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Journal of Proteomics 132 (2016) 1–12

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Journal of Proteomics

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Unveiling the elusive and exotic: Venomics of the Malayan blue coralsnake (Calliophis bivirgata flaviceps)

Choo Hock Tan a,⁎, Shin Yee Fung b,⁎⁎, Michelle Khai Khun Yap b, Poh Kuan Leong a,Jia Lee Liew a, Nget Hong Tan b

a Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysiab Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

⁎ Correspondence to: C.H. Tan, Department of PharmUniversity of Malaya, 50603 Kuala Lumpur, Malaysia.⁎⁎ Correspondence to: S.Y. Fung, Department ofMoleculaUniversity of Malaya, 50603 Kuala Lumpur, Malaysia.

E-mail addresses: [email protected], tanch@[email protected] (S.Y. Fung).

http://dx.doi.org/10.1016/j.jprot.2015.11.0141874-3919/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 7 September 2015Received in revised form 9 November 2015Accepted 15 November 2015Available online 17 November 2015

The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia wasinvestigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein familieswere detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipaseA2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms ofNew World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteomealso revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reportedhigh level of adenosine in the venom. Also detected were 5′-nucleotidase (0.3%), hyaluronidase (0.1%) andcysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2,phosphodiesterase, 5′-nucleotidase and acetylcholinesterase activities but not L-amino acid oxidase activity.The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50 = 62.14 ±0.87 μg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic actionwith phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent an-tivenom from Taiwan. Together, thefindings provide insights into the composition and functions of the venomofthis exotic oriental elapid snake.Biological significance: While venoms of the New World coral snake have been extensively studied, literaturepertaining to the Old World or Asiatic coral snake venoms remains lacking. This could be partly due to the inac-cessibility to the venom of this rare species and infrequent cases of envenomation reported. This study identifiedand profiled the venom proteome of theMalayan blue coral snake (C. b. flaviceps) through SDS-PAGE and a high-resolution nano-LCMS/MS method, detailing the types and abundance of proteins found in the venom. Thebiological and toxic activities of the venom were also investigated, offering functional correlation to the venomproteome studied. Of note, the venom contains a unique toxin profile predominated with phospholipase A2

and cytotoxin with no detectable post-synaptic neurotoxin. The venom is moderately lethal to mice and thefatal effect could be cross-neutralized by a heterologous elapid bivalent antivenom from Taiwan. The findingsenrich snake toxin databases and provide insights into the composition and pathogenesis of the venom of thisexotic species.

© 2015 Elsevier B.V. All rights reserved.

Keywords:Calliophis bivirgata flavicepsVenomicsMalayan blue coral snakeMaticotoxinLabel-free proteomicsShotgun proteomics

1. Introduction

Coral snakes are slender, vibrantly colored and less aggressive elapidsnakes inhabiting forested areas. They can be subdivided into two distinctgroups based on their native macro-biogeographical distributions in the

acology, Faculty of Medicine,

rMedicine, Faculty ofMedicine,

.edu.my (C.H. Tan),

west and the east of the globe. Three genera are currently recognized ineach macro-biogeographical group of coral snakes: Leptomicrurus,Micruroides and Micrurus comprise the New World (American) coralsnakes, whereas Calliophis, Hemibungarus and Sinomicrurus constitutetheOldWorld (oriental or Asiatic) coral snakes [1,2]. Phylogenetic studiesindicated that the NewWorld coral snakes represent the descendants ofan ancestor from the oriental group, dispersed presumably via the Beringland bridge during the Miocene [1]. Despite their elusive and shy nature,coral snakes are proteroglyphous (front-fanged) venomous species capa-ble of causing severe toxicity in envenomation, albeit fatality is extremelyuncommon. In Central America, bites by Micrurus sp. constitute 1–2% ofall snakebite cases [3].Most caseswere reported as dry bites, but systemictoxicity could infrequentlymanifest as neuromuscular paralysismediated

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2 C.H. Tan et al. / Journal of Proteomics 132 (2016) 1–12

through the neurotoxins in the venom, resulting in respiratory failure [3].Venoms of the New World coral snakes have been the subject ofmuch research interest for decades; in recent years, venomic studiesof the various Micrurus species expanded tremendously [5–9]. Incontrast, the toxinological and proteomic studies of the venoms oforiental coral snakes are still lacking, hindering the understandingof the pathogenesis and treatment strategy for Asiatic coral snakeenvenomation.

In Asia, the fossorial, burrowing coral snakes distribute widely in theIndian subcontinent, Southeast Asia (including islands of Indonesia andthe Philippines), south China, and Taiwan to as far as the Ryukyu Islandsof Japan. Bites by Asiatic coral snakes have not been well documentedand appeared rare presumably due to infrequent human encounters.Nonetheless, the potential toxicity of the bites has placed this group ofsnakes in Category 2 of medically important venomous snakes by theWHO in most countries [4]. On morphology, the oriental coral snakesgenerally possess brilliant colors striking to the eyes, a phenotypebelieved to be of aposematic purpose for survival. They are generallyelusive with minimal human encounters, thus limiting the researchopportunity into the medical importance of these species. Continuousefforts by herpetologists have nonetheless led to the currently re-cognized systematics, where the Old World coral snakes are groupedinto three paraphyletic clades; the tropical mainland clade (genus:Calliophis) has themost number of species. Calliophis bivirgata (synony-mized with Maticora bivirgata), is a more common species of orientalcoral snake distributed in Southeast Asia. There are three subspeciesknown (withmajor distribution): C. b. flaviceps (Malayan Peninsula, Su-matra, Thailand), Calliophis bivirgata tetrataenia (Borneo) and Calliophisbivirgata bivirgata (Java). The subspecies found in theMalayan Peninsu-la and Thailand, Calliophis bivirgata flaviceps, can grow up to 1–1.6 m inlength [5]. It is the only species of oriental coral snakes reported to havecaused human fatalities. There were at least 2 fatal envenomationcases reported in Malaysia, where a young child died 2 h afterbeing bitten at the finger web at the base of the thumb in 1956,and a man died within 5 min after being bitten twice on his lefttoes in 1985 [6,7]. Notwithstanding the fact that bites by this specieswere rare, the rapid lethal effect from the bite and the unavailabilityof specific antivenom against the venom [4] pose a medically impor-tant issue in the management of snake envenomation in this region.This is in fact relevant in developing countries like Malaysia, as rapidurbanization has resulted in humans encroaching into the naturalhabitats of snakes. In addition, the noticeably increased interest forexotic species among snake keepers and hobbyists also flags analarming sign of untoward complication.

The anatomy of the venom apparatus of C. bivirgata is known to beunique among many snakes with the reason unknown: its venomglands are exceptionally long, extending beyond the jaw for one-thirdthe length of the body [8]. An earlier study on its venom property byTakasaki et al. [9] revealed that the venom is comprised predominantlyof phospholipases A2 and lethal cytotoxin homologues. In the presenceof phospholipase A2, two of the cytotoxins also exhibited weak indirecthemolytic activity. The median lethal dose (LD50) of this venom wasreported to be 0.81 μg/g (i.v.) in a rodent model [5]. In large part,research on the toxinology of the venom remains fragmented and thescientific data is in paucity, presumably due to the elusive nature ofthe species and the difficulty in obtaining the venom. In view of itspotential medical importance and the unavailability of specificantivenom, there is an urgency to investigate the toxinology of thevenom and its cross-neutralization by commercially available heter-ologous antivenoms. The current study aimed to fill the knowledgegap through unraveling the venom proteome of the species in corre-lation to its biochemical and pharmacological activities includinglethality neutralization. It is hoped that the findings would providedeeper insights into the pathophysiology and treatment strategy ofenvenoming by this exotic oriental coral snake, besides enrichingthe snake toxin databases for future study.

2. Materials and methods

2.1. Venoms and antivenoms

C. b. flaviceps venomwas a pooled sample from several milkings of asub-adult specimen captured at a low hill area in Pahang State, CentralMalaysia. The identification of the snake and milking of the venomwere performedbyone of the authors (CHT), and the venomwas lyoph-ilized and kept at −20 °C. Identification of the snake was based on itskey characteristics: bluish-black dorsum, red-colored ventral surfacecontinuous from head to tail, and a pair of sky blue stripes runninglaterally along each side of the body. The entirely red ventral surfacedistinguished it from the similar-looking red-headed krait (Bungarusflaviceps). Venoms of Bungarus multicinctus, Micrurus fulvius and Najaatra used in a comparative studywere purchased from Latoxan (Valencia,France).

Two polyvalent elapid antivenoms were used in this study:(a) Neuro Polyvalent Snake Antivenom (NPAV; lyophilized; batchno. NP00109, exp. date 5th Oct. 2014; manufactured by QueenSaovabha Memorial Institute, Bangkok, Thailand), a purified F(ab)′2obtained from serum of equines hyperimmunized against a mixture offour venoms of Thai origin:Naja kaouthia (monocled cobra),Ophiophagushannah (king cobra), Bungarus candidus (Malayan krait) and Bungarusfasciatus (banded krait), and (b) Taiwan neurotoxic bivalent antivenom(NBAV; batch no. FN10001; exp. date 31stMarch 2016), a purified equineF(ab)′2 raised against the venoms of B. multicinctus and N. atra ofTaiwanese origin, manufactured by the Center for Disease Control,Taipei, Taiwan ROC.

ICR mice (20–25 g) were supplied by the Laboratory Animal Centre,Faculty ofMedicine, University ofMalaya. The protocol of animal studieswas based on the Council for International Organizations of MedicalSciences (CIOMS) guidelines on animal experimentation [10] and wasapproved by the Institutional Animal Care and Use Committee of theUniversity of Malaya (ethics clearance number: 2014-09-11/PHAR/R/TCH).

2.2. Material

Ammonium bicarbonate, dithiothreitol (DTT) and iodoacetamidewere purchased from Sigma-Aldrich (USA). MS grade trypsin protease,Spectra™ Multicolor Broad Range Protein Ladder (10 to 260 kDa), andHPLC grade solvents used in the studies were purchased from ThermoScientific™ Pierce™ (USA). Millipore ZipTip® C18 Pipette Tips were ob-tained from Merck, USA. Other chemicals and reagents of analyticalgrade were purchased from Sigma-Aldrich (USA).

2.3. SDS PAGE and in-gel trypsin digestion

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was conductedaccording to Laemmli [11], calibrated with the Thermo ScientificPageRuler Prestained Protein Ladder (10–170 kDa). The venom(100 μg) was loaded onto a 15% gel, and the electrophoresis was per-formedunder reducing condition at 80 V for 2.5 h. Proteinswere stainedwith Coomassie Brilliant Blue R-250 and a total of 10 gel sections werecut across the sample lane. Each gel sectionwas then excised into small-er pieces (~1 × 2mm) and stored in a 600 μL micro-centrifuge tube. In-gel digestion was carried out using an in-gel tryptic digestion kit (Ther-mo Scientific). The gel pieces were destained for 30 min at 37 °C withshaking, and the in-gel proteinswere then reducedwith reducing bufferat 60 °C for 10 min, and alkylated in the dark at room temperature for1 h. Washing was repeated with destaining buffer at 37 °C for 15 minfollowed by dehydration using 50% acetonitrile for 15 min. Gel pieceswere then allowed to air-dry for 5–10min, and subsequently incubatedwith activated trypsin solution at room temperature for 15min. Digestionbuffer was then added to the tube and the sample was incubated at 30 °Covernight with shaking. The digested mixtures were then separated, and

3C.H. Tan et al. / Journal of Proteomics 132 (2016) 1–12

the peptides were further extracted by incubating the gel pieces in1% trifluoroacetic acid for 5 min. The digested protein samples weredesalted and concentrated with the Millipore ZipTip® C18 Pipette Tips(Merck, USA).

2.4. Nano ESI-liquid chromatography and tandem mass spectrometry(nano-ESI-LCMS/MS)

The digested peptide eluates were then subjected to nano-electrospray ionization (ESI) MS/MS analysis, using the Agilent 1200HPLC-Chip/MS Interface, coupled with an Agilent 6520 Accurate-MassQ-TOF LC/MS system. Samples were loaded in a large capacity chip300 Å, C18, 160 nL enrichment column and 75 μm × 150 mm analyticalcolumn (Agilent part no. G4240-62010) with a flow rate of 4 μL/minfrom a capillary pump and 0.3 μL/min from a nano pump of Agilent1200 series. Injection volume was adjusted to 1 μL per sample and themobile phases were 0.1% formic acid in water (A) and 90% acetonitrileinwaterwith 0.1% formic acid (B). The gradient appliedwas: 3–50% solu-tion B for 30 min, 50–95% solution B for 2 min, and 95% solution B for5 min using Agilent 1200 series nano-flow LC pump. Ion polarity wasset to positive ionizationmode. Drying gasflow ratewas 5 L/min and dry-ing gas temperature was 325 °C. Fragmentor voltage was 175 V and thecapillary voltage was set to 1995 V. Spectra were acquired in MS/MSmode with an MS scan range of 110–3000 m/z and an MS/MS scanrange of 50–3000m/z. Precursor charge selectionwas set as doubly, triplyor up to triply charged statewith the exclusion of precursors 922.0098m/z (z= 1) and 121.0509 (z= 1) set as reference ions. Data was extractedwith anMH+mass range between 600 and 4000 Da and processed withAgilent Spectrum Mill MS Proteomics Workbench software packages.Carbamidomethylation of cysteine was set as a single modification. Thepeptide finger mapping was modified to specifically search against anon-redundant NCBI database with taxonomy set to Serpentes (taxid:8570). From the LCMS/MS, protein identifications were validated withthe following filters: protein score N 11, peptides score N 6 and scoredpeak intensity (SPI) N 60%. Only results with “Distinct Peptide” identifica-tion of 2 or greater than 2 are considered significant.

2.5. Estimation of gel intensity by densitometry and relative proteinabundance by LCMS/MS

The electrophoresis gel containing Coomassie blue-stained proteinbandswas scanned by densitometry (Thermo Scientific PiercemyImageAnalysis Software) for the relative intensity of proteins in each gel sec-tion along the electrophoretic lane. Estimation of venom protein abun-dance through LCMS/MS technique was adapted from Aird et al. [12]and Tan et al. [13,14]. From the mass spectrometry analysis, the meanion intensity, also called the mean spectral intensity (MSI) of each pro-tein identifiedwas determined by dividing its summed spectral intensi-ty with the number of spectra. The ratio of a protein within a gel sectionwas determined based on its mean ion spectral intensity (MSI) relativeto the total mean spectral intensity of all proteins from the gel section.To obtain the relative abundance of a protein in a gel section, the ratioof the proteinwas adjusted according to the relative intensity of the cor-responding gel section (as determined by densitometry using ThermoScientific Pierce myImage Analysis Software). The generic equation issummarized below:

Relative abundance of protein A in gel section 1

¼ Mean spectral intensity of protein A in section 1Total mean spectral intensity in section 1

� �

� Relative gel intensity of section 1 %ð Þ:

The total abundance of a protein throughout the SDS PAGE lane isthe sum of its relative abundances from all gel sections.

2.6. Enzymatic assays

The venom solution stocks for enzymatic assays were prepared at aconcentration of 1 mg/mL. The enzymatic activities were assayed incomparison for the venoms of C. b. flaviceps, B. multicinctus, M. fulviusand N. atra.

2.6.1. L-amino acid oxidase (LAAO) assay

L-amino acid oxidase activity was determined as described by Tanet al. [15]. Fifty microliters of 0.00075% of horseradish peroxidase (1000units/mg) was added to 0.9 mL of 0.2 M triethanolamine buffer, pH 7.6,containing 0.1% L-leucine and 0.0075% o-dianidisine and incubated forthree minutes at room temperature. Fifty microliters of venom solutionwas added and the increase in absorbance at 436 nm was measured.The molar absorption coefficient is 8.31 × 103 cm−1 M−1.

2.6.2. 5′-Nucleotidase assayThe 5′-nucleotidase activity was measured according to Heppel and

Hilmore [16]. One hundred microliters of venom solution was added toan assay mixture containing 0.5 mL of 0.02 M 5′-AMP (pre-adjusted topH 8.5), 0.5 mL of 0.2 M glycine buffer, pH 8.5 and 0.1 mL of 0.1 MMgSO4. Themixturewas incubated at 37 °C for 10min, and the reactionwas terminated by adding 1.5 mL of 10% trifluoroacetic acid. The quan-tity of inorganic phosphate released was determined by ascorbic acidmethod [17]. Briefly, 1 mL of ascorbic acid reagent (equal parts of 3 Msulfuric acid, 2.5% ammonium molibdate, 10% ascorbic acid and water)was added to the above mixture. The mixture was left at room temper-ature for 30 min and the absorbance at 820 nm was then determined.A standard curve of known inorganic phosphate concentrations wasconstructed. The enzyme activity was expressed as nmole of phosphaterelease per minute.

2.6.3. Phospholipase A2 (PLA) assayPhospholipase A2 activity was determined by the acidimetric meth-

od [18]. The egg yolk substrate suspension was prepared bymixing onepart chicken egg yolk, one part 18 mM calcium chloride, and one part8.1 mM sodium deoxycholate. The pH of the substrate suspension wasadjusted to 8.0 with sodium hydroxide. The suspension was stirred toensure good mixing. One hundred microliters of venom solution wasadded to 15 mL of the substrate suspension and the rate of decrease inpH was recorded using a pH meter. A decrease of 1 pH unit of the eggyolk suspension corresponded to 133 μmol of fatty acids released.

2.6.4. Phosphodiesterase (PDE) assayPhosphodiesterase activity was determined by a method modified

from Lo et al. [19]. One hundred microliters of sample was addedto an assay mixture containing 0.5 mL of 2.5 mM calcium bis-p-nitrophenylphosphate, 0.3 mL of 0.01 M magnesium sulfate and0.5 mL of 0.17 M veronal buffer, pH 9.0. The hydrolysis of the substratewas monitored by measuring the rate of increase of absorbance at400 nm. The extinction coefficient is 8100 cm−1 M−1.

2.6.5. Acetylcholinesterase assayThe assay was performed according to the method described by

Ellman et al. [20]. One hundred microliters of venom solution wasadded to an assay mixture contained 0.8 mL of 0.025 M sodium phos-phate buffer, pH 7.5, 0.05 mL of 0.0125 M acetylthiocholine iodide and0.05 mL of 6.66 mM dithionitrobenzoate (DTNB) containing 1 mg/mLsodium bicarbonate. The hydrolysis of the substrate was measured bythe increase in absorbance at 412 nm. The molar extinction coefficientis 13,600 cm−1 M−1. The enzyme activity was expressed as μmole ofproduct released/minute.


2.7. Determination of the cytotoxicity activity of C. b. flaviceps venom usingcell viability assay

Mousefibroblast cells (ATCC®CRL-2648)were culturedwithDMEM(ATCC®) containing 10% fetal bovine serum, 4 mM L-glutamine,4500 mg/L glucose, 1 mM sodium pyruvate and 1500 mg/L sodium bi-carbonate in humidified 95% air, 5% CO2, 37 °C incubator, in a 75 cm2 tis-sue culture flask. Approximately 80% confluent cells were harvested byadding 5 mL of trypsin–EDTA solution to detach the adherent cells,followed by centrifugation at 1250 rpm for 5 min. Cells at optimal den-sity were seeded into a 96-well microtitre plate and incubated for 24 hfor cells to attach. After that cells were treated with C.b. flaviceps venomfor another 48 h. The viability of the cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.After 48 h incubation, 10 μL of MTT solution (5 mg/mL in PBS) wasadded into the treated cells and allowed to incubate for 4 h. Formazancrystals resulting from MTT reduction were dissolved by 100 μL DMSOper well. Absorbance was then measured at 570 nm using a microtitreplate reader (Bio-Rad). Vehicle-control wells with cells only anddiluent-control wells with similar DMSO concentrations as treatedcells were included. All experiments were performed in triplicate. Thepercentage of cell viability was calculated using the following formula:

% cell viability ¼ Abs570nm control�Abs570nm treatment

Abs570nm control� 100%:

The IC50 (50% inhibitory concentration, μg/mL) was defined as theamount of the venom that induced 50% inhibition in the cell viabilitycompared to the control. The IC50 value was determined by usingGraphPad Prism (version 6.0). The cytotoxicity of three other elapidvenoms including N. atra venom, B. multicinctus venom and M. fulviusvenom were also examined for comparison.

2.8. Determination of the myotoxicity of venom

The myotoxic activity of C. b. flaviceps venom was determinedusing a rodent model as described by Tan et al. [13]. A dose of 5 μgvenom (dissolved in 100 μL saline) was injected intramuscularlyinto the gastrocnemius of mice (n = 3). The negative controlgroup (n = 3) received intramuscular injection of saline withoutvenom. The mice were monitored for 3 h under urethane anesthesia.Subsequently, blood was collected through cardiac puncture and themice were euthanized by cervical dislocation. Sera obtained weresent for total creatine kinase (CK) analysis in an independent pa-thology service laboratory.

Fig. 1. (A) SDS-PAGE of Calliophis bivirgata flaviceps venom; (B) relative intensity of gelsections. C. b. flaviceps venom(100 μg)was separated by SDS-PAGE (15% gel) under reduc-ing condition. The resulting gel lane was divided into 7 sections and analyzed for relativeintensity using Pierce myImageAnalysis Software (Thermo Scientific). Prestained proteinmolecular mass standard was loaded on the left lane (M, in kDa).

2.9. Determination of venom lethality and neutralization by heterologousantivenom

Median lethal dose, LD50, of the venom was determined by intrave-nous (caudal veins) injection into ICRmice (n=4 for each dosage). Thesurvival ratio was recorded after 48 h. Experiments with preincubationof venomand antivenomprior to injectionwere conducted as describedby Tan et al. [21]. Briefly, a challenge dose at 2.5 or 5 LD50 of the venomdissolved in saline was pre-incubated with various dilutions of thereconstituted antivenom at 37 °C for 30 min. The venom–antivenommixture was subsequently centrifuged at 10,000 ×g and the superna-tant was injected intravenously into the mice (n = 4 for each dosage).The median lethal dose (LD50), median effective dose (ED50) and the95% confidence intervals (C.I.) were calculated using the probit analysismethod of Finney [22] with BioStat 2009 analysis software (AnalystSoftInc., Canada). Potency of antivenom was assessed according to Moraiset al. [23].

3. Results and discussion

3.1. SDS-PAGE profile of C. b. flaviceps

The SDS-PAGE of C. b. flaviceps venom revealed that the venom iscomprised of mainly low molecular-mass proteins of 6–15 kDa (sec-tions 6 and 7; Fig. 1A), with an estimated abundance of approximately70% of the total venom proteins (Fig. 1B). Proteins of higher molecularmass (N20 kDa) were observed as light bands in sections 1 to 5. Thisis similar to the SDS-PAGE findings of most elapid venoms, includingthe New World coral snakes [24–26], Asiatic kraits [27] and cobras[28] as well as the hydrophid sea snake [29].

3.2. 1D SDS-PAGE shotgun proteomic analysis of C. b. flaviceps venom

The shotgun LCMS/MS profiling of the venom revealed a total of 23distinct proteins. These proteins were assigned according to the res-pective gel sections in Table 1. The proteins were further clusteredinto families (Table 2); of these, the first twenty were considered tobe toxins/putative toxins. Phospholipase A2 (PLA2) is the proteinfamily with the highest abundance (41.1%), followed by cytotoxin(CTX, 22.6%), snake venom metalloproteinase (SVMP, 18.7%), vespryn(14.6%) and several other toxins with lower abundance such as phospho-diesterase (PDE, 1.3%), cysteine-type endopeptidase inhibitor (cystatin,0.6%), 5′-nucleotidase (5′NUC, 0.3%) and hyaluronidase (0.1%). In addi-tion, there were three non-toxin proteins which exist in a very smallquantity (Table 2; Fig. 2).

The venom proteome revealed for the C. b. flaviceps specimen in thisstudy shares some common features with those of the American coralsnakes. Themost prominent common feature is the dominant presenceof three-finger toxins and phospholipases A2. For most AmericanMicrurus venoms, these two toxin families usually constitute 60–90%of total venom proteins [24–26,30]. However, a striking differencenoted between the venom proteome of C. b. flaviceps from the NewWorld coral snakes was the absence of postsynaptic neurotoxin. Ourfindings on this specimen concurs with the earlier study by Takasakiet al. [9] which also found no post-synaptic neurotoxins in the venomspooled from two C. b. flaviceps of Thailand, indicating this is probably a

Table 1Assignment of the in-gel digested proteins (gel section 1–7) of Calliophis bivirgata flaviceps (Malaysia) venom by nano-ESI-LCMS/MS analysis.

Section Spectra/distinctpeptides

Distinct summedMS/MS searchscore

Species with thehomologousprotein

Databaseaccessionnumber

Protein namea z m/zmeasured(Da)

Sequence

Section 11 10/9 144.28 Micrurus fulvius 537444868 Phosphodiesterase 1 2 524.298 (K)YGPVSGQVIK(S)

2 749.9098 (R)VMEVLQWLDLPR(A)2 752.8858 (K)SMEAIFLAHGPGFK(E)3 780.3962 (R)NPVWWGGQPIWHTATYQGLK(A)2 554.2798 (K)NPFYSPSPAK(E)2 678.3379 (K)AATYFWPGSEVK(I)2 454.742 (K)FDSEAIVK(N)2 759.9108 (R)LWNYFHSTLLPK(Y)2 546.7952 (R)TLGMLMEGLK(Q)4 666.8266 (R)NLHNCVNLILLADHGMEAISCNR(L)2 498.2453 (K)NVPEDFFK(F)3 376.2143 (K)RLHFANNVR(I)4 583.556 (K)AKRPDFLTLYIEEPDTTGHK(Y)2 472.2659 (K)QPLSETLR(L)2 339.6947 (R)AVYPTK(T)3 500.2758 (R)VMEVLQWLDLPR(A)2 498.2453 (K)NVPEDFFK(F)3 711.3627 (K)RPDFLTLYIEEPDTTGHK(Y)

2 5/4 66.76 Micropechisikaheka

633276509 P-III snake venommetalloprotease

2 860.8865 (R)AAKDDCDLPEICTGR(S)3 574.2627 (R)AAKDDCDLPEICTGR(S)2 694.8598 (K)YIEFYVVVDNK(M)2 725.8024 (K)DDCDLPEICTGR(S)2 330.1818 (R)IPCAAK(D)2 578.2222 (K)CGDGMVCSNR(H)3 506.2756 (K)KYIEFYVVVDNK(M)2 758.9084 (K)KYIEFYVVVDNK(M)

3 3/3 43.5 Crotalusadamanteus

(0)324156 Phosphodiesterase 1 2 842.4473 (R)DVELLTGLDFYSALK(Q)3 647.0255 (R)VRDVELLTGLDFYSALK(Q)2 472.2659 (K)QPLSETLR(L)

4 3/2 37.51 Demansiavestigiata

118151738 Metalloproteinase 2 649.2803 (R)SAECPTDSFQR(N)2 330.1818 (K)IPCAAK(D)2 649.278 (R)SAECPTDSFQR(N)

5 2/2 29.71 Cryptophisnigrescens

145982768 Nigrescease-1 2 330.1818 (K)IPCAAK(D)2 404.2121 (R)IFGEWR(E)

6 2/2 27.76 Ophiophagushannah

565308117 Metalloproteinase (hypotheticalprotein)

2 607.8322 (R)NDNAQLLTGIR(F)3 500.2885 (R)KRNDNAQLLTGIR(F)


565305842 Vespryn (hypothetical protein) 2 403.7145 (K)HFFEVK(Y)2 465.7582 (R)EWAVGLAGK(S)

Section 28 18/15 215.93 Ophiophagus

hannah565318847 Sulfhydryl oxidase 1 2 872.4535 (K)IYMADLESAVLYSLR(I)

2 722.8758 (R)IEAAVFPNLEGER(L)2 773.4134 (K)TIVLQSEWEEALK(N)3 492.2659 (K)DFVSVLVQHFAGR(L)2 536.7768 (R)SIPGLEDWR(S)3 624.6708 (R)KIYMADLESAVLYSLR(I)3 581.9705 (K)IYMADLESAVLYSLR(I)2 499.2164 (K)FMWDENR(V)2 514.764 (R)SQYTNFLR(N)2 375.7338 (R)VLMFLK(W)2 401.7338 (R)NLPGVFR(R)2 637.8302 (R)DEAVLWLWSR(H)2 579.824 (R)VLQSNEELVK(Q)2 657.8734 (R)RVLQSNEELVK(Q)2 737.8923 (K)DFVSVLVQHFAGR(L)2 773.4139 (K)TIVLQSEWEEALK(N)2 366.2068 (K)NIESLR(G)2 384.7245 (K)IQWPPK(S)

9 10/10 146.15 Micrurus fulvius 537444870 Ecto-5′-nucleotidase 1 2 958.007 (K)VLLPSFLAAGGDGYYMLK(G)2 653.3437 (R)VPTYVPLEMEK(T)2 453.7299 (K)VFPAMEGR(V)3 348.2078 (K)IIALGHSGFK(E)2 430.2487 (K)IINVGSEK(V)2 653.8629 (R)QVPVVQAYAFGK(Y)2 725.3651 (R)VVSLNVLCTECR(V)2 476.2622 (K)VVYDLSQK(A)2 476.2806 (K)VGIIGYTTK(E)3 411.2151 (R)EVVHFMNSLR(Y)2 628.3813 (K)FPILSANIRPK(G)3 508.6136 (R)HGQGTGELLQVSGIK(V)2 358.6831 (R)SPIDER(A)

(continued on next page)


Table 1 (continued)






Sequence

10 8/7 85.21 Acanthophiswellsi

476538125 SVMP-Aca-4 2 330.1816 (R)IPCAAK(D)2 391.1875 (K)SFGEWR(E)2 633.3468 (K)SVAVIQDYSKR(T)2 555.2974 (K)SVAVIQDYSK(R)2 694.8593 (K)YIEFYVVVDNK(M)2 578.2209 (K)CGDGMVCSNR(Q)3 506.2751 (K)KYIEFYVVVDNK(M)2 758.9067 (K)KYIEFYVVVDNK(M)2 414.2542 (R)ETVLLPR(R)2 542.2444 (R)QCVDVQTAY(–)



3 574.2618 (R)AAKDDCDLPEICTGR(S)2 725.8021 (K)DDCDLPEICTGR(S)2 330.1816 (R)IPCAAK(D)2 578.2209 (K)CGDGMVCSNR(H)2 414.2542 (R)ETVLLPR(K)2 356.6709 (K)LYCEK(T)

12 5/5 65.14 Python bivittatus 602631303 Multiple inositol polyphosphatephosphatase 1

2 513.2888 (K)LLPFTYSGK(T)3 755.0573 (R)IVPYAANLLFVLYHCDQAR(S)2 479.2805 (R)FPSLLAPGR(R)2 553.809 (K)VLEYLNDLK(Q)2 478.793 (K)VQILLNEK(L)

13 4/4 56.03 Micrurus fulvius 537444868 Phosphodiesterase 1 2 524.2954 (K)YGPVSGQVIK(S)2 749.9086 (R)VMEVLQWLDLPR(A)2 546.7931 (R)TLGMLMEGLK(Q)2 339.6944 (R)AVYPTK(T)


118151738 Metalloproteinase 2 649.2792 (R)SAECPTDSFQR(N)2 330.1816 (K)IPCAAK(D)2 349.7335 (K)TVLLPR(K)

15 3/3 45.3 Pseudechisporphyriacus

145982756 Porphyriacase-1 2 330.1816 (K)IPCAAK(D)2 391.1875 (K)SFGEWR(E)2 471.1904 (K)GQGCGFCR(I)


145982768 Nigrescease-1 2 330.1816 (K)IPCAAK(D)2 404.2127 (R)IFGEWR(E)2 356.6709 (R)LYCEK(G)

17 2/2 23.54 Crotalusadamanteus

(0)330517 Phosphodiesterase 1 2 498.2432 (K)NVPEDFFK(F)2 454.7404 (K)FDSEAIVK(N)

Section 318 6/5 78.27 Bitis arietans 113203681 Hyaluronidase 3 635.3108 (K)HSDSNAFLHLFPESFR(I)

2 622.3384 (R)NDQLLWLWR(D)2 497.2861 (R)YKVDLDLK(T)4 508.7588 (R)KHSDSNAFLHLFPESFR(I)2 905.9972 (R)DSTALFPSIYLETILK(S)2 301.1921 (R)MIPLK(T)2 905.9972 (R)DSTALFPSIYLETILK(S)3 331.8601 (R)YKVDLDLK(T)



2 758.9092 (K)KYIEFYVVVDNK(M)2 860.8868 (R)AAKDDCDLPEICTGR(S)2 725.8034 (K)DDCDLPEICTGR(S)3 574.2613 (R)AAKDDCDLPEICTGR(S)3 506.2759 (K)KYIEFYVVVDNK(M)2 578.2235 (K)CGDGMVCSNR(H)2 498.2416 (K)FKGAGAECR(A)2 330.1824 (R)IPCAAK(D)2 394.2293 (R)KIPCAAK(D)2 384.1825 (K)CIMSTR(R)3 506.2746 (K)KYIEFYVVVDNK(M)2 694.8587 (K)YIEFYVVVDNK(M)2 480.7269 (–)TNTPEQDR(Y)2 725.8009 (K)DDCDLPEICTGR(S)2 414.2556 (R)ETVLLPR(K)2 758.9059 (K)KYIEFYVVVDNK(M)2 758.9059 (K)KYIEFYVVVDNK(M)


565318847 Sulfhydryl oxidase 1 2 536.7772 (R)SIPGLEDWR(S)2 514.7635 (R)SQYTNFLR(N)2 401.7343 (R)NLPGVFR(R)2 657.8721 (R)RVLQSNEELVK(Q)2 579.8239 (R)VLQSNEELVK(Q)2 366.2074 (K)NIESLR(G)

21 9/5 69.83 Acanthophiswellsi

476538125 SVMP-Aca-4 2 758.9092 (K)KYIEFYVVVDNK(M)3 506.2759 (K)KYIEFYVVVDNK(M)2 330.1824 (R)IPCAAK(D)3 506.2746 (K)KYIEFYVVVDNK(M)2 694.8587 (K)YIEFYVVVDNK(M)2 414.2556 (R)ETVLLPR(R)


Table 1 (continued)






Sequence

2 758.9059 (K)KYIEFYVVVDNK(M)2 758.9059 (K)KYIEFYVVVDNK(M)2 555.2971 (K)SVAVIQDYSK(R)


118151738 Metalloproteinase precursor 2 649.2804 (R)SAECPTDSFQR(N)2 649.2772 (R)SAECPTDSFQR(N)2 330.1824 (K)IPCAAK(D)2 394.2293 (R)KIPCAAK(D)2 349.7311 (K)TVLLPR(K)

23 4/4 53.43 Hoplocephalusbungaroides

476539268 SVMP-Hop-46, partial 2 330.1824 (K)IPCAAK(D)2 391.1877 (K)SFGEWR(E)2 394.2293 (R)KIPCAAK(D)2 414.2556 (R)ETVLLPR(K)



3 500.2903 (R)KRNDNAQLLTGIR(F)2 607.8316 (R)NDNAQLLTGIR(F)2 607.8311 (R)NDNAQLLTGIR(F)3 457.5911 (K)RNDNAQLLTGIR(F)

25 2/2 35.35 Naja mossambica 172046653 Snake venom metalloproteinase-disintegrin-like mocarhagin

2 535.287 (K)SVAVVQDHSK(S)2 498.2416 (K)FKGAGAECR(A)2 330.1824 (K)IPCAAK(D)


145982768 Nigrescease-1 2 404.2136 (R)IFGEWR(E)2 330.1824 (K)IPCAAK(D)2 404.2139 (R)IFGEWR(E)2 404.2132 (R)IFGEWR(E)

27 2/2 27.72 Drysdaliacoronoides

336042218 MTP4 2 391.1877 (R)SFGEWR(N)3 637.9245 (R)NGHPCQNNEGYCYNGK(C)

Section 428 5/5 52.16 Acanthophis

wellsi476538125 SVMP-Aca-4 2 694.8586 (K)YIEFYVVVDNK(M)

2 330.1827 (R)IPCAAK(D)2 555.2931 (K)SVAVIQDYSK(R)2 414.2547 (R)ETVLLPR(R)3 506.2778 (K)KYIEFYVVVDNK(M)


565318847 Sulfhydryl oxidase 1 2 536.776 (R)SIPGLEDWR(S)2 722.8742 (R)IEAAVFPNLEGER(L)2 773.4113 (K)TIVLQSEWEEALK(N)2 401.7346 (R)NLPGVFR(R)


118151738 Metalloproteinase precursor 2 649.279 (R)SAECPTDSFQR(N)2 330.1827 (K)IPCAAK(D)2 349.7319 (K)TVLLPR(K)



2 607.8307 (R)NDNAQLLTGIR(F)2 500.2889 (R)KRNDNAQLLTGIR(F)2 457.5952 (K)RNDNAQLLTGIR(F)

32 2/2 29.55 Naja mossambica 172046653 Snake venommetalloproteinase-disintegrin--like mocarhagin

2 535.2894 (K)SVAVVQDHSK(S)2 498.2425 (K)FKGAGAECR(A)2 330.1827 (K)IPCAAK(D)


145982768 Nigrescease-1 2 404.2133 (R)IFGEWR(E)2 330.1827 (K)IPCAAK(D)

34 2/2 24.43 Micrurus fulvius 537465633 Endonuclease domain-containing1 protein

2 545.8055 (K)LAQLYNVNR(V)2 428.7187 (R)YQNLYR(F)

35 2/2 23.18 Crotalus horridus 521752401 Ecto-5′-nucleotidase 2 476.2772 (K)VGIIGYTTK(E)2 430.2492 (K)IINVGSEK(V)



3 574.2614 (R)AAKDDCDLPEICTGR(S)2 330.1827 (R)IPCAAK(D)

Section 537 2/2 23.73 Micropechis

ikaheka633276509 P-III snake venom

metalloprotease3 574.2608 (R)AAKDDCDLPEICTGR(S)2 330.1814 (R)IPCAAK(D)


145982768 Nigrescease-1 2 330.1814 (K)IPCAAK(D)2 404.2147 (R)IFGEWR(E)


118151738 Metalloproteinase precursor 2 330.1814 (K)IPCAAK(D)2 349.7292 (K)TVLLPR(K)

Section 640 4/2 40.25 Maticora

bivirgatus129429 Phospholipase A2 2 2 678.2751 (R)SFVDYGCYCGK(G)

2 678.2759 (R)SFVDYGCYCGK(G)2 913.9759 (–)NLYQFGNMIKCTIPK(R)2 913.9824 (–)NLYQFGNMIKCTIPK(R)


116242842 Ohanin 2 403.7127 (K)HFFEVK(Y)2 465.7568 (R)EWAVGLAGK(S)

Section 742 4/4 71.59 Naja kaouthia (0)319553 Cystatin precursor 2 580.3083 (K)YYLTMELVK(T)

2 650.3704 (R)IVEAQSQVVAGAK(Y)3 749.3747 (K)AAAFAVQEYNTLSANTHYFK(E)2 738.3949 (R)FQVWSRPWLEK(T)

(continued on next page)


Table 1 (continued)






Sequence


116242842 Ohanin 2 749.3642 (R)FSSSPCVLGSPGFR(S)2 749.365 (R)FSSSPCVLGSPGFR(S)2 403.716 (K)HFFEVK(Y)2 416.7392 (R)VLGSPGFR(S)2 403.715 (K)HFFEVK(Y)2 465.759 (R)EWAVGLAGK(S)2 465.7585 (R)EWAVGLAGK(S)

44 4/2 45.66 Maticorabivirgatus

117738 Cytotoxin homolog maticotoxin A 2 578.3009 (–)LICYNTPFK(D)2 782.828 (K)TCAEGENLCYYGK(K)2 578.2999 (–)LICYNTPFK(D)2 578.2997 (–)LICYNTPFK(D)

Abbreviations: PLA2, phospholipase A2; SVMP, snake venom metalloproteinase; PDE, phosphodiesterase; 5′-NUC, 5′-nucleotidase. Cysteine residues determined in MS/MS analysis arecarbamidomethylated. Protein identifications were validated with the following filters: protein score N 11, peptides score N 6 and scored peak intensity (SPI) N 60%. Only results with“Distinct Peptide” identification of 2 or greater than 2 are considered significant.

a Refers to protein names derived from the protein database based on best homology match and BLAST search.


unique phenotype of the species across Thailand and PeninsularMalaya.Formost NewWorld coral snakes, the three-finger toxins consistmostlyof postsynaptic neurotoxins and a lesser amount of cytotoxins; how-ever, the three-finger toxin family of the C. b. flaviceps specimen inthis study comprises solely of cytotoxin. It is also noted that snakevenom lectins and CRISPs, both of which have been reported in theNew World coral snake venoms, were not detected by the nano-LCMS/MS used in this study.

PLA2 constitutes 41.1% of the venomprotein abundance. An isoformofPLA2 detected in the venom proteome of the specimen has an N-terminal

Table 2Summary of venomproteins ofMalaysia Calliophis bivirgata flaviceps assigned by protein familieproteins.

Family Protein namea Gel section

Snake venom metalloproteinase (SVMP)1 P-III snake venom metalloprotease, partial 1–52 Metalloproteinase precursor 1–53 SVMP-Aca-4 2–44 SVMP-Hop-46 partial 35 Snake venom metalloproteinase-disintegrin-like mocarhagin 3–46 Nigrescease-1 1–57 Porphyriacase-1 28 Metalloproteinase 1,3–49 MTP4 3

Vespryn/ohanin1 Ohanin 6–72 Vespryn 1

Phosphodiesterase (PDE)1 Phosphodiesterase 1 1–22 Phosphodiesterase 1 13 Phosphodiesterase 1 2

Phospholipase A2 (PLA2)1 Phospholipase A2 2 6

Cytotoxin maticotoxin1 Cytotoxin homolog maticotoxin A 7

Hyaluronidase1 Hyaluronidase 3

Cystatin1 Cystatin precursor 7

5′ Nucleotidase1 Ecto-5′-nucleotidase 1 22 Ecto-5′-nucleotidase 4

Sulfhydryl oxidase1 Sulfhydryl oxidase 1 2–4

Endonuclease1 Endonuclease domain-containing 1 protein 4

Multiple inositol polyphosphate phosphatase 11 Multiple inositol polyphosphate phosphatase 1 2

a Refers to protein names derived from the protein database based on homology match andb Refers to the number of distinct peptides thatwasmatched to the protein identified in one

Table 1.

sequence (residues 1–32) likely identical to that of PLA2 isoenzyme II iso-lated from Maticora (Calliophis) bivirgata venom by Takasaki et al. [9](matched sequence: N1LYQFGNMIKCTIPKRXXXRSFVDYGCYCGKG32).This is an N-type PLA2 with a Cys11 residue critical in forming the charac-teristic disulfide bond between the 11th and 77th residues for Group IPLA2 [31]. Snake venom PLA2s exhibit various biological activities; somePLA2 enzymes from the New World coral snake venoms have beenshown to causemyotoxicity and presynaptic neurotoxicity [26,30]. How-ever, PLA2 isoformswith low toxicity and non-lethal effect in experimen-tal animals are not uncommon [29]. An earlier study by Takasaki et al. [9]

s. Data were derived fromESI-nano-LCMS/MS analysis of peptide ions from in-gel digested

Database accession number Distinct peptides matchedb Relative abundance (%)

18.7633276509 2–6 4.61118151738 3–5 3.93476538125 5–9 0.57476539268 4 0.45172046653 2 0.12145982768 2–4 4.38145982756 3 0.41565308117 2–4 3.87336042218 2 0.32

14.6116242842 2–6 14.4565305842 2 0.2

1.3537444868 4–10 0.57(0)324156 3 0.74(0)330517 2 0.02

41.1129429 4 41.1

22.6117738 4 22.6

0.1113203681 6 0.1

0.6(0)319553 4 0.6

0.3537444870 10 0.2521752401 2 0.1

0.4565318847 3–18 0.4

0.04537465633 2 0.04

0.04602631303 5 0.04

BLAST search.ormore gel sections. Sequences of peptides and their corresponding proteins are shown in

Fig. 2. Venom proteome of Calliophis bivirgata flaviceps. The venom proteome of C. b. flaviceps as revealed by label-free 1D SDS-PAGE shotgun proteomics. The three dominant proteinfamilies are PLA2, cytotoxin and SVMP. Abbreviation: PLA2, phospholipase A2; SVMP, snake venom metalloproteinase; PDE, phosphodiesterase; HYA, hyaluronidase.


demonstrated that the PLA2 isoforms isolated from the venomof this spe-cies were non-lethal in mice but acted synergistically with some of thevenomcytotoxins, potentiating the hemolytic activity [9]. The cytolytic ef-fect of the PLA2s was notably feeble without cytotoxins (and vice versa),with a drop in activity by almost 90% magnitude. Thus, while the patho-genic role of C. b. flaviceps venom PLA2 deserves further investigation, itappears that the enzyme acted to enhance the effect of cytotoxin in thepathogenesis of C. b. flaviceps envenoming.

The second major component of C. b. flaviceps venom revealedin this study is cytotoxin (22.6%). The tryptic peptides werebest matched to the N-terminal sequence of maticotoxin A(L1ICYNTPFKDXXKTCAEGENLCYYGKK27), a cytotoxin homolog se-quenced and reported previously for this species [9]. MaticotoxinA has an intravenous LD50 (i.v.) of 0.65 μg/g but was reported tobe devoid of cytotoxicity on Fogh–Lund amnion cells [9]. Interestingly,the toxin exhibited hemolytic activity which was enhanced in the pres-ence of PLA2, supporting the importance of the synergistic interaction be-tween these twomajor groups of toxins in the venom. In this study, threeother cytotoxin isoforms (maticotoxins C, D1 and D2, inferred fromHPLCelution pattern) reported by Takasaki et al. [9] were not detected specifi-cally, for the fact that there are still no available database peptide se-quences of the three toxins thus far (not sequenced by the previousresearchers). These cytotoxins, nonetheless, demonstrated similar activityas maticotoxin A (feeble hemolytic activity greatly enhanced by PLA2),and likely share similar sequences withmaticotoxin A, to which the tryp-tic peptides might have matched in the current proteomic study.

Snake venom metalloproteinases (SVMPs) constituted 18.7% of thevenom abundance. Of the nine subtypes detected, the main form is aP-III SVMP (4.6%). Generally, viper and pit viper venoms are character-ized by a high content of SVMP whereas elapid venoms contain a lesseramount (including the New World coral snakes) with the exception ofO. hannah venom [12,24,28,32–34]. Compared to other elapid venoms,the SVMP content of C. b. flaviceps venom is relatively higher, implyingthat this toxin family may play an important pathogenic role in thevenom. It is well-established that different SVMPs exhibit varied phar-macological activity such as hemorrhagic, fibrinogenolytic, cytotoxic,platelet aggregation inhibitory as well as pro-inflammatory activities[35]. The toxicity of SVMPs present in C. b. flaviceps venom awaitsfurther investigation.

Vespryn/ohanin protein was found mostly in the low molecularweight sections 6 and 7 of the SDS-PAGE (Fig. 1), and accounts for14.6% of the total venom proteins. The ohanin/vespryn is a toxin ofapproximately 12 kDa first found in the venom of O. hannah [36],

and recently has been increasingly reported from other snakes in-cluding the Asiatic cobra N. kaouthia [28], Australian elapids [37]as well as the Central American coral snake Micrurus nigrocinctus[30]. This group of toxin is non-lethal to mice but capable of induc-ing hyperalgesic and hypolocomotive effects in laboratory animals[36]. Vespryn present in C. b. flaviceps venom likely has the putativefunction of hypolocomotion that aids the snake in prey handling.

Another interesting aspect of the venom proteome of this specimenis the relatively high content of phosphodiesterases (PDEs) (1.3%). Gen-erally, viperid and crotalid venoms are known to exhibit higher PDE ac-tivity than elapid venoms [38]. Three forms of the enzyme weredetected in the venom. PDE catalyzes the hydrolysis of phosphodiester(widely found in nucleic acids) to phosphomonoester [39], causing anincrease in the extracellular levels of adenosine and other purine deriv-atives thatmay lead to hypotension, inflammation and even blockade ofneurotransmitters. The relatively high content of this enzyme in thevenom of this C. b. flaviceps specimen may be associated with the pres-ence of abundant (endogenous) adenosine reported previously in thevenom of the Thai species [9]. Another related enzyme family, 5′-nucle-otidase (two forms detected), was also identified at amuch lower abun-dance (0.1% of venom proteins). The venom 5′ nucleotidase reportedmay have the putative function of generating purines, particularly aden-osine. The products of these nucleoside-releasing enzymes can inducehypotension and thus aids in prey immobilization. The introduction ofpurines in envenoming strategy has a universal advantage that it is im-possible for prey organisms to develop resistance against them, as pu-rine nucleosides are endogenous regulatory compounds (such ascAMP) [40].

The use of LCMS/MS also revealed a minute amount of a cysteine-type endopeptidase inhibitor (cystatin, 0.6%) and hyaluronidase(0.1%) in the venom proteome of C. b. flaviceps. The role of cystatinin snake venom is not known, but it could be useful to inhibitauto-proteolytic activity of toxins stored in the glandular lumens,or extracellular matrix proteases in prey. Hyaluronidase is a glycosi-dase involved in facilitating the spread and absorption of toxins. It isan ancillary venom component that is capable of augmenting thetoxic effect of a venom [41].

Besides, three proteins with very low abundances not known tobe toxins were also detected in the venom (b1% of total venom pro-teins). The three proteins belong to the families sulfhydryl oxidase-1, multiple inositol polyphosphate phosphatase-1 and endonucleasedomain-containing 1 protein. These are likely cellular proteins withendophysiological functions, detected by the sensitive LCMS/MS.

Fig. 3. The cytotoxic effects of Calliophis bivirgata flaviceps and three other elapid venomsagainst CRL-2648 fibroblast cell lines. The cells were treated with the respective venomsfor 48 h and cell viability was determined by MTT assay.


3.3. The enzymatic activities of C. b. flaviceps venom

Table 3 shows the venom enzymatic activities of the current C. b.flaviceps specimen in comparison to three other elapids (M. fulvius,B. multicinctus and N. atra). Among the four venoms, C. b. flavicepsvenom exhibited the lowest enzymatic PLA2 activity (210 μmol/min/mg). Nonetheless, PLA2 enzymatic activity may not proportionally cor-relate its toxic effect [31]. The exceptionally high activity of PDE thatacts primarily as an enzyme is in agreement with the relatively highPDE content in the venom proteome. Like the venom of the NewWorld coral snakeM. fulvius, it exhibited very low acetylcholinesterase,although the enzyme was not detected in its venom proteome. Ace-tylcholinesterase is generally a minor toxin enzyme in some snakevenoms. A previous study by Tan and Ponnudurai [42] reported itsenzymatic activity in severalMicrurus sp. venoms, although its pres-ence has not been all reported in the venom proteomes of someMicrurus species studied recently [24,25,30,43]. The enzymaticassay in this study also did not detect LAAO activity, consistentwith the absence of the enzyme revealed in the venom proteome.Of note, the fresh and lyophilized venom of this snake appearedcrystal-white (Fig. 1A) much like that of sea snake venom, indicat-ing the negligible amount or the absence of LAAO that usuallygives the yellowish color to venom with its flavin content. On theother hand, the enzymatic assays also indicated that the twominor or negligible enzymes (acetylcholinesterase and LAAO) incoral snake venoms likely have a more important function in cobraand krait venoms, particularly in the latter which are known tohave a much higher level of acetylcholinesterase among varioussnake lineages [44].

3.4. Cytotoxicity of C. b. flaviceps venom

The cytotoxicity of C. b. flaviceps venom on the mouse fibroblast cellline, CRL-2648 was investigated by MTT assay. Fig. 3 and Table 2 showthe cytotoxic effects of C. b. flaviceps venom aswell as three other elapidvenoms. Among the four elapids, the venoms ofN. atra and C. b. flavicepsexhibited dose-dependent cytotoxic activity, with the cobra venombeing significantly more potent than C. b. flaviceps (IC50 of 10.47 ±0.52 μg/mL v.s. 62.14±0.87 μg/mL, p b 0.05). However, therewas no re-markable cytotoxic effect for the venoms of B.multicinctus andM. fulviusagainst the CRL-2648 cell lines. The cytotoxicity of C. b. flaviceps venomis presumably due to the action of the venom cytotoxin and PLA2, eventhough an earlier study [9] suggested thatmaticotoxin A alone, purifiedfromM. bivirgata venom, did not exhibit cytotoxicity on human amnioncells. The relatively weaker cytotoxicity of C. b. flaviceps venomcompared toN. atra venom is perhaps partly due to the difference in cy-totoxin content between the two venoms: venoms of most cobras in-cluding N. atra have a very high cytotoxin content (N30–60%) [28,45,46] compared to that of the venom of C. b. flaviceps specimen used inthis study (approximately 20%). It should be noted that while the NewWorld coral snake venom examined here (M. fulvius) was not cytotoxicto themouse fibroblast cell lines, remarkable venom cytotoxicity for the

Table 3Enzymatic activities and cytotoxicity of the venoms of Calliophis bivirgata flaviceps and three o

Venoms PLA2

(μmol/min/mg)PDE(nmol/min/mg)

LAAO(nmol/mi

C. b. flaviceps 210.8 ± 10.3 37.3 ± 13.1 NAM. fulvius 742.9 ± 10.2 6.1 ± 0.7 1.9 ± 1.1B. multicinctus 790.8 ± 70.0 6.7 ± 0.3 12.2 ± 1.8N. atra 766.9 ± 70.6 22.8 ± 0.8 18.4 ± 2.2

NA: No activity.Abbreviations: PLA, phospholipase A2; PDE, phosphodiesterase; LAAO, L-amino acid oxidase; 5For cytotoxicity assay: The cells (CRL 2648 fibroblast)were treatedwith the various venoms for 4S.D. (n = 3). N.D.: Not detected.

venoms of severalMicrurus species has been reported on human cancercell lines [47].

3.5. Myotoxicity of C. b. flaviceps venom

Myotoxicity was evident in mice experimentally envenomed withthe venom. The serum creatinine kinase (CK) was 8820 ± 818 U/L,compared to the level of 364±72U/L in the control group that receivedsaline injection (p b 0.01). Creatinine kinase is a marker for venommyotoxicity, its elevation implies the occurrence of myolysis ormyonecrosis in snake envenomation [4]. The myotoxic effect of C. b.flaviceps venom is consistent with the observed cytotoxicity of thevenom, presumably induced by the venom cytotoxin and PLA2. Experi-mentally, cytotoxicity with elevated serum creatinine kinase level hasbeen noted in mice envenomed by a few New World coral snakes,such as M. nigrocinctus [48] and Micrurus spixii venom [49], althoughthis is rarely reported in a clinical setting. In envenoming cases, coralsnake bites generally produce a minimal local effect without local ne-crosis, however, myalgia suggestive of myotoxicity has been describedin bite cases of M. nigrocinctus [3] and another Calliophis sp. of OldWorld coral snakes (unpublished). Our findings indicate that muscletenderness and plasma creatinine kinase should be monitored clinicallyin C. b. flaviceps envenomation.

3.6. Venom lethality and neutralization by heterologous antivenoms

The i.v. median lethal dose (LD50) of the venom inmice was 0.70 μg/g(95% C.I.: 0.65–0.82), which is comparable to the value reported byChanhome et al. (0.80 μg/g) [5]. At a challenge lethal dose of 5 LD50,

both the Thai neuro polyvalent antivenom (NPAV) and the Taiwanneurotoxic bivalent antivenom (NBAV) failed to protect the mice.

ther elapids (Micrurus fulvius, Bungarus multicinctus, Naja atra).

n/mg)5′NUC(nmol/min/mg)

ACE(nmol/min/mg)

Cytotoxicity(IC50 μg/mL)

279.1 ± 6.2 4.9 ± 1.2 62.14 ± 0.8789.3 ± 46.7 13.9 ± 5.8 NA

158.3 ± 4.1 1396.7 ± 592.2 NA750.0 ± 91.3 439.7 ± 106.1 10.47 ± 0.52

′NUC, 5′nucleotidase; ACE, acetylcholinesterase.8 h and the cell viabilitywasdetermined byMTT assay. All values are expressed inmean±


The antivenoms were tested as they represent the ideal regionalantivenoms for elapids in Southeast/East Asia, including Naja sp.and Bungarus sp. Nevertheless, at the lower challenge dose of 2.5LD50, NBAV was able to cross-neutralize the lethal effect of thevenom with an ED50 (effective dose at 50% survival rate) of 97.6 μLantivenom, equivalent to a potency of 0.2 mg venom/mL antivenom.The findings indicated the presence of common antigen(s) betweenthe venom of the C. b. flaviceps specimen in this study, and thevenoms of the two Taiwanese elapids (B. multicinctus and N. naja,venoms of which were used to produce the bivalent antivenom).Admittedly, the use of venom from a single C. b. flaviceps specimenoffers limited evidence on the potential benefit of NBAV. Furtherstudy is thus anticipated to elucidate the venom antigenicity andto validate the use of heterologous antivenom in cross-neutralizingthe venom of Calliophis sp.

4. Concluding remarks

This is the first report of the venomics of an OldWorld/oriental coralsnake in correlation to its venom toxinology. C. b. flaviceps venomdisplays a proteomic profile that is predominated with phospholipaseA2 and three-finger toxin (collectively ~60% of total protein abundance),much similar to the venoms ofmost elapids (includingNewWorld coralsnakes of Micrurus sp.). However, it varies distinctly from the venomsof many other elapid snakes (such as Naja, Bungarus, Ophiophagus,Micruru, Hydrophis sp.) by the peculiar absence of post-synaptic neuro-toxins, as examined in this study with the use of nano-LCMS/MS.Although the venom used in this study was sourced from a single spec-imen (withmultiplemilkings), these salient features of highly abundantPLA2 and unique cytotoxin (maticotoxin), along with the absence of α-neurotoxins are in good agreementwith a toxin isolation study reportedin 1991 [9]. The compatible findings also indicate that the principaltoxin profile of this species is likely conserved between Thailand andPeninsular Malaya, although further studies using a more representa-tive venom pool of multiple snakes from the respective areas will benecessary to validate this. Experimentally, the venom proteome cor-relatedwell with its functions on enzymatic, cytotoxic,myotoxic and le-thal activities. Although the bite by this exotic species is uncommon, thecross-neutralization of its lethal effect by the Taiwanese neurotoxicbivalent antivenom is probably of therapeutic significance, pendingfurther clinical validation. On the other hand, the venom proteomeunveiled for this exotic snake provides enrichment to the snake toxindatabases, which may be found useful in drug discovery and optimiza-tion of antivenom formulation in the future.

Transparency document.The Transparency document associated with this article can be

found in the online version.

Acknowledgments

This work was supported by UMHigh Impact Research Grant UM.C/625/1/HIR/MOE/E000040-20001 from the Ministry of EducationMalaysia and UMRG grant number RG 346-15AFR from the Universityof Malaya.

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