × 10−81 2.00 × 10−22, 2.00 × 10−36 9.00 × 10−40, 1.00 × 10−54 6.00 × 10−16 3.00 × 10−4

Inflammation Protease

Various Enzymes

Hemostasis Impairment

Phospholipase

Other

and phosphatidylethanolamine-binding protein 4.

proteins identified as putative venom proteins were C-type lectins. Additionally, there were three proteins identified from the venom fractions that were not identified in the 2D gel that were homologous to a kazal-type serine protease inhibitor, a tissue factor pathway inhibitor,

Fig. 3. 2D gel of crude S. horrida venom with boxes enclosing the spots with homology to putative venom proteins. Black indicates spots with homology to stonustoxin, red indicates spots with homology to C-type lectins, orange indicates spots with homology to the stonefish hyaluronidase, and white indicates the spots with homology to peroxiredoxin. Numbers indicate other proteins identified in the gel as denoted in Table 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 3 Proteins identified by ProteinPilot from the 2D gel of crude S. horrida venom. Name

2D Mass

Function

ID on Fig. 3

Adenosylhomocysteinase

~40 kDa

1

Hydroperoxide isomerase Carbonic anhydrase Gastrotropin

~41–37, 30–35 kDa ~26 kDa ~16 kDa, < 15 kDa

Glyceraldehyde-3-phosphate dehydrogenase Triosephosphate isomerase Glutathione peroxidase Thioredoxin Nucleoside diphosphate kinase Superoxide dismutase Golgi-assiciated plant pathogenesis related protein Unidentified protein with C2 superfamily domain

~30–35 kDa ~24 kDa ~26–24, 18, ≤15 kDa ≤15 kDa ≤15 kDa ~15 kDa ~15 kDa ~25 kDa

Inhibits S-adenosyl-L-methionine-dependent methyl transferase, moderates methylation Catalyzes isomerisation of hydroperoxides, involved in lipid metabolism Reversible hydration of carbon dioxide, maintains acid-base balance Stimulates gastric acid and pepsinogen secretion, involved in enterohepatic bile acid metabolism Plays a role in glycolosis and nuclear functions Involved in gluconeogenesis and carbohydrate synthesis Protects tissues from oxidative damage Oxidoreductase enzyme that act as antioxidant Catalyzes production of nucleoside triphosphates Antioxidant Involved in inflammatory processes C2 domains are involved in targeting proteins to cell membranes

2 3 4 5 6 7 8 9 10 11 12

was identified in venom fractions that eluted between 40 and 50% acetonitrile in the major chromatogram peaks. The gastrotropin ORF did not contain a signal sequence, as is consistent with gastrotropins from other species. When compared to gastrotropin sequences found in the genetic data of other fish species and the human sequence, there was high homology across all sequences (Fig. 7A). Notable changes in the S. horrida sequence of highly conserved residues when compared to the other gastrotropin sequences include Glu > Val7, Tyr > His15, Lys > Asp34, Glu > Asp38, Thr > Arg47, Trp > Leu48, Gln > Lys50, and Arg > Met118. Homology modelling of the S. horrida sequence using the human structure showed that, while there were some minor secondary structure differences, the stonefish protein was predicted to maintain the beta-barrel structure in which bile-acids are known to bind in human gastrotropin (Fig. 7B).

3.3.3. Golgi-associated plant pathogenesis related protein Golgi-associated plant pathogenesis related proteins (GAPR) are part of the CAP protein superfamily, which also includes cysteine-rich secretory proteins and allergen 5. They modulate immune response and positively regulate epithelial cell migration. A protein with homology to human GAPR-1 was identified in both the 2D gel and HPLC trace of S. horrida venom. The S. horrida sequence was ~45 residues longer at the N-terminal than the human protein (Uniprot accession number GAPR1_HUMAN) and two predicted GAPR proteins from the Atlantic salmon (Uniprot accession number B5X2W7_SALSA) and sablefish (Anoplopoma fimbria, Uniprot accession number C3KJ99_ANOFI). Additionally, the salmon and sablefish GAPRs were more homologous to the human protein than they were to the stonefish sequence (Fig. 8). The most homologous putative venom protein, cysteine-rich venom protein (CRVP) from the Lowland copperhead snake Austrelaps superbus, and the Venom Allergen 5 from the yellow hornet, Dolichovespula arenaria (Uniprot accession numbers CRVP_AUSSU and VA5_DOLAR, respectively) also showed longer N-terminals than the GAPR sequences but neither were homologous to the N-terminal S. horrida sequence.

Fig. 4. The RP-HPLC chromatogram at 214 nm of crude S. horrida venom from which venom fractions were collected.

3.3. S. horrida venom proteins 3.3.1. C-type lectins There were five C-type lectins identified from the 2D gel plus an additional two identified from the HPLC fractions. Their sequences ranged in length from 74 to 177 residues and while SignalP [43] identified signal sequences for four of the sequences, there were no identified signal sequences for the other three sequences (Fig. 5). The stonefish lectins showed great variability to each other and to the putative fish venom lectin Nattectin from T. nattereri. However, there were several highly conserved residues across all the sequences, especially the cysteine residues, probably related to the maintenance of the overall protein structure. Phylogenetic analysis of the stonefish lectin sequences, along with Nattectin and other homologous fish sequences, clearly showed the stonefish lectins cluster together in a well-supported clade (Fig. 6). The closest sequence to the stonefish lectins was identified in whole genome sequencing of the Spotted green pufferfish, Tetraodon nigroviridis (Accession number Q4S497). Nattectin, while not in a well-resolved clade, was clearly shown by the phylogenetic analysis to be a different type of C-type lectin to the stonefish lectins.

3.3.4. C2 superfamily domain containing protein A protein with no BLAST hit against the Swissprot database with an approximate mass of 25 kDa was identified from the 2D gel of S. horrida crude venom. It was shown to contain a C2 domain, which binds to phospholipids and targets cell membranes. A second BLASTp search of the contig sequence against all available non-redundant proteins showed the protein to be homologous to predicted perforin-like proteins found in the genomic data of various teleost fish species. An 18residue signal sequence was predicted by the SignalP v4.1 server and 49.5% of the remaining sequence was found at the protein level with a confidence of 95% or higher (Fig. 9).

3.3.2. Gastrotropin An S. horrida transcriptome contig with homology to gastrotropin (also known as fatty acid binding protein 6), which binds bile acid and is involved in their metabolism, was identified in the 2D gel in spots < 15 kDa with a pI above 7, as well as in spots of 25–26 kDa, indicating the possibility of dimer formation. Additionally, the protein 17

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Fig. 5. Alignment of C-type lectins identified in S. horrida. Alignment is coloured on a gradient based on consensus with the most conserved residues coloured the darkest. Identified signal sequences are underlined in black and Cysteine residues are identified by red boxes. All lectins were identified in the RP-HPLC fractions, while those also found in the 2D gel are denoted with a *. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

protein is known to modulate several signaling pathways. The S. horrida protein was also homologous to PEBP4s found in the genomes and transcriptomes of several fish species available in the UniProt database [45]. When modelled against rat PEBP1, the stonefish protein lacked several of the secondary structural characteristics from the model protein, including several helices.

3.3.5. Kazal-type serine protease inhibitor A Kazal-type serine protease inhibitor was identified in the RP-HPLC fraction of S. horrida venom that eluted between 53 and 57 min (approximately 26.8–28.6% solvent B). The sequence was shown to have a 33 residue signal sequence on Signal P and only a five residue segment of the remaining sequence was not found in the MS/MS data (Fig. 10). While the sequence was most homologous to the housekeeping serine protease inhibitor Kazal-type (SPINK) 6 from mouse, it also showed homology to the toxin Turripeptide Pal9.2 from the white giant-turris snail (Polystira albida). All three shared a –C-C-C-C-C-C– cysteine framework, although the loop lengths of the S. horrida peptide were closer to those of the mouse peptide than to the Turripeptide.

4. Discussion 4.1. Sequencing and transcriptome Sequencing of the S. horrida venom gland on the high throughput Illumina next generation sequencing platform resulted in two sets of paired-end reads containing > 2.2 × 107 raw reads each. When assembled using Trinity software this gave almost 180,000 protein contigs. In contrast to sequencing of the stingray Neotrygon kuhlii venom gland which resulted in only 4584 contigs [26], the number of stonefish contigs is more congruent with the 147,881 and 105,191 contigs found from sequencing the venom glands of the Potamotrugon amandae and P. falkneri stingrays, respectively [28]. The sequencing of the venom gland of the more closely related teleost fish, Pelteobagrus fulvidraco (Chinese yellow catfish), resulted in 210,413 contigs [24]. The number of contigs was very close to that of the scorpionfish Scorpaenopsis cirrosa, though considerably more than the numbers found in other scorpionfish species S. neglecta and S. possi [23]. Whereas there was a large range of contig lengths identified (224–17,970) the average length was relatively short at 1088, less than the average contig lengths for either P. amandae or P. falkneri [28], but longer than the average length of the three scorpionfish species [23]. Despite the large number of contigs from the S. horrida venom gland, the majority had no homology to putative proteins from the SwissProt database. A further 40.3% were identified by BLASTp to be homologous to non-toxin housekeeping proteins used in general cellular function and maintenance, and 0.4% of contigs were recognised as putative venom proteins. Both P. amandae and P. falkneri also had similar levels of contig hits with putative venom proteins from ToxProt being 1.7% and 1.8% respectively [28]. Interestingly, these are much

3.3.6. Tissue factor pathways inhibitor Tissue factor pathways inhibitors (TFPI) are composed of three connected Kunitz domains. They have inhibitory activity against trypsin, plasmin, factor Xa, and factor VIIa/tissue factor [44]. The protein with homology to human TFPI-2 was identified from the RPHPLC fractionation of crude S. horrida venom maintained this structure, and showed high homology to both the human TFPI-2 (Uniprot accession number TFPI2_HUMAN) and those found fish species (Fig. 11A). Additionally, the stonefish sequence showed a 39% sequence homology to bitisilin-3 (Uniprot accession number VKT3_BITGA) a protein from the Gaboon adder (Bitis gabonica) venom gland consisting of two linked Kunitz domains. All three Kunitz domains of the stonefish protein were identified to be 52 residues in length (Fig. 11B). The first domain had 52% and 44% sequence identities with the second and third domains respectively, while the second and third domains had a 40% sequence identity with each other. When BLAST searched individually, the domains showed homology with actitoxins, Kunitz-type serine protease inhibitors from sea anemones that also act on voltage gated potassium channels. 3.3.7. Phosphatidylethanolamine-binding protein An S. horrida protein with homology to human phosphatidylethanolamine-binding protein (PEBP) 4 was recognised in the venom fractions (Fig. 12). The function of PEBP4 is not entirely understood but the 18

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Fig. 6. Unrooted consensus tree of fish C-type lectin sequences showing posterior probability support values. Lectins are labelled as fish type followed by the associated UniProt accession number. Stonefish lectins are located in the red box, while Nattectin is located in the blue box. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

in this study could have resulted in tissue damage. Additional transcriptomes of the stonefish venom gland at different points in regeneration could therefore lead to an increase in venom related proteins, which would be complemented by comparison to transcriptomes from non-venomous tissue. Interestingly, there were transcriptome ORFs that showed homology to a broad range of putative animal toxin families including various enzymes, proteins promoting inflammation or hemostatic disruption, neurotoxins, and enzyme inhibitors. This was similar to the venom gland transcriptomes of three scorpionfish species, which matched transcripts to putative toxins such as venom metalloproteinases, translationally controlled tumor proteins, and neuropeptides from anemone and sea snails [23]. However, only a small number of these families were actually identified in the S. horrida venom through proteomic methods. As stonefish venom is purely defensive, it has evolved solely to deter predators and is therefore unlikely to aid in prey capture

lower than the proportion of toxins found in other venom gland transcriptomes. For example, a study of three Pseudonaja snake species found that the percent of transcriptome toxins ranged between 24 and 27% [32] and a study of the scorpion Centruroides tacomanus found 53% of venom gland transcripts to represent putative toxins [46]. One possibility for this is that the venom glands in fish species are much less defined than those in other animals, and are closely associated with the surrounding epidermal tissue, resulting in the inclusion of this tissue in venom gland samples. Additionally, by milking the venom several days prior to collecting the venom gland tissue, there was significant damage to the gland tissue that likely resulted in the up-regulation of proteins required to repair and rebuild the venom gland before the toxins could be produced again at high levels. While this is a common practice when collecting venom gland tissue in other animals [31,32], the injection methods of these species require minimal damage to the toxin-producing tissue, whereas the mechanical pressure used to milk the stonefish 19

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Fig. 7. A) Alignment of gastrotropin sequences from S. horrida, zebrafish, Japanese killifish, Senegalese sole, Atlantic salmon, and human (Uniprot accession numbers Q61MW5_DANRE, H2LLF7_ORYLA, A0A1B0SZT2_SOLSE, B5X688_SALSA, and FABP6_HUMAN, respectively.) Alignment is coloured on a gradient based on conservation with the most highly conserved residues coloured the darkest. B) NMR structure of human gastrotropin in complex with the bile-acid salts glycocholate and glycochenodeoxycholate (PDB accession number 2mm3) and homology model of the stonefish gastrotropin using the human protein as a model.

in the venoms of snakes [49], jellyfish [50,51], and the stingray N. kuhlii [26], as well as at high frequencies in the venom gland transcriptomes of P. falkneri and P. amandae [28]. The peroxiredoxin identified in the venom of the western diamondback rattlesnake Crotalus atrox was a 36-residue peroxiredoxin-4 [49], as were those found in jellyfish and the freshwater stingray transcriptome [28,51] whereas the peroxiredoxin from N. kuhlii was a peroxiredoxin-6 with a mass around 20–25 kDa on a 2D gel [26] which corresponds to the mass observed in S. horrida venom. In humans, peroxiredoxin-6 is involved in redox regulation of cells and regulates phospholipid turnover. While the role of peroxiredoxin in venoms has not been well studied, it has been suggested that they maintain redox hemostasis and may lead to diversification of toxins through disulfide bond engineering [49,50]. Based on the proteomic data from this study, and with the possible exception of SNTX, C-type lectins appear to be the most abundant S. horrida venom components. Seven separate lectin sequences with masses ranging from 8800 to 17,500 Da were identified in the venom proteome. C-type lectins have previously been identified as active components in the venoms of a wide range of snake species, as well as in insects, where they disrupt blood coagulation [48]. Additionally, a Ctype lectin called nattectin from T. nattereri has been shown to have hemagglutination activity, as well as to cause neutrophil mobilisation in mice [30], and to bind to RGD-dependent integrins which leads to improved integrin-mediated cell adhesion and apoptosis resistance in HeLa cells [52]. Interestingly, while showing some homology to nattectin, phylogenetic analysis revealed the stonefish lectins to be in a distinctly separate clade. However, they were related to other C-type lectins identified in the genomic data of several other non-venomous fish species such as pufferfish and gar. Therefore, it appears that C-type

as is seen in other animal venoms. Instead, it simply aims to cause predators to search for food elsewhere, which may explain the relatively low number of toxin families identified in the venom, as we would anticipate only a small number of highly effective toxins would be needed to achieve this goal. On the other hand, it is possible that some of the toxin contigs are expressed in the venom at levels too low for analysis by the methods used or were otherwise incompatible with the proteomic methodology of this study.

4.2. Putative venom proteins While there were a large number of contigs that were homologous to stonustoxin subunits, only those representing the base α- and βsubunits were strongly observed in the venom proteome. However, while gel spots corresponding to the whole subunits were identified, there were also many spots that corresponded to smaller fragments of the SNTX subunits. Interestingly, while there were many proteases found in the venom gland transcriptome, none were identified in the milked venom, which is congruent with the lack of protease activity observed in crude S. horrida venom previously observed [47], suggesting that proteolysis after milking was highly unlikely to be responsible for the SNTX fragmentation. The only other protein component previously identified from S. horrida venom, the hyaluronidase Q801Z8, was also identified in both the transcriptome and proteomic data in this study. Hyaluronidases are common in animal venoms where they act as spreading factors to aid in the dissemination of toxins throughout the body [48]. A peroxiredoxin-6 of approximately 24 kDa was identified in the 2D gel of S. horrida venom. Peroxiredoxins have previously been identified 20

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Fig. 8. A) Alignment of GAPR sequences from S. horrida, atlantic salmon, sablefish, and human, along with CRVP from A. superbus and Venom Allergen 5 from D. arenaria. Alignment is coloured on a gradient based on conservation with the most highly conserved residues coloured the darkest. B) Human GAPR-1 (PDB accession number 5vhg) and the stonefish homology model based on it.

While some of them, such as actin and heat-shock proteins are considered unlikely to lend any toxic activity to the venom, several may act as toxic components. Adenosylhomocysteinase has previously been identified in the venom of the Neoponera villosa ant [54], although it was deemed to be a housekeeping protein in that study. Adenosylhomocysteinase modulates protein methylation and could therefore play a role in posttranslational modification of venom components. Similarly, nucleoside diphosphate kinase has been observed in snake venom but was not deemed to be an active venom component [55]. However, because they have been assumed to be housekeeping proteins, they were never tested for toxic activities. Other enzymes found in S. horrida venom such as hydroperoxide isomerase, trioesphosphate isomerase, and carbonic anhydrase 1 have not previously been reported in animal venom. While these may simply be cellular housekeeping proteins, it is still possible that they may lend activity to the stonefish venom. Glyceraldehyde-3-phosphate dehydrogenase was also identified in N. villosa venom [54], as well as in the venom of the Asian Wasp, Vespa affinis [56]. The wasp glyceraldehyde-3-phosphate dehydrogenase was determined to act as a major allergen in the venom and may play a similar role in S. horrida venom, possibly contributing to the edema and erythema observed in those stung by stonefish [11]. Interestingly, while peroxiredoxin is accepted as a putative venom

Fig. 9. Sequence of the protein with no known BLAST hits containing a C2 domain. The predicted signal sequence found using the SignalP-4.1 server is underlined and italicised, while the identified CD domain is in bold. Fragments identified from the 2D gel with a by Protein Pilot with a confidence of 95% or higher are highlighted in yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

lectins underwent convergent recruitment events in toadfish and stonefish venoms. Several C-type isolectins (Sp-CL 1–5) were also identified in the venom of the scorpionfish Scorpaena plumieri, which were shown to have hemagglutinating activity [53]. In accordance with these findings, we predict that S. horrida C-type lectins contribute to venom activity via hemagglutinating activity and contribute to the inflammation observed in instances of stonefish venom stings. 4.3. Other venom proteins In addition to the putative venom proteins observed in the venom proteome, a number of other proteins and enzymes were also identified. 21

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Fig. 10. A) Alignment of the Kazal-type serine protease inhibitor found in S. horrida venom with the most homologous serine protease inhibitor from mouse (Uniprot accession number ISK6_MOUSE), from the snakehead murrel fish Channa striata (Uniprot accession number A0A0D6DQK7_CHASR), and the most homologous putative venom protein, Turripeptide Pal9.2 from P. albida (Uniprot accession number TU92_POLAB). Alignment is coloured on a gradient based on conservation with the most highly conserved residues coloured the darkest. Signal peptides are underlined in black and red boxes are placed around the mature peptide cysteine residues. B) Human SPINK-6 (PDB accession number 2n52) and the stonefish homology model based on it including predicted 1–5, 2–4, 3–6 cysteine connectivity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

superbus CRVP with the highest homology to the S. horrida sequence is predicted to block smooth muscle contraction possibly by acting on voltage-gated calcium channels based on sequence similarity (Uniprot). Despite GAPR proteins being known to have high sequence variability, the S. horrida sequence was noticeably different to the human and two fish sequences. However, the homology model generated using the human protein showed a very similar structure, though notably lacking the extended N-terminal sequence from the stonefish protein. It is possible that these differences allow it to act as a venom toxin, possibly in similar ways to putative venom proteins like CVRPs or Allergen 5. A protein containing a membrane-targeting C2 superfamily domain and with homology to predicted fish perforin-1-like proteins was found in the S. horrida venom. Perforins cause cytotoxicity by oligomerising to form pores in cell membranes. In fish they have been linked to several different genes and are utilised by certain T cells to kill targeted cells [70]. While perforin-1 is generally over 500 residues in length and contains both a membrane-attack complex/perforin (MACPF) domain and a C2 domain, the mature stonefish sequence was comprised of 123 residues and lacked the MACPF domain. However, the S. horrida sequence was a similar length to a number of predicted perforin-1-like proteins from the genetic data of various teleost fish with which it showed homology. Interestingly, the most well studied S. horrida venom component SNTX is a large, dimeric perforin toxin accredited with much of the venom activity [9,21]. However, this smaller perforin-like protein may also contribute to the cytolytic activity seen in the venom. The serine protease inhibitor Kazal-type found in S. horrida venom showed the most homology to housekeeping protease inhibitors found in other fish and mammalian species. Homology modelling against the most homologous human SPINK6 also showed the structure to be highly conserved. In regular cellular function, the homologous SPINK6s are selective for kallikreins and operate in skin barrier function, as well as mediating regulation of desquamation [71,72]. The stonefish peptide also showed homology to Turripeptide Pal9.2 from a sea anemone, which is predicted to act as an ion-channel neurotoxin based on similarity (Uniprot). While the homology model shows that the S. horrida peptide maintains the characteristic 1–5, 2–4, 3–6 cysteine connectivity

component, other antioxidants are not. Glutathione peroxidase, thioredoxin, and superoxide dismutase were all detected in the venom of S. horrida along with peroxiredoxin. Glutathione peroxidase has previously been identified in snake venom [57,58], as has thioredoxin [59]. Superoxide dismutase has been found in the venoms of scorpions [60], wasps [61], and ants [54]. Stonefish have been reported to be able to survive extended periods of time with limited water during low tides [5], during which times high levels of antioxidants would be necessary to avoid oxidative damage to the venom proteins and gland tissue. While the antioxidants found in S. horrida venom may act in this manner, it is also possible that they contribute to venom activity as in the case of two Leptopilina parasitoid wasp species where the venom superoxide dismutase acted as an immune suppressive [61]. Gastrotropin is expressed in various digestive system organs and is involved in bile acid metabolism and binds to bile acid salts [62]. It has been identified in the intestinal tract of several fish species [63–65]. It was identified in both the 2D gel and RP-HPLC fractions of S. horrida venom, and appeared to be relatively abundant, suggesting an important role in the venom. It is unclear what that role is, however homology modelling shows that the S. horrida protein maintains a high similar beta-barrel structure to that seen in the human protein. It is possible that the gastrotropin holds a similar function to that of the antioxidants, maintaining homeostasis and protecting the venom proteins and gland tissue from molecules that might cause degradation during environmental extremes. Conversely, the gastrotropin could be playing another role, but more study is needed to truly glean its function. GAPR proteins are found across a range of animals and contain a relatively high degree of sequence variability [66]. Their function is not entirely understood but they are associated with the immune system [67]. They are also closely related to putative venom proteins such as CRVPs and Allergen 5. Allergen 5 is found in insect venoms and elicits a strong immune response [68], whereas CRVPs are common in snake venoms. The function of some CRVPs remains unknown, but others have been found to inhibit smooth muscle contraction or to act on cyclic nucleotide- or L-type Ca+-gated ion channels [69]. The A. 22

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Fig. 11. A) Alignment of the TFPI found in S. horrida venom with the most TFPIs from human and the sablefish (Uniprot accession number C3KHI5_ANOFI), as well as bitisilin-3. Alignment is coloured on a gradient based on conservation with the most highly conserved residues coloured the darkest. Signal peptides are underlined in black and red boxes are placed around the mature peptide cysteine residues. B) Alignment of the three Kunitz domains identified in the stonefish TFPI (domains labelled D1-D3, starting from the N-terminus) along with two examples of homologous actitoxins (Uniprot accession numbers VKT2_ANTAF and VKT8_ANEVI respectively). C) Human carboxypeptidase inhibitor (PDB accession number 4BD9) and the stonefish TFPI homology model based on it. In the homology model, Kunitz domain 1 is coloured orange, domain 2 is coloured yellow, and domain 3 is coloured pink. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of three linked Kunitz-type domains that act as anticoagulants [77]. Kunitz-type serine protease inhibitors are found in a variety of animal venoms where they have a range of activities including anticoagulation [48]. Often the Kunitz-type toxins are relatively short and composed of a single domain with three conserved disulfide bonds with 1–6, 2–4, 3–5 cysteine connectivity, which leads some of them to have dual activity as protease inhibitors and as potassium channel modulators [78]. A protein composed of two linked Kunitz domains was discovered in the venom gland cDNA library of the adder B. gabonica, however its activity was not explored [79]. The individual Kunitz-domains from the S. horrida TFPI showed between 40 and 52% identity with each other and in the homology model were shown to have very similar structures. Interestingly, the individual domains were also homologous to actitoxins, some of which have dual activity as protease inhibitors and voltage-gated potassium channel inhibitors. However, the size of the stonefish TFPI means that it likely acts only as a protease inhibitor, contributing an anticoagulant activity to the venom. Phosphatidylethanolamine-binding protein 4 appears to have multiple functions including anti-apoptotic activity via inhibition of the

that is also seen in Pal9.2, the loop lengths between the two peptides vary with a (8)C(7)C(7)C(10)C(2)C(14)C(2) pattern for the stonefish sequence and (5)C(3)C(7)C(10)C(6)C(13)C pattern for Pal9.2 with the second and third loops being highly conserved. However, variation in the other loops could result in significant conformational differences. While serine protease inhibitors are common in animal venoms, they are often Kunitz-type inhibitors. However, a Kazal-type serine protease inhibitor was identified in the venom of the Apis cerana honeybee that acted as an antimicrobial via its inhibitor activity [73]. Additionally, the first Kazal-type inhibitor-like proteins from snake venoms were recently discovered in arboreal pit vipers [74,75]. Further analysis of a Kazal-type inhibitor-like protein from Bothriechis schlegelii revealed that it was homologous with SPINK1 but lacked significant toxic activity, leading to the hypothesis that it could function to inhibit serine proteinases in the venom gland [76]. However, as the S. horrida protein was homologous to SPINK6, it is likely that it maintains kallikrein inhibitory activity, but may also act as an ion channel targeting neurotoxin. Tissue factor pathway inhibitors are protease inhibitors composed 23

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Fig. 12. A) Alignment of the PEBP4 found in S. horrida venom with the human PEBP4 (Uniprot accession number PEBP4_HUMAN) and a PEBP4 from the turquoise killifish Nothobranchius furzeri (Uniprot accession number A0A1A8A2K5_NOTFU). Alignment is coloured on a gradient based on conservation with the most highly conserved residues coloured the darkest. Signal peptides are underlined in black. B) Rat PEBP1 (PDB accession number 2IQY) and the stonefish homology model based on it. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

venoms. Instead, S. horrida venom is composed largely of C-type lectins and SNTX. Additionally, a number of proteins that do not represent recognised toxins were identified in the venom. Further investigation into proteins that have previously been found in animal venoms but overlooked as assumed housekeeping proteins is warranted. Other proteins, including phosphatidylethanolamine-binding protein 4 and gastrotropin, have not previously been reported in venoms but may add unique activities to S. horrida venom. This highlights the lack of understanding of S. horrida venom composition (and fish venoms in general) and how the different components add to the physiological effects of envenomation, opening up a number of avenues for further study.

Raf-1/MEK/ERK pathway [80,81]. It is a ubiquitous protein in vertebrate species, including fish species. Unlike PEBP1–3, PEBP4 possesses a signal sequence, defining it as a secretory protein [81]. The S. horrida PEBP4 sequence was homologous to the human protein, as well as a PEBP4 from the killifish N. furzeri, although both fish proteins lack the 25-residue chain seen at the C-terminus of the human protein. The strong sequence identity indicates the stonefish protein to have a similar activity, however it is unclear whether it acts as a toxin in S. horrida venom or is simply a cellular housekeeping protein. Interestingly, out of the seven C-type lectins identified in the S. horrida venom, four were identified to have signal sequences, while three lacked signal sequences. Additionally, this study confirmed that both SNTX subunits lack signal sequences [18], while the previously identified venom hyaluronidase signal sequence was confirmed [15]. As with putative gastrotropin proteins, the gastrotropin-like stonefish sequence lacked a signal sequence, but several other proteins found in the venom retained signal sequences, as is seen with their homologous housekeeping counterparts. Morphological study of the stonefish venom gland showed that the venom-secreting cells had neither Golgi apparatus nor rough endoplasmic reticulum, denoting a holocrine type secretion that is congruent with the lack of signal sequences associated with SNTX and some other venom proteins [7]. However, the presence of several venom proteins possessing signal sequences does not align with these observations. The venomic study of the Chinese Yellow Catfish also found that not all identified venom proteins had predicted signal sequences [24]. These results highlight the current lack of knowledge into fish venom toxin production and processing, which should be studied further. Overall, S. horrida venom appears to have a unique composition compared to other animal venoms, with only four putative venom proteins identified in the proteome, none of which were small cysteinerich neurotoxic peptides that are abundant in most other well-studied

Conflict of interest None declared.

Acknowledgements We thank Dr. Bruno Madio for his expert guidance in 2D electrophoresis. This study was supported by the University of Queensland's UQ International Scholarship (RZ), a Fellowship from the National Health and Medical Research Council (PFA), and by the Australian Research Council (DECRA Fellowship grant number DE160101142 to EABU).

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jprot.2019.04.002. 24

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