1 s2.0 S1874391914003807 Main

J O U RN A L OF P ROT EO M IC S 1 1 0 ( 2 01 4 ) 2 0 9 22 9
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Combined venom gland cDNA sequencing and

venomics of the New Guinea small-eyed snake,
Micropechis ikaheka
Owen Paivaa,1 , Davinia Plab,1 , Christine E. Wrightc,d , Markus Beutlerc,d , Libia Sanzb ,
Jos Mara Gutirreze , David J. Williamsa,d,, Juan J. Calveteb,
a
School of Medicine & Health Sciences, University of Papua New Guinea, Boroko, NCD, Papua New Guinea
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientficas (CSIC), Valencia, Spain
c
Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia
d
Australian Venom Research Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia
e
Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, San Jos, Costa Rica
b
AR TIC LE I N FO
ABS TR ACT
Article history:
The venom arsenal of the New Guinea small-eyed snake, Micropechis ikaheka, was investigated by
Received 1 June 2014
a joint cDNA sequencing and venomics approach. Twenty-seven full-length DNA sequences
Accepted 14 July 2014
encoding novel venom proteins were recovered in this study. Using this cDNA dataset we
Available online 8 August 2014
achieved locus-specific resolution for 19 out of the approximately 50 reverse-phase- and SDSPAGE-separated venom proteins. The venom proteome of M. ikaheka is dominated by at least 29
Keywords:
D49-phospholipase A2 (PLA2) and 14 short and long neurotoxins of the three-finger toxin (3FTx)
Micropechis ikaheka
family. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins.
Snake venomics
Two PIII-metalloproteinase (SVMP) molecules (7.6%), three CRISP isoforms (1.8%), and a single
Venom gland cDNA
Kunitz-type inhibitor, vespryn, 5-nucleotidase, serine proteinase and LAO molecules, none of
Pharmacological effects of
which represents more than 0.7% of the total venom proteome, complete the protein arsenal of
snake venom
M. ikaheka. In concordance with clinical observations, this venom composition points to a central
Three-finger toxin
role for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed by
Phospholipase A2
this species. PLA2 molecules represent the main myotoxic components of M. ikaheka
venom. In addition, the estimated LD50 for mice of the reverse-phase-isolated 3FTx
(0.22 mg/kg) and PLA2 (1.62 mg/kg) enriched fractions, strongly suggests that these two
toxin classes contribute synergistically to venom lethality, with the 3FTxs playing a
dominant role. The high structural and functional conservation exhibited by M. ikaheka
and Australian elapid venoms may underlay the positive clinical outcomes of envenoming
resulting from bites by M. ikaheka that have been documented through the use of bioCSL
polyvalent antivenom.
Correspondence to: D.J. Williams, Charles Campbell Toxinology Centre, School of Medicine & Health Sciences, University of Papua New
Guinea, P.O. Box 168 Konedobu, NCD 125, Papua New Guinea.
Correspondence to: J.J. Calvete, Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain. Tel.: +34 96 339 1778;
fax: +34 96369 0800.
E-mail addresses: david.williams@unimelb.edu.au (D.J. Williams), jcalvete@ibv.csic.es (J.J. Calvete).
1
These authors have contributed equally to this work and both should be considered first author.
http://dx.doi.org/10.1016/j.jprot.2014.07.019
1874-3919/ 2014 Elsevier B.V. All rights reserved.
210
Biological significance
The poorly understood venom proteome of the New Guinea small-eyed snake, Micropechis
ikaheka, a large and powerfully built elapid endemic to Papua New Guinea and Indonesian
West Papua province, was investigated through a combined venomics and venom gland
transcriptomics approach. Although M. ikaheka accounts for only a small proportion of
snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by
this snake. Major effects of envenomings include life-threatening post-synaptic neuromuscular blockade resulting in respiratory paralysis, myotoxicity, severe bleeding, hypotension
and cardiovascular abnormalities. We have investigated the contribution of 3FTxs and PLA
molecules in venom lethality, myotoxicity, and cardiovascular function. Our work provides2
important correlations between venom composition and its pharmacological activity. In
conjunction with the antivenomics work reported in the companion paper, our study may
contribute to improve treatment outcomes for snakebite victims of M. ikaheka.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Molecular evidence indicates a rapid late-Miocene radiation of
Australasian venomous snakes [1] that began 1215 million
years before present (MYBP). Over this time the AustraloMelanesian Hydrophiinae radiation has generated over 160
species recognized in ~ 50 genera of terrestrial and marine
elapids [2]. However, research into Australasian elapid venoms
has mainly focused on the genera of greatest clinical significance, including Acanthophis, Hoplocephalus, Notechis, Oxyuranus,
Pseudechis, Pseudonaja and Tropidechis.
The New Guinea small-eyed snake or Ikaheka snake,
Micropechis ikaheka [3] (Fig. 1), is a basal endemic species that
has variously been considered a sister taxon to the Australian
Pseudechis, Pseudonaja, Oxyuranus and Demansia and a sister
taxon to the Laticauda [4]. The latter study estimated the appearance of Micropechis at 11.5 MYBP and considered it basal
to all other hydrophiines. This species is large (average length,
1.21.7 m; maximum length, 2.12.3 m), heavyset and extremely excitable when disturbed. It is restricted entirely to Papua
New Guinea and Indonesian Papua province on the islands of
New Guinea where it is widely distributed throughout the
mainland and several of the offshore islands along the Madang
and Sepik coasts, including Karkar Island and Manam Island
(Madang province), Mushu, Kairiru, Walis, Tarawai, Tumleo and
other small islets (Sanduan & East Sepik provinces), and the Aru
Islands to the west [5] (Fig. 1). Wild-caught snakes sometimes
regurgitate partly digested snakes, and in captivity the species
shows a preference for snakes and lizards, but can occasionally
be conditioned to accept rodents. It has been said to prey on a
Fig. 1 The New Guinea smalleyed snake. Distribution of the New Guinea small-eyed snake (Micropechis ikaheka) in Papua
New Guinea and Irian Jaya (West Papua). Picture: M. ikaheka from Kaviak Plantation, Karkar Island, Madang province (Map &
photo: David Williams).
wide variety of small ground dwelling animals, particularly

lizards, frogs, small rodents and other snakes, including its own
species [6,7]. Specimens have been collected in a variety of
habitats, at elevations from sea level to over 1500 m, ranging
from the hot, humid lowland floodplains with grasslands,
rainforests, swamps and marshes through wet monsoonal
forests into cool, moist highland montane forests. Favoured
manmade microhabitats include copra, cocoa and oil palm
plantations, as well as village gardens.
This fossorial species is shy and inoffensive unless disturbed, and while seldom seen by day, it is often encountered at
night when it emerges from shelter to forage for food, and can
be very defensive if touched, stepped on, or able to sense movement nearby. It is a medically important species that has been
implicated in human fatalities in both PNG and Indonesian
Papua [8]. One of its favoured habitats is old coconut husk piles
in and around copra plantations. M. ikaheka shelter in these
piles of decaying husks during the day [9] and bites typically
occur when plantation workers disturb snakes entering or
leaving these refugia, as people arrive for work, or are walking
home in the mornings or late afternoons. The New Guinea
small-eyed snake accounts for only a small proportion of
snakebites on the mainland, whereas approximately 40% of
snakebites on Karkar Island could be attributed to bites by M.
ikaheka [8]. The subcutaneous lethal dose of M. ikaheka venom per
25 g mouse has been reported to be 3.4 g [10]. This venom has
strong neurotoxic and myotoxic activities, as well as anticoagulant, platelet-aggregation-inhibiting, and insulin-secretionstimulating effects [1113]. In vivo studies have focused on the
cardiovascular and haematological effects of this venom. It has
been reported to cause pulmonary hypertension and depression
of cardiac output accompanied by increased free-haemoglobin
plasma levels (more than 50-fold) [11]. Envenoming by M. ikaheka
is known to provoke life-threatening post-synaptic neuromuscular blockade resulting in respiratory paralysis, frequently accompanied by myotoxicity resulting in rhabdomyolysis and
myoglobinuria, and sometimes coagulopathy and spontaneous
severe and life-threatening bleeding, hypotension and cardiovascular abnormalities [8,11].
The venom composition of M. ikaheka is poorly understood,
and has been studied by only a handful of researchers. Four
groups of IB PLA2s (MiPLA-1, MiPLA-2 [PDB code 1PWO], MiPLA-3
[1OZY], and MiPLA-4 [1P7O]), which exhibited different levels of
myotoxic and anticoagulant activities, have been isolated from
M. ikaheka venom [1417], and the effects of venom on skeletal
muscle and venom-induced contraction of smooth muscle have
been shown to be dependent on PLA2 activity [18]. Some toxin
molecules potentially involved in the clinical effects of M. ikaheka
venom have been isolated and characterized structurally and
functionally. Thus, blood clotting alterations have been attributed to the action of mikarin, a single-chain Ca2+-independent
metalloproteinase, and group I prothrombin activator of 47 kDa
isolated from the venom of M. ikaheka [19] (UniProtKB/Swiss-Prot
accession code P0DJ43) and to the anticoagulant activity of some
PLA2 [15,16]. Several short- and long-chain post-synaptic neurotoxins of 68 kDa have also been isolated from M. ikaheka venom.
One of these, mikatoxin, has been found to produce neuromuscular paralysis through irreversible nicotinic AChR antagonism
[20,21]. However, the overall composition and relative abundance of the toxins of M. ikaheka venom, necessary figures to
211
establish structurefunction correlations, have not been reported yet. To fill this gap, we have conducted a combined study of
venom gland cDNA sequencing and venom proteomics of M.
ikaheka, together with the analysis of the toxin profile of venom
fractions. In conjunction with the functional venomics and
antivenomics work reported in the companion paper [22], our
work provides important correlations between the composition
of the venom and its pharmacological activity that might
improve treatment outcomes for snakebite victims.
2. Materials and methods

2.1. Ethical approval
Experiments involving mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals
(CICUA) of the University of Costa Rica, and adhere to the
International Guiding Principles for Biomedical Research
Involving Animals of the Council of International Organizations of Medical Sciences (CIOMS). For studies undertaken
with rat tissues, the University of Melbourne Animal Ethics
Committee approved experiments in accordance with the
Australian Code for the Care and Use of Animals for Scientific
Purposes (8th edition, 2013, National Health and Medical
Research Council, Canberra). Venomous snakes maintained
at the University of Papua New Guinea in Port Moresby, PNG,
are collected, kept and used in experiments in accordance
with approvals from the UPNG SMHS Ethics Committee, and the
PNG Government's Department of Environment & Conservation.
2.2. Venom and venom glands

A pool of venom from M. ikaheka was obtained from adult specimens maintained alive at the Charles Campbell Toxinology
Centre's Serpentarium, at the University of Papua New Guinea
(UPNG) School of Medicine & Health Sciences. The venom was
frozen at 80 C, lyophilized later, placed in 5 mL, capped glass
vials and stored at 80 C prior to use. A M. ikaheka specimen
was euthanized and the two venom glands were carefully
dissected out, immediately placed in RNAse later (Bio-Rad) and
stored at 80 C.
2.3. RNA isolation and complementary DNA (cDNA) template

synthesis
The snap frozen venom glands were later partially thawed,
weighed, placed in a 5 mL test tube and homogenized with a
polytron prior to RNA isolation. RNA isolation was carried out
by the Tri Reagent method, using 1 mL of Tri reagent solution
(Sigma-Aldrich, Castle Hill, Australia) per 50100 mg venom
gland tissue and following the manufacturer's protocols. To
confirm successful isolation of RNA from M. ikaheka venom
glands, electrophoresis of a small quantity of the RNA pellet
sample solution was carried out on a 1% Tris-Acetate EDTA
(TAE) agarose gel run at 70 V for about 45 min or until the
tracking dye reached halfway through the gel. Following
confirmation of RNA isolation on TAE agarose gel, RNA was
precipitated in isopropanol, washed with 70% ethanol and
finally resuspended in DEPC-treated water. First strand cDNA
212
was then synthesized from 1 g of total RNA with an oligo(dT)

1218 primer via reverse transcription with 200 U of Superscript II RNAse H Reverse Transcriptase (Invitrogen, Mt.
Waverly, Australia). Ethanol was then precipitated from the
sample and the template cDNA was resuspended in sterile
water and stored at 20 C until used.
2.4. Amplification of toxin sequences from M. ikaheka venom

gland cDNA
out on 1% TAE agarose gels and single gel bands positive for
cDNAs of interest were excised. Each excised gel band was then
purified using the QIAEX II gel extraction kit (QIAGEN, Hilden,
Germany) and the product cloned using the pGEM-T vector
system (Promega, Madison, Wisconsin) in either Top10 or D5H
Escherichia coli cloning systems. Multiple clones were then sequenced with an ABI Big Dye Terminal cycle sequence ready
reaction kit (Perkin-Elmer, Norwalk, USA).
2.5. Reverse-phase and SDS-PAGE analyses

Specific forward (F) and reverse (R) primers designed from
transcripts encoding sequences previously identified from
screens of cDNA libraries prepared from venom glands of
various Australian elapid snakes [2327] were: CRISP (F:
5-GGA GTT ACA CTG GGG CTC-3; R: 5-ACT GAA TGG GAG
ATC AGC-3); 5-nucleotidase (5-NUC) (F: 5-ATG ACA ACT
TCT TGG AGT G-3; R: 5-TTA TTC TTC TTC TTC CTC-3); shortchain neurotoxins (SNTXs) (the single-gene-specific primer
5-GGT CGT CGA TGG ATG AGA GCA AAA CTC-3); long-chain
neurotoxins (LNTXs) (F: 5-ATG AAA ACT CTG CTG CTG
ACC-3; R: 5-GTC GAG ATG TCA AAG ACG CA-3); serine
protease (SP) (F: 5-ATG GCT CCT CAA CTA CTC CTC TG-3; R:
5-TTA GAG CCG ACC AGT GCT TGA CTC-3); PLA2 (F: 5-TGC
TTG CAG CTT CAC CAC TGA C-3; R: 5-TCC TCG CGC TGA AGC
CTC TCA AA-3); snake venom metalloproteinase (SVMP) (F:
5-ATG GCT CCT CAA CTA CTC CTC TG-3; R: 5-TTA GAG CCG
ACC AGT GCT TGA CTC-3); and vespryn (VES) (F: 5-ATG CTC
CTG TTC ACA CTA TGC T-3; R: 5-TTA AAG ATT TGT GAG
TGA AAC ACC T-3). PCR amplifications were carried out from
the M. ikaheka cDNA template using AmpliTaq Gold Taq
polymerase (Applied Biosystems, Foster City, USA), to identify
full-length coding sequences. PCR reaction mixtures were made
up to a final volume of 25 L containing approximately 200 ng of
cDNA template, 25 pmol of the forward and reverse primers,
1 unit of AmpliTaq Gold (Applied Biosystems, Foster City, CA) in
1 buffer, 2.25 mM MgCl2, and 200 M deoxyribonucleoside
triphosphates (dNTPs). The PCR amplification (cycling) conditions for each protein of interest were carried out separately on a
2720 Thermocycler (Applied Biosystems) according to methods
by St. Pierre and colleagues (2005a, 2005b, 2007a, and 2007b): for
CRISP, the reaction was thermocycled at 95 C for 8 min
followed by 20 cycles of denaturation (95 C for 20 s), annealing
(51 C for 20 s) and extension (72 C for 30 s), and 15 cycles of
95 C for 20 s, 54 C for 20 s, and 72 C for 60 s, and a final
extension step at 72 C for 3 min; for 5-NUC, 95 C for 60 s
followed by 30 cycles of 95 C for 20 s, 52 C for 20 s, 72 C for
150 s, and a final extension at 72 C for 3 min; for SNTX and
LNTX, 95 C for 8 min followed by 30 cycles of 95 C for 20 s,
59 C for 20 s, 72 C for 40 s, and a final extension at 72 C for
3 min; for SP, denaturation at 95 C for 8.5 min followed by
35 cycles of 95 C for 30 s, 52 C for 60 s, 72 C for 3 min, and a
final extension step at 72 C for 12 min; for PLA2, 95 C for 8 min
followed by 30 cycles of 95 C for 20 s, 61 C for 20 s, 72 C for
40 s, and a final extension step at 72 C for 12 min; for SVMP,
95 C for 8.5 min followed by 35 cycles of 95 C for 30 s, 52 C for
60 s, 72 C for 3 min, a final extension step at 72 C for 12 min;
and for VES, 95 C for 1 min followed by 30 cycles of 95 C for
20 s, 52 C for 20 s, 72 C for 2.5 min, and a final extension step
at 72 C for 3 min. All PCR reactions were run with appropriate
no template controls. Following PCR, the PCR products were run
For venomics characterisation, 22.5 mg of crude, lyophilized

venom proteins was dissolved in 200 L of water containing
0.1% trifluoroacetic acid (TFA), centrifuged to remove debris,
and separated by reverse-phase HPLC using an ETTANTM LC
HPLC system (Amersham Biosciences) and a Lichrosphere
RP100 C18 column (250 4 mm, 5 m particle size) eluted at
1 mL/min with a linear gradient of 0.1% TFA in water (solution
A) and acetonitrile (solution B) (5% B for 10 min, followed by 5
15% B over 20 min, 1545% B over 120 min, and 4570% B over
20 min). Protein detection was carried out at 214 nm with a
reference wavelength of 400 nm. Fractions were collected manually, dried in a vacuum centrifuge (Savant), redissolved in
water, and submitted to SDS-PAGE analysis in 12% polyacrylamide gels, under reducing conditions. Gels were stained with
Coomassie blue R-250.
2.6. Characterization of the venom proteome

Protein bands of interest were excised from a Coomassie
Brilliant Blue-stained SDS-PAGE gel and subjected to in-gel
reduction (10 mM dithiothreitol) and alkylation (50 mM
iodoacetamide), followed by overnight sequencing-grade
trypsin digestion (66 ng/L in 25 mM ammonium bicarbonate,
10% acetonitrile; 0.25 g/sample) in an automated processor
(using a Genomics Solution ProGest Protein Digestion Workstation) following the manufacturer's instructions. Tryptic digests
were dried in a SpeedVac, redissolved in 15 L of 0.1% formic
acid in water, and submitted to LC-MS/MS. To this end, tryptic
peptides were separated by nano-Acquity UltraPerformance
LC (UPLC) using BEH130 C18 (100 m 100 mm, 1.7 m
particle size) column in-line with a Waters SYNAPT G2 High
Definition Mass Spectrometry System. The flow rate was set to
0.6 L/min and the column was developed with a linear gradient
of 0.1% formic acid in water (solution A) and 0.1% formic acid in
acetonitrile (solution B), isocratically 1% B for 1 min, followed by
112% B for 1 min, 1240% B for 15 min, and 4085% B for 2 min.
Doubly and triply charged ions were selected for collisioninduced dissociation (CID) MS/MS. Fragmentation spectra were
interpreted (a) manually (de novo sequencing), (b) using the
on-line form of the MASCOT program at http://www.
matrixscience.com against the NCBI non-redundant database, and (c) processed in Waters Corporation's ProteinLynx
Global SERVER 2013 version 2.5.2. (with Expression version
2.0) against the species-specific venom gland cDNA-derived
toxin sequences. MS/MS mass tolerance was set to 0.6 Da.
Carbamidomethyl cysteine and oxidation of methionine were
selected as fixed and variable modifications, respectively.
The relative abundances (expressed as percentage of the
total venom proteins) of the different protein families were
calculated from the relationship of the sum of the areas of the

reverse-phase chromatographic peaks containing proteins
from the same family to the total area of venom protein
peaks in the reverse-phase chromatogram [28,29].
2.7. Fractionation of M. ikaheka venom into 3FTx- and PLA2rich fractions

Two mg-aliquots of M. ikaheka whole venom were fractionated by reverse-phase HPLC using a Teknokroma Europa C18
(0.4 cm 25 cm, 5 m particle size, 300 pore size) column
and an Agilent LC 1100 High Pressure Gradient System
equipped with DAD detector and micro-Auto-sampler. The
flow rate was set to 1 mL/min and the column was developed
with a linear gradient of 0.1% TFA in water (solution A) and
acetonitrile (solution B), isocratically (5% B) for 5 min, followed by 525% B for 10 min, 2545% B for 60 min, and 4570% for
10 min. Protein detection was carried out at 214 nm with a
reference wavelength of 400 nm. Fractions eluting between 8
and 12 min and between 13 and 25 min, enriched in 3FTxs
and D49-PLA2 molecules, respectively, and were separately
pooled and used to correlate venom lethality and myotoxic
activity to particular toxin classes. The toxin profile of the 3FTxand D49-PLA2-enriched fractions was checked by analytical
reverse-phase HPLC using a Discovery BIO Wide Pore C18
(15 cm 2.1 mm, 3 m particle size, 300 pore size) column
and an Agilent LC 1100 High Pressure Gradient System equipped
with DAD detector. The flow rate was set to 0.4 mL/min and the
column was developed with a linear gradient of 0.1% TFA in
water (solution A) and 0.1% TFA in acetonitrile (solution B):
isocratically (5% B) for 1 min, followed by 525% B for 5 min, 25
45% B for 35 min, and 4570% for 5 min. Protein detection was
carried out at 214 nm with a reference wavelength of 400 nm.
2.8. Estimation of the median lethal dose (LD50) of venom

fractions
Various doses of the 3FTx or the PLA2 fractions, dissolved in a
total volume of 100 L of 0.14 M NaCl, 0.04 M phosphate, pH 7.2
(PBS) were injected into groups of 4 CD-1 mice (1618 g) by the
intravenous route. Deaths occurring during 24 h were recorded,
and the Median Lethal Dose (LD50) was estimated by probit
analysis.
2.9. Myotoxic activity of the PLA2 fraction

Groups of four CD-1 mice (1820 g) were injected, intramuscularly in the right gastrocnemius, with two doses of the
PLA2 fraction (30 or 60 g), dissolved in 50 L of PBS. Control
mice were injected with 50 L of PBS alone. Three hours after
injection, a blood sample was collected into heparinised
microcapillary tubes. The creatine kinase (CK) activity of plasma
was determined using a commercial kit (CK LIQUI-UV, Stanbio
Lab., Texas, USA). Activity was expressed in units/L [30].
2.10. Inhibition of PLA2, lethal and myotoxic activities by pbromophenacyl bromide (pBPB)
To assess the role of PLA2 activity in the lethal and myotoxic
effects of this venom, 1 mL of a solution of venom (3 mg/mL
213
in 0.1 M Tris, 0.7 M EDTA, pH 8.0, buffer) was incubated with

150 L of a solution of pBPB (3 mg/mL ethanol). Incubation
was carried out at room temperature for 24 h. Controls
included venom solution incubated with ethanol without
pBPB, and pBPB solution incubated with buffer Tris. Abrogation of PLA2 activity was assessed by incubating various
amounts of control or inactivated venom, for 20 min at 37 C,
with chicken egg yolk suspension in a 0.1 M Tris, 10 mM
CaCl2, 1% Triton X-100, pH 8.5, buffer. Then, fatty acids were
extracted and titrated with 0.018 M NaOH [31]. The samples
were also tested for lethal and myotoxic activities, as described
above. In the case of myotoxicity, in addition to plasma CK
activity quantification, mice were euthanized by CO2 inhalation
and samples of injected gastrocnemius muscles were dissected
out and immersed in 10% formalin solution for fixation. After
routine processing for embedding in paraffin, 58 m sections
were obtained and stained with haematoxylineosin for microscopic observation [30].
2.11. Pharmacological effects in rat isolated cardiac and

vascular tissues
Male SpragueDawley rats (339 5 g) were exposed to 5%
isoflurane (Baxter Healthcare Pty. Ltd.; NSW, Australia) and
95% O2. When deeply anaesthetised, they were killed by rapid
heart excision. Tissues were immediately placed in Krebs'
physiological salt solution (PSS) and bubbled with carbogen
(O2 95%; CO2 5%) at pH 7.4. PSS is composed as follows (in
mmol/L): NaCl 119; KCl 4.69; MgSO47H2O 1.17; KH2PO4 1.18;
glucose 11 (5.5 for mesenteric artery); NaHCO3 25; CaCl26H2O
2.5; and EDTA 0.026.
2.12. Mesenteric arteries

Third order mesenteric arterial branch ring segments (2 mm
length; 327 5 m i.d.) were dissected from the mesenteric
fan. Ring segments were set up in MulvanyHalpern isometric myograph baths (620 M; Danish Myo Technology; Aarhus,
Denmark). Force of contraction was measured via a PowerLab 4/
30 amplifier and computer (Chart v5.5.6 for Mac; ADInstruments
Pty. Ltd.; Bella Vista, NSW, Australia). Vessel segments were
normalized to 100 mm Hg and adjusted to a passive tension
equivalent to a transmural pressure of 90 mm Hg [32]. Artery
viability was determined by potassium depolarisation with
depolarising isotonic high K+-containing physiological saline
solution (KPSS), in which Na+ in Krebs' solution was replaced by
K+ ([K+]KPSS = 124 mM), and noradrenaline (10 M). Tissues were
then washed with physiological saline solution (PSS) and
allowed to return to their resting contractile tone. Responses
were expressed as % of KPSS maximum contraction.
For assessment of venom-induced relaxation, arteries were
pre-contracted with endothelin-1 (ET-1 tone, 13 nM) to 7080%
of KPSS maximum contraction. Venom was added in cumulative
half-log10 increments (starting at 0.9 g/mL) allowing time for
each response to reach plateau between additions. Tissues were
pre-treated for 30 min with vehicle (PSS), indomethacin (3 M),
atropine (3 M), N-nitro-L-arginine methyl ester (L-NAME;
100 M) or glibenclamide (3 M) prior to addition of venom.
For assessment of intramural sympathetic nerve-mediated
responses, arteries were subjected to a selective 2-adrenoceptor
214
inhibition protocol to enhance the contractile responses to

electrical field stimulation, i.e. to prevent auto-inhibition of
neurotransmitter release by presynaptic 2-adrenoceptors
[33]. The vessels were electrically stimulated by square wave
electrical field stimulation via platinum electrodes connected
to a low-output-resistance stimulator (Grass SD9/S88, Quincy, MA, USA). A control stimulation episode, without venom,
consisting of 3 trains (3 s burst, 0.25 ms pulse width; 30 V) at 3
different frequencies 6.25, 12.5 and 25 Hz was performed.
M. ikaheka venom was then added (90, 270, 360 and 450 g/mL).
Each venom concentration was incubated for 10 min followed
by a stimulation episode. Finally, the venom was washed out (3
times within 10 min) and a final stimulation episode was
applied. Contractions caused by electrical stimulation were
expressed as % of KPSS maximum contraction.
2.13. Rat right and left atria

The beating heart was placed in an acrylic bottom Petri dish
filled with cold PSS (full glucose) and bubbled with carbogen.
The left and right atria were then separated and pierced by
two stainless steel hooks (made from 30 G needles) at the
opposing ends of each atrium and connected to an acrylic
organ bath leg and an isometric force transducer (Grass
Instruments FT03C) in 20 mL organ baths filled with 15 mL
PSS and continuously bubbled with carbogen at 37 C. Force
transducers were connected to a 6-channel amplifier (Octal
Bridge Amp, ADInstruments). The input was recorded with
Chart (v5.5.6) for Mac.
Both atria were passively stretched to a force of 1 g (9.81 mN).
Electrical field stimulation at 1 Hz, for 0.3 ms at 150% threshold
voltage (minimum voltage that causes contraction) via two
platinum field electrodes connected to an electrical stimulator
(S88, Grass Instruments) was applied to stimulate the left
atrium, whereas the right atrium beats spontaneously. After a
60 min resting period with washouts every 30 min a stable
baseline was established. Isoprenaline (0.1 M) was applied to
check atrial viability. Venom was then added in cumulative
half-log10 increments (starting at 0.009 g/mL) allowing time for
the response to plateau between additions without or with
30 min pre-treatment with the -adrenoceptor antagonist
propranolol (1 M) or calcitonin gene-related peptide (CGRP)
antagonist CGRP837 (1 M). To assess the role of phospholipase
A2 activity, venom was treated with p-bromophenacyl bromide
(pBPB) using the method of Daz-Oreiro and Gutirrez [34].
Separate aliquots of venom were kept overnight at room temperature to serve as controls. The right atrial rate was expressed
in beats/min, whereas the left atrial force was shown as change
from the resting force (g).
2.14. Statistical analysis

Data are expressed as the mean 1 standard error of the
mean (SEM) of n experiments. For mesenteric arteries, the
initial tone was compared between venom treatment groups
by one-way ANOVA. Maximum relaxation to venom in each
pre-treatment group was compared to the venom alone
group using one-way ANOVA, with Dunnett's post hoc test
(Prism 6; GraphPad Software, San Diego, CA, USA). Relaxation
within each group was analysed with repeated measures
(RM) ANOVA; a Dunnett post hoc test was used to compare

each concentration with baseline. The pre- and post-treatment
right atrial rates were compared within groups by Student's
paired t-test; post-treatment baselines were compared between
groups using one-way ANOVA, with Dunnett's post hoc test. The
sigmoidal concentrationresponse curves were fitted using
Prism 6 for each individual experiment (where applicable). The
EC50 (the concentration required to cause 50% of the maximum
response) and maximum responses of each treatment group
were compared to the control group (venom alone) by one-way
ANOVA, with Dunnett's post hoc test. Average SEM within
tissues was calculated by RM ANOVA using the pooled estimate
of error from the residual mean square as [error mean square/
number of tissues]0.5 after subtracting the sums of squares
between tissues and between concentrations from the total
sum of squares for each treatment [35]. A p value < 0.05 was
considered significant.
3. Results and discussion

3.1. Novel full-length sequences of M. ikaheka venom gland
cDNA clones
Twenty-seven full-length DNA sequences encoding novel
venom proteins were identified from the M. ikaheka venom
gland cDNA library. The translated full-length amino acid
sequences include three cysteine-rich secretory proteins
(CRISPs; AHZ0882022: 238 residues), one 5-nucleotidase (5
NUC; AHZ08799: 559 amino acids), two serine proteinases
(SPs; AHZ0880001: 242 amino acids each), three short
neurotoxins of 62 residues (SNTXs; AHZ0881618), three
long neurotoxins (LNTXs; AHZ0882325: 8384 residues),
twelve D49-phospholipase A2 (PLA2) molecules (AHZ08804
12, AHZ0881415: 124 amino acid residues; AHZ08813: 119
residues), a PIII snake venom metalloprotease [PIII-SVMP;
AHZ08819: 613 residues] and two vespryn isoforms (AHZ08802
03: 218 amino acids) differing in just an amino acid position
within the propeptide sequence.
The primers used to generate the M. ikaheka venom gland
cDNA library were designed by other workers [2327] and are
based on sequences in the venoms of Australian elapid snakes.
These primers were chosen because they have been previously
used successfully against non-target Australo-Papuan elapid
templates. For example, the serine protease primers were
designed by St Pierre et al. [25] from a Pseudonaja textilis Factor
X-like protease (AY631238), where the forward primer matches
positions 131153 of the target template and any unintended
templates, and the reverse primer matches positions 15221499
of the target template and positions 15341511 on potential
unintended templates. The products produced by the primers
span a conserved calcium-binding EGF-like domain containing
the Ca2+ binding sites, another EGF-like Factor Xa inhibitory site,
as well as cleavage sites and part of the active sites in the
trypsin-like serine protease domain. The deduced protein sequence of the M. ikaheka serine proteases AHZ08800 and
AHZ08801 has greatest similarity (90%) to A8QL56, - and
-fibrinogenase (OhS1) from the Asian king cobra (Ophiophagus
hannah) venom and to Q5MCS0, a fibrinogenolytic serine
protease known as harobin from spine-bellied sea snake
(Hydrophis hardwickii) venom. Similarly while PLA2 primers

were designed by St Pierre et al. 2005 [23] from an Oxyuranus
scutellatus PLA2 sequence, the PLA2 sequenced in this current
study from an M. ikaheka cDNA library has greatest identity
not to PLA2 from O. scutellatus, but to 1OZY and 1PWO both
known PLA2s from M. ikaheka. A novel PLA2 (AHZ08813) generated
from the same primers had 80% identity and 86% similarity to
P00609 (toxin II-5) from Notechis scutatus venom, rather than an O.
scutellatus PLA2. We feel these examples underscore the fact that
the primer source species has not influenced the results reported
in the current study.
A 238 amino residue M. ikaheka CRISP [AHZ08822] comprised of a 19 residue propeptide and a 219 mature protein is
identical to kaouthin-2 [P84808], a CRISP from Asian monocellate
cobra (Naja kaouthia) venom [36]. The other two CRISP sequences
[AHZ08820 and AHZ08821] differed from each other only in a
valine for alanine substitution at position 2 in the mature peptide.
The closest (8789% identity) reported homologs of these CRISP
molecules are ETE62137 (O. hannah), ACE73577 and ACE73578
(Bungarus candidus), and kaouthin-2 [P84808] (N. kaouthia).
The full-length protein sequence of a 5-nucleotidase from
M. ikaheka [AHZ08799] exhibits 99% identity to the venom
gland 5-nucleotidase from the black whip snake, Demansia
vestigiata [ABK63558], and 95% identity to a cytosolic purine
5-nucleotidase [XP_003218558] from the American anole lizard
(Anolis carolinensis), but only 2629% identity with three partial
5-nucleotidase sequences [ETE69544, ETE58819, ETE67404] from
the venom of the king cobra (O. hannah).
The two highly homologous serine proteinases [AHZ08800
01] show highest similarity (90%) to the - and -fibrinogenase
OhS1 from the venom of king cobra (O. hannah) [A8QL56], and
the fibrinogenolytic serine protease harobin from the spinebellied sea snake (H. hardwickii) [Q5MCS0].
The cDNA-encoded full-length SNTXs and LNTXs share an
identical 21-residue propeptide sequence (1MKTLLLTLVVVTIVC
LDLGYT21). The three short neurotoxins [AHZ0881618] show
7071% amino acid sequence identity (7879% similarity) to
three Pseudechis post-synaptic 3FTxs (A8HDJ4 from the redbellied black snake, Pseudechis porphyriacus; BAN63794 and
A8HDJ3 from the mulga snake, Pseudechis australis), and to the
post-synaptic short neurotoxin II [P10457] from the yellowipped sea krait, Laticauda colubrina. On the other hand, two of the
M. ikaheka long neurotoxins [AHZ08823 and AHZ08824] exhibit
highest protein sequence identity (8285%) to the post-synaptic
-elapitoxin-Nss2a [P01384] from the venom of the Australian
tiger snake N. scutatus, while the third long neurotoxin [AHZ
08825] amino acid sequence exhibits 79% identity to the postsynaptic long neurotoxin ABX58167 from the Australian pygmy
copperhead, Austrelaps labialis.
PLA2 toxins are an important component of the venom of
many Australian elapid snakes. Twelve PLA2 clones [AHZ08804
15] were amplified and sequenced. The translated mature
sequences of AHZ0880412 and AHZ0881415 comprise 124
amino acid residues and display high identity to previously
reported M. ikaheka venom PLA2s [14,15,17]. Thus, AHZ08808,
AHZ08804, AHZ08809, AHZ08807, AHZ08806, and AHZ08810 are,
respectively, 100%, 99%, 99%, 90%, 89%, and 88% identical
to MiPLA3 [1OZY], while AHZ08812, AHZ08815, AHZ08805,
AHZ08811 and AHZ08814 share 94%, 93%, 90%, 88% and 88%
amino acid sequence identity with MiPLA2 [1PWO]. MiPLA-J
215
[AHZ08813] represents a new acidic PLA2 composed of 119

amino acid residues that exhibit greatest similarity (80%
identity; 86% similarity) to a basic 119 amino acid residue
neurotoxic PLA2 (toxin II-5) from N. scutatus venom [P00609]
[37,38].
The vespryn (Venom PRY-SPRY domain-containing proteins)
precursor isoform sequences, AHZ08802 and AHZ08803, display
high homology (>90% identity) to a number of vespryn molecules
from Australian elapid snakes, including Cryptophis nigrescens
[ABW74872 and ABW74873], P. porphyriacus [ABW74875], Drysdalia
coronoides [AEH95532-34], P. australis [ABW74876], N. scutatus
[ABW74866], D. vestigiata [ABK63561], and others.
We obtained the complete amino acid sequence of a PIIISVMP [AHZ08819] which has 84% similarity to microlepidotease1 from Oxyuranus microlepidotus [ABQ01137], textilease-1 from P.
textilis [ABQ01140], and scutellatease-1 from O. scutellatus
[ABQ01136]. Other PIII-SVMPs with high similarity included
carinatease-1 (83%) from Tropidechis carinatus [ABQ01132],
scutatease-1 (82%) from N. scutatus [ABQ01138], australease-1
[81%] from P. australis [ABQ01134] and porphyriacase-1 [80%]
from Pseudechis porphyriacus [ABQ01133]. The PIII-SVMP [AHZ
08819] sequence also exhibits sequence similarity with peptide
sequences of the prothrombin activator, Mikarin, isolated from
M. ikaheka venom [14] (Fig. 2).
3.2. The venom proteome of M. ikaheka

The venom of M. ikaheka was separated by RP-HPLC into 30
fractions (Fig. 3A). Each chromatographic fraction was analysed
by SDS-polyacrylamide gel electrophoresis (Fig. 3A, insert) and
electrospray ionization (ESI) mass spectrometry (Table 1).
Protein bands were excised from SDS-polyacrylamide gel and
submitted to venomics analysis [28,29]. Production spectra were
interpreted manually or processed in MASCOT or ProteoLinx
against the NCBI non-redundant database and the speciesspecific venom gland cDNA-derived toxin sequences. The data,
listed in Table 1, identified ~50 protein species belonging to nine
different snake venom protein families, whose relative abundances are displayed in Fig. 3B.
The venom proteome is dominated by a diversity of D49PLA2 and 3FTx molecules. These protein classes represent,
respectively, 80% and 9.2% of the total venom proteins. We
detected 29 PLA2s and 14 3FTx bands, including unique
peptide evidence for eight PLA2 [AHZ08804, 05, 07, 0913] and
five 3FTx [AHZ0881618, 2425] venom gland amplified cDNAs
(Table 1). Although peptide ions matching AHZ08806 and
AHZ08808 were sequenced, we were unable to unambiguously
identify these proteins because the MS/MS-derived peptide
sequences did not bear the resolution capable of distinguishing
amongst individual isoforms.
PIII-SVMP fragments with identity to a novel SVMP [AHZ
08819] and the partial sequence of a PIII-SVMP prothrombin
activator Mikarin [P0DJ43] accounted for 7.6% of the total
venom proteome. Most tryptic peptides sequences gathered
from the PIII-SVMP molecules eluted in RP-HPLC peaks 24, 25,
26, 27 and 30 are included in both Mikarin and AHZ08819
(Fig. 2). However, two of them, m/z 736.7(2+) EAQCDSGECCEK
and 577.7(2+) CGDGMVCSNR are only present in AHZ08819
(Fig. 2), indicating that this novel PIII-SVMP, rather than
Mikarin, may represent the major PIII-SVMP of the M. ikaheka
216
Fig. 2 Alignment of the partial amino acid sequences of Mikarin [P0DJ43] and the full-length sequence of AHZ08819 (this
work). Non-identical positions are highlighted in red, and internal tryptic peptide sequenced by MS/MS that match the
cDNA-deduced amino acid sequence of AHZ08819 are shown in boldface and underlined. MS/MS-derived peptide sequences
that correspond neither to P0DJ43 nor to AHZ08819 are displayed in lower case.
venom sampled. In addition, minor tryptic peptide ions sequenced from the 52 kDa band of fractions 2530 exhibited
similarity to other elapid SVMPs (Table 1), suggesting that, in
addition to Mikarin and AHZ08819, M. ikaheka venom contains
uncharacterized PIII-SVMP(s).
Three CRISP isoforms, and single Kunitz-type inhibitor,
vespryns, 5-nucleotidase, serine proteinases, and L-amino
acid oxidase (LAAO) molecules, complete the protein arsenal
of M. ikaheka venom (Table 1, Fig. 3B). None of these toxin
families is more than 1.8% of the total venom proteome of M.
ikaheka. Except for the LAAO molecule, the fragments of
which can be assembled into a partial sequence with 40%
identity to LAAO from O. scutellatus [Q4JHE3] and N. scutatus
[Q4JHE2] and 39% to LAAO from Bungarus fasciatus [A8QL52]
and B. multicinctus [A8QL51], and a novel BPTI/Kunitz-type
inhibitor with a sequence PADTGPCKFPAFYNPVQR that had
76% identity to several Kunitz-type inhibitors from Austrelaps
superbus [B5KL3839] Drysdalia coronoides [ACR7849798 ACR7
8500, F8J2F3] P. porphyriacus [B5KL31] and P. rossignolii [BAJ
76674] which were not represented in the pool of cDNA clones
amplified from M. ikaheka venom gland, all the other minor
proteins correspond to a full-length putative toxin translated
from the venom gland cDNA library.
Australia and Papua New Guinea are home to a rich
biodiversity of venomous terrestrial and marine snakes of
the family Elapidae, which include species possessing some
of the most toxic venoms in the world [5]. Neurotoxicity

leading to respiratory paralysis represents the predominant
mechanism of prey immobilization and death caused by
most Australian and Papuan snakes [12,39]. Clinical observations indicate that human envenoming by M. ikaheka is
predominantly characterized by neurotoxicity, i.e. muscle
paralysis, systemic myotoxicity and haematological alterations [8]. The venom arsenal of M. ikaheka strongly suggests
a central role for neurotoxins in the envenomation strategy
developed by the New Guinea small-eyed snake. Catalytically active (D49) PLA2 proteins are present in the venom of
many terrestrial and marine Australo-Papuan elapid snakes
and typically exhibit presynaptic neurotoxic activity, myotoxic
activity, or both, although some forms exhibit antiplatelet
activity [4043]. Members of the three-finger toxin (3FTx) family
are also abundant proteins in the venoms of many elapids
(cobras, kraits and mambas), hydrophiids (sea snakes), and
colubrids [41,4448]. Despite their pronounced structural similarity, the proteins of this family are amongst the most
functionally diverse groups of snake venom toxins, exhibiting
a wide variety of pharmacological effects including neurotoxic,
cytotoxic, cardiotoxic, anticoagulant, and antiplatelet activities
[47,49]. The -neurotoxins found in Australian elapids are postsynaptically acting short or long-chain neurotoxins. Short and
long-chain neurotoxins have similar effects and bind with high
affinity to nicotinic acetylcholine receptors of skeletal muscle
217
Fig. 3 Characterisation of the venom proteome of M. ikaheka. Panel A, elution profiles of 2 mg of M. ikaheka venom proteins by
RP-HPLC, and electrophoretic analysis of -mercaptoethanol-reduced proteins eluted in each peak (insert). Protein bands were
submitted to in-gel tryptic digestion followed by LC-nESI-MS/MS tryptic ion sequencing, as described in Materials and methods
summarized in Table 1. Panel B, relative occurrence of proteins from different toxin families in the venom of M. ikaheka. CRISP,
cysteine-rich secretory protein; LAO, L-amino acid oxidase; PIII-SVMP, snake venom Zn2+-metalloproteinase (SVMP) of classes
III; DC, disintegrin-like/cysteine-rich fragment of PIII-SVMP; 5NT, 5-nucleotidase; SP, serine proteinase; vespryn, venom PRY
SPRY domain-containing protein; 3FTX, three-finger toxin; Kunitz, Kunitz-type serine proteinase inhibitor; D49-PLA2,
D49-phospholipase A2. For details of the individual proteins consult Table 1.
cells. In addition, L-type Ca2+ channel antagonists of the 3FTx

family may act synergistically with muscarinic three-finger
toxins to promote hypotension [50].
Vespryns (Venom PRYSPRY domain-containing proteins)
are a recently identified family of proteins [5153]. Ohanin
isolated from king cobra venom is a well-characterized vespryn
molecule, which is known to induce hypolocomotion and
hyperalgesia in mice [54]. A primary function of L-amino acid
oxidases (LAAO) is probably to promote prey hypotension by
activating soluble guanylate cyclase in the presence of superoxide dismutase [50]; CRISPs are widely distributed molecules in
snake venoms (reviewed by Yamazaki and Morita [55]; but

consult also [56]). Neurotoxic cysteine-rich secreted proteins
have been identified and characterized from the Australian
elapids P. australis and P. porphyriacus. These molecules target
cyclic nucleotide-gated ion channels and inhibit smooth
muscle contraction [57,58].
First reported in 1938 [59], 5 nucleotidases have since been
found in a number of snake venoms [60]. These hydrolytic
enzymes play a central role in liberating adenosine which
may help in prey immobilization [50]. The identification of
free purines as endogenous constituents of venoms has
Spot ID
Mapp/M + H +
m/z
Peptide sequence
Mi1
cDNA sequence
0.4
6682.6
6609.2
6668.6
0.7
2.1
7.5 kDa
6910.8
0.05
15 kDa
0.05
7 kDa
0.1
16 kDa
0.1
7 kDa
0.4
7 kDa
457.6
517.7
769.3
840.3
749.9
749.8
840.4
1427.6
749.8
840.4
618.3
503.2
701.4
3
2
2
2
2
2
2
1
2
2
2
2
2
941.3
569.8
659.3
570.3
642.8
566.7
688.3
601.8
549.3
671.3
695.3
688.3
497.3
941.3
601.8
548.7
734.7
1010.5
507.7
695.3
757.9
649.8
720.3
757.9
516.8
2638.2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
Nter:
TWNGIHSITER
GCGCPTVKR
LCCNQQSSQPK
GISLMCCHADECNN
KTWNGIHGSITER
KTWNGIHGSITER
GISLMCCHADECNN
KTWCDAWCGSR
KTWNGIHGSITER
GISLMCCHADECNN
SCSGGETSCYK
(171.1)PADTGPCK
FPAFYYNPVQR
LICYLSPKDTQIAPP
FCXTESWCDGFCGSR
NGENXCFKR
GCAATCPEAKPR
NGENICKFKR
SWCDAWCGSR
(217,2)QTCPPADK
VDXGCAATCPXAK
GGSGTPVDELDR
LFICNCDR
AFTDYGCYCGK
VDXGCAATCPTPK
VDXGCAATCPXAK
LICYLSPK
FCXTESWCDGFCGSR
GGSGTPVDELDR
LFICNCDR
YIEANNHIDPKR
GILSRPYVNTYAYDCTK
FVCNCDAK
VDXGCAATCPTPK
(199.2)EXGCAATCPXPK
TWCDAWCGSR
TCPPGENLCYTK
VVELGCAATCPIPK
ICLSTPDVK
HYEDVICCSTDNCNPFPTRPR
AHZ0881618
AHZ0881718
AHZ08817
AHZ0881718
AHZ08816 [2283]
AHZ08818 [2283]
AHZ0882425 [2292]
AHZ08825 [1888]
~AHZ08823
~AHZ0881618
AHZ08825
~AHZ0882425
~AHZ08825
AHZ0882324
AHZ0880409, 1112, 1415
AHZ0880407, 0912, 1415
AHZ0880412, 1415
~AHZ0882324
AHZ0882324
~AHZ08823
AHZ0880409, 1112, 1415
AHZ0880407, 0912, 1415
AHZ08805
~AHZ0880415
~AHZ0882324
AHZ0882324
AHZ0882425
AHZ0882325
AHZ0882324
AHZ0882325
AHZ08824
Protein family
NCBI/UniProtKB
~P80548
~P01434, ~ACY68694
1NTN_A, P01382
B5KL39, ACR78500
~B5KL31, BAJ76674
~P0C8R6
~A8HDK7
~P29179
ADN67572
~P29179
~P82662
~Q8UW28
0512217A, P01384
1OZY_A, 1PWO_A, 1P7O_A
1PWO_A
~Q9W7J5, A8HDK7K9
0512217A, P01384
~P01384
~A8HDK7
3FTx
3FTx
3FTx
3FTx
3FTx
3FTx
(short
(short
(short
(short
(short
(short
-neurotoxin)
-neurotoxin)
-neurotoxin)
-neurotoxin)
-neurotoxin)
-neurotoxin)
3FTx (long -neurotoxin)

BPTI/Kunitz-type inhibitor
BPTI/Kunitz-type inhibitor
3FTx (short -neurotoxin)
3FTx (weak neurotoxin)
3FTx (-neurotoxin)
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
1PWO_A
D49-PLA2
AAB33760, AAZ22635, 37, 4041, 43 D49-PLA2
~Q9W7J5, A8HDK7K9
0512217A, P01384
1NTN_A, P01382, Q53B57
A1IVR7R9, F8J2E2
0512217A, P01384
~BAN66265
~P01384
7826.6
7552.3
Best match
218
Table 1 Assignment of the reverse-phase fractions from the venom of the New Guinean small-eyed snake, Micropechis ikaheka (Mi-), isolated as in Fig. 2a, to protein families
by nESI-MS/MS collision-induced dissociation of peptide ions obtained from in-gel digested protein bands separated by SDS-PAGE (insert in Fig. 2a). X = Ile or Leu; Mox,
methionine sulphoxide. Cysteine residues are carbamidomethylated. Nter, N-terminal sequence determined by automated Edman degradation. Apparent molecular masses
(Mapp in kDa) were estimated from SDS-PAGE analyses of -mercaptoethanol-reduced samples. Quasimolecular isotope-averaged masses (M + H+) were determined by
ESI-MS (QTrap2000), and those matching masses calculated for full-length cDNA sequences are underlined.
8, 9
4.1
17 kDa
1.2
17 + 7 kDa
2.2
1415.5 kDa
1.1
7 kDa
0.3
10a
4.8
25 kDa
13,898.5
14,014.1
2
2
2
2
2
2
2
1
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
886.8
802.9
866.4
668.8
998.4
747.8
1011.3
601.8
524.3
1055.5
2
2
2
2
2
2
2
2
2
2
840.4
532.8
1011.3
656.8
549.3
450.2
601.8
1
2
2
2
2
2
2
Nter:
10b
4.7
11
5.2
12
7.2
13,367.1
[14,039.1, 13908.7]
14039.2
Nter:
TP(146.2)TCPPGEVXCFTK
APYIDANNR
TCPPGENLCYTK
VVELGCAATCPIPK
LICYLSPK
VDXGCAATCPTPK
AVCDCDVEAAECFAR
GTMYDYYCSSDGPYCR
TPYNNDFYNIDTK
IHDDCYADAEK
GGSGTPVDELDR
TWCDAWCGSR
TCPPGENLCYTK
HYEDVICCSTDNCNPFPTRPR
NGENXCFKR
GCAATCPEAKPR
LICYLSPK
YFVEVGEECDCGPPQVCR
DDCDLPEICTGR
CPTDSFQR
EDLDYG(Mox)VEPGTK
C(I-16)ALWGPGVK
CTIPGREPLLAFSNYGCYCGK
APYIDANNR
GGSGTPVDELDR
LFICNCDR
EPLLAFSNYGCYCGK
APYIDANNR
GILSRPYVNTYAYDCTK
NLIQFSYLIQCANHG
AVCDCDVEAAECFAR
TPYNNDFYNIDTK
TPYNNDFYNIDTKK
IHDDCYADAEK
GTMYDYYCSSDGPYCR
PYVNTYAYDCTK
GILSRPYVNTYAYDCTK
GGSGTPVDELDR
APYIEANNR
GILSPGYVNTYSYDCTDGK
NLYQFRNMIICTIPDR
NLYQFR
GKITCNDQK
GILSRPYVNTYAYDCTK
YIEANNHIDPK
LFICNCDR
TAAMCFAK
GGSGTPVDELDR
AHZ0882325
AHZ08812, AHZ08815
AHZ0882325
AHZ0882324
~AHZ0882324
AHZ08813
AHZ08813
AHZ08813
AHZ08813
AHZ0880409, 1112, 14-15
AHZ0882425
AHZ0882325
AHZ08824
~AHZ08825
~AHZ08819
~AHZ08819
AHZ08819
~AHZ08819
AHZ08819
AHZ08806, 12, 15
AHZ08812, AHZ08815
AHZ0880409, 1112, 1415
AHZ0880407, 0912, 1415
AHZ0880412, 1415
AHZ08812, AHZ08815
AHZ08805
AHZ08813
AHZ08813
AHZ08813
AHZ08813
AHZ08813
AHZ08813
AHZ08805
AHZ08805
AHZ0880409, 1112, 1415
AHZ0880607, 10
~AHZ08812
AHZ08805, 11, 14
AHZ08805, 11, 14
AHZ08805
AHZ08805
AHZ08805
AHZ0880407, 0912, 1415
AHZ0880412, 1415
AHZ0880409, 1112, 1415
~B2BRQ6
~1PWO_A
A1IVR7R9, F8J2E2
0512217A, P01384
~P0C8R6
~Q9W7J5, A8HDK7K9
~ACY68711
~ABK63573, ~ACY68711
~Q8UUH8H9
~P00609, ~F8J2D2
1NTN_A, P01382, Q53B57
A1IVR7-R9, F8J2E2
~P01384
~P29179
ADN67572, P85520
~P01384
~A8QL49
P0DJ43, ~ABQ01137
ABQ0113235, 39
~P0DJ43, ~ABQ01132
~3K7L_A, ~Q9PVK7
1P7O_A
~1PWO_A
1OZY_A, 1P7O_A
~1PWO_A
~1PWO_A
ACY68711, ~2114420A
~ACY68711
~Q8UUH8H9
~Q8UUH8H9
~P00609, ~F8J2D2
~ABK63573, ~ACY68711
~1PWO_A
~1PWO_A
1PWO_A
~1PWO_A
~1PWO_A
~1PWO_A
~1OZY_A, ~1PWO_A
~1PWO_A
1PWO_A
(continued on next page)
219

D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
3FTx (weak toxin)
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
876.9
517.3
720.3
757.9
497.3
695.3
886.8
1994.7
802.8
668.8
601.8
649.8
720.3
2638.2
569.8
659.3
497.3
1100.9
725.8
506.2
728.8
542.3
1237.5
517.3
601.8
549.3
889.9
517.3
1011.3
220
Table 1 (continued)
Spot ID
Mapp/M + H +
m/z
Peptide sequence
Mi-
cDNA sequence
13
2.7
14
2.5
15 kDa
13773.4
13848.8
4.9
14026.1
16 kDa
17
3.1
18
13.2
0.4
20
1.5
13775.7
21
11.4
13903.3
13875.4
13932.9
22
1.8
16 kDa
23431.6
2992.4
3006.6
1209.1
689.1
524.3
549.3
1210.6
1749.6
1778.8
1
1
2
3
2
2
2
1
1
524.3
549.3
1210.6
875.3
580.8
524.3
601.7
549.3
1210.6
524.3
601.7
549.3
1210.6
2
2
2
2
2
2
2
2
2
2
2
2
2
1011.3
549.3
2962.3
889.8
597.3
2
2
1
2
2
Nter:
13848.8
19
2
2
2
2
2
2
3
2
2
2
2
2
2
2
Nter:
13877.9
14016.6
23 + 16 kDa
13773.7
594.8
741.4
1068.9
628.8
524.3
601.8
807.4
684.8
580.8
1209.1
873.8
601.7
549.2
615.7
Nter:
GGSGTPVDDXDR
(244.1)APYXEANN(219)K
GILSPGYXNTYSYDCDNGK
APYIEANN(384.2)
APYIEANNR
GGSGTPVDELDELR
GILSPGYXNTYSYDCDNGK
(C-16)KLFICNCDR
GGSGTPVDDLDK
CTIPGIEPLLAFSNYGCYCGK
CCQTHDYCYDEAK
GGSGTPVDELDR
LFICNCDR
GGDGTPVDELDR
NLYQFRNMIICTIP
NMIICTIPDREPLLAFSNYGCYCGK
LFICNCDRTAAMCFAKAPYIEANNR
GILSGPSFNTYAYDCTDGK
APYIEANNR
LFICNCDR
CCQTHDYCYDEAK
EPLLAFSNYGCYCGK
NLIQFRKMIKCTIPG
APYIEANNR
LFICNCDR
CCQTHDYCYDEAK
GGSGTPVDDLDK
APYIEANNR
GGSGTPVDELDR
LFICNCDR
APYIEANNR
GGSGTPVDELDR
LFICNCDR
NLLQFRKMIKCTIPG
GILSRPYVNTYAYDCTK
LFICNCDR
EPLLAFSNYGCYCGKGGSGTPVDELDR
YLYVCQYCPAGNIR
QIVDKHNALR
AHZ08810
AHZ0880607, 10
AHZ08811
AHZ0880607, 10
AHZ08806
AHZ08806, 11
AHZ08811
AHZ0880407, 0912, 14-15
AHZ08810
AHZ08804, 0710
AHZ0880607, 10
AHZ0880409, 1112, 14-15
AHZ0880407, 0912, 1415
~AHZ0880409, 1112, 1415
AHZ08805, 11, 14
AHZ08805, 11, 14
AHZ0880607, 10
AHZ08804, 0710
AHZ08804, 0809
AHZ0880607, 10
AHZ0880407, 0912, 1415
AHZ08804, 0710
AHZ0880607, 10
AHZ0880412, 1415
AHZ08804, 0809
AHZ0880607, 10
AHZ0880407, 0912, 1415
AHZ08804, 0710
AHZ0880607, 10
AHZ08810
AHZ0880607, 10
AHZ0880409, 1112, 1415
AHZ0880407, 0912, 1415
AHZ08804, 0710
AHZ0880607, 10
AHZ0880409, 1112, 1415
AHZ0880407, 0912, 1415
AHZ08804, 0710
AHZ08804, 0809
AHZ08805
AHZ0880407, 0912, 1415
AHZ0880409, 1112, 1415
AHZ0882021 [28235]
AHZ0882021 [28235], AHZ08822 [26237]
Protein family
NCBI/UniProtKB
AET85561, AAZ29512
1PWO_A
1PWO_A, 1P7O_A
1PWO_A
1PWO_A
1PWO_A, 1P7O_A
1PWO_A, 1P7O_A
AET85561, AAZ29512
1OZY_A
~1OZY_A, ~ABK63564
~P59069, ~ADF50037
~1PWO_A
~1PWO_A
~1PWO_A
1OZY_A
1OZY_A
1PWO_A
1OZY_A
~1OZY_A, ~ABK63564
1OZY_A, 1P7O_A
1OZY_A
1PWO_A
1OZY_A
~1OZY_A, ~ABK63564
AET85561, AAZ29512
1PWO_A
1OZY_A
1PWO_A
1OZY_A
1OZY_A
1PWO_A
1OZY_A, 1P7O_A
~2DDB_A, ACN93671
P81993, ACN93671
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
D49-PLA2
CRISP
CRISP
15
Best match
23
0.3
23523.7
35 kDa
23 kDa
16 kDa
24
52 kDa
<0.1
37 kDa
24, 25
<0.1
29 kDa
25, 26
3.0
52 kDa
27
3.2
48 kDa
28, 29
0.3
56 kDa
CSFAHSPPHLR
EWAVGLAGK
YLYVCQYCPAGNIR
DSIATPY
VIQAWYDENKK
HFFEVK
EWAVGLAGK
TVKNVGVPQVVPDNPER
DDCDLPEICTGR
TNTPEQDRYLQVK
EVVKF(Mox)NSXR
VGXXGYTTK
VPTYVPLEK
GVIAGNSNVICPSDSR
NILCAGVLEGGK
TNTPEQDRYLQVK
SACCNAATCK
DDCDLPEICTGR
VAPDICFTYNQK
EAQCDSGECCEK
AAKDDCDLPEICTGR
CPTDSFQR
VQGCGFCR
CGDGMVCSNR
TNTPEQDRYLQVK
SACCNAATCK
DDCDLPEICTGR
VAPDICFTYNQK
VQGCGFCR
CGDGMVCSNR
NGHPCQNNKGYCYNGK
CPIMTNQCIALWGPGVK
VTXXEASER
YPVKPSEEGK
SASQXYR
RXYFEPPXPPK
FWEADGXHGGK
TSADXVXNDXSXXHQLPK
EXQAXCYPSMXK
XYFAGEYTAR
VHGWXDSTXK
RVWEVK
YDTYSTK
TLSYVTADYVXVCSSSR
TSADXVXNDXSXXHQXPK
XXEEXKR
NDDXFSYEK
STTDLPSR
AHZ0882021
AHZ0880203
AHZ0882021
AHZ0882021
AHZ0882021
AHZ0880203
AHZ0880203
AHZ0880203
AHZ08819
AHZ08819
AHZ0880001
AHZ0880001
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
[28235]
[28235]
[28235]
[28235]
ACN93671, P84808
P82885
~2DDB_A, ACN93671
~2DDB_A
~ACN93671
P82885
P82885
~ABW74874
P0DJ43
P0DJ43
~ F8S0Z7
~AHJ80886
~BAN89427
ETE66745
A8QL53
P0DJ43
P0DJ43
P0DJ43
~ABQ01133
P0DJ43
P0DJ43
P0DJ43
~ABQ01133
P82942
3K7L_A
Q4JHE3, Q4JHE2
CRISP
Vespryn
CRISP
CRISP
CRISP
Vespryn
Vespryn
Vespryn
PIII-SVMP
PIII-SVMP
5-nucleotidase
5-nucleotidase
5-nucleotidase
Serine protease
Serine protease
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
LAAO
(continued on next page)
221
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
<0.1
654.3
465.8
888.9
447.2
697.3
403.7
465.8
924.9
725.8
796.4
619.8
476.3
523.3
549.3
615.9
796.4
571.7
725.8
728.3
736.8
860.9
505.7
492.2
577.7
796.4
571.7
725.8
728.3
492.2
577.7
955.9
1945.1
509.2
567.3
412.7
678.9
608.8
659.7
726.9
595.8
578.3
408.7
439.2
960.9
989.1
451.3
565.8
438.7
222
Spot ID
Mapp/M + H+
m/z
Peptide sequence
Best match
Mi30
2530
cDNA sequence
1.1
52 + 48 kDa
52 kDa
637.6
505.7
796.4
736.8
725.8
505.7
577.7
664.4
671.3
414.2
3
2
2
2
2
2
2
2
2
2
NGHPCQNNKGYCYNGK
CPTDSFQR
TNTPEQDRYLQVK
EAQCDSGECCEK
DDCDLPEICTGR
CPTDSFQR
CGDGMVCSNR
XNXEPDVSVTXK
XNXEPEVSVTXK
ETVXXPR
Protein family
NCBI/UniProtKB
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
AHZ08819
ABQ01132
P0DJ43
~F8RKV9
~F8RKV9
~ADD14036
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
PIII-SVMP
Table 1 (continued)
further supported the role of purinergic signalling in envenomation. Purines are known to potentiate venom-induced
hypotension and paralysis via purinergic receptors. In addition,
ATP released from skeletal muscle by the myotoxic action of
PLA2s acts as a danger signal stimulating purinergic receptors
to enhance and spread both the muscle damage caused by the
myotoxins and pain [61]. High concentrations of adenosine
generated by the combined action of venom myotoxins and
nucleotidases may play an important role in envenomation
via its hypotensive, paralyzing and anticoagulant activities
[62].
3.3. Lethal activity of M. ikaheka venom fractions

The finding that the venomics profile of M. ikaheka is strongly
based on PLA2s and 3FTxs prompted us to investigate the role
of these toxin classes in venom lethality in mice. To this end,
the venom was fractionated by reverse-phase HPLC and
fractions eluting between 8 and 12 min (3FTxs) and between
13 and 25 min (PLA2s) were separately pooled (Fig. 4) and used
to assess lethality and myotoxic activity. The 3FTx fraction
had an estimated LD50 of 0.22 mg/kg (95% confidence limits:
0.030.41 mg/kg) by the i.v. route. Mice injected with doses of
0.59 mg/kg or higher died within one hour of injection. Animals
receiving this fraction showed signs of respiratory and limb
paralysis. On the other hand, the PLA2 fractions showed a lower
toxicity, with an estimated LD50 of 1.62 mg/kg (95% confidence
limits: 0.902.51 mg/kg). These mice also showed signs of respiratory difficulties, although the time of death was more
223
prolonged (more than 12 h) when compared to mice injected

with the 3FTx fraction.
The LD50 of the M. ikaheka PLA2 venom fraction was much
higher than those of typical presynaptically active PLA2s from
Australian elapid venoms, such as notexin [63], textilotoxin
[64], and taipoxin [65]. Hence, although mice injected with the
pooled PLA2s of M. ikaheka venom developed signs of neurotoxicity, i.e. limb paralysis and respiratory distress, these PLA2 molecules do not seem to play a predominant role in the overall
lethal effect of the venom. Furthermore, inhibition of PLA2 by
chemical modification with pBPB affected the venom's lethality
only to a partial extent. LD50s of native and pBPB-inhibited venom
were 0.62 mg/kg (95% confidence limits: 0.430.88 mg/kg), and
1.24 mg/kg (95% confidence limits: 0.871.60 mg/kg). Hence, our
results support the hypothesis that post-synaptically-acting
3FTxs play the predominant role in the neurotoxic effect induced
by M. ikaheka venom. However, despite their lower toxicity
as compared to 3FTxs, PLA2s might contribute to the overall
lethality of the venom. The estimated LD50 of M. ikaheka
venom was 0.62 mg/kg. As judged from proteomic analysis,
this amount of venom contains 0.057 mg of 3FTxs and
0.5 mg of PLA2s. These figures represent 26% and 30% of the
estimated LD50 of the 3FTx and the PLA2 fractions, respectively, thus suggesting that these two toxin classes may
contribute synergistically to venom lethality. This hypothesis is
supported by the antivenomics outcome reported in the
accompanying paper [22], showing that Australian antivenoms
exhibited strong immunorecognition of -neurotoxins of the
3FTx family and neutralized the lethal, i.e. neurotoxic, and
Fig. 4 RP-HPLC fractionation of M. ikaheka venom into 3FTx- and PLA2-rich fractions. Panel A, reference analytical
reverse-phase HPLC separation of 75 g of whole venom proteins of M. ikaheka. Fractions eluting between 8 and 12 min
(3FTxs) and between 13 and 25 (D49-PLA2s) were separately pooled and used to assess lethality and myotoxic activity. Panel B,
reverse-phase HPLC separation of 75 g of pooled 3FTxs from M. ikaheka venom eluted between 8 and 12 min in panel A. Panel
C, reverse-phase HPLC separation of 50 g of pooled PLA2s from M. ikaheka venom eluted between 13 and 25 min in panel A.
224
myotoxic activities of M. ikaheka venom, but exhibited poor

neutralisation of PLA2 and anticoagulant activities.
3.4. Myotoxic activity is associated with the PLA2 venom

fraction
Intramuscular injection of 30 and 60 g of PLA2 fraction in the
gastrocnemius induced prominent myotoxicity, reflected in
increments in plasma CK activity 3 h after injection (Fig. 5).
Mice injected with PBS, 30 g PLA2 and 60 g PLA2 showed CK

activities of 240 30 U/L, 8029 1255 U/L, and 13,572 1552 U/L,
respectively. On the other hand, judging from both the plasma
CK activity (Fig. 5A) and the histological morphology of injected
muscle (compare panels B and C of Fig. 5), the incubation of
M. ikaheka venom with pBPB for 24 h completely abrogated PLA2
activity. Thus, mice receiving native venom showed widespread
areas of necrosis in the gastrocnemius muscle (Fig. 5B), whereas a
normal histological pattern of muscle tissue was observed in
mice injected with inactivated venom (Fig. 5C). Myotoxic PLA2
molecules are known components of Australian elapid venoms
[6668]. Myotoxic PLA2s have been isolated and characterized
from M. ikaheka venom [14,15,18]. Our results indicate that these
enzymes are abundant components of M. ikaheka venom and
that myotoxicity is due to the direct action of PLA2 molecules on
muscle fibres. Myotoxic PLA2s are likely to play a key role in
human envenoming, since rhabdomyolysis with myoglobinuria
have been described in cases of M. ikaheka envenoming [8].
3.5. Pharmacological effects in rat isolated cardiac and

vascular tissues
Fig. 5 Inhibition of the myotoxic activity of M. ikaheka venom

by chemical modification with pBPB. Groups of mice (n = 4) were
injected intramuscularly in the right gastrocnemius with either
20 g native venom (venom), 20 g venom incubated with
pBPB to abrogate PLA2 activity (venom inh), or PBS (saline).
Injection of the PBS and pBPB controls did not induce any
evidence of toxicity in mice. Plasma creatine kinase (CK) activity
was quantified 3 h after injection. As shown in panel A,
myotoxicity was completely abrogated upon inhibition of PLA2
activity. Panels B and C show, respectively, light micrographs of
haematoxylineosin stained sections of skeletal muscle
injected with either 20 g native venom native venom or 20 g
venom treated with pBPB. Notice the abundance of necrotic
muscle fibres in (B) (arrows) whereas fibres with normal
appearance are observed in (C). Bars represent 100 m.
Previous in vivo findings showed that venom-induced vasorelaxation correlates with systemic hypotension in piglets
intravenously injected with M. ikaheka venom [11]. In rat
small mesenteric arteries (327 5 m internal diameter), M.
ikaheka venom (9270 g/mL) did not cause contraction, even
when vessels were pre-contracted to 1020% of the maximum
contraction to KPSS (n = 4; data not shown); this small precontraction is used to detect activity of weak constrictor agents.
To examine vasorelaxation, arteries were pre-contracted with
endothelin-1 (13 nM) to 7080% of KPSS maximum contraction.
Venom caused a significant concentration-dependent relaxation
(from 9 to 270 g/mL; P < 0.05, RM ANOVA; Fig. 6A); the maximum relaxation was 61 4% KPSS (n = 5). Acetylcholine 10 M
was added at the end of the experiments (on top of the maximum
venom concentration of 270 g/mL) and relaxed vessels
further to 79 4% KPSS comparable to the acetylcholineinduced maximum relaxation in vessels treated only with
vehicle ( 80 6% KPSS; n = 5; data not shown) indicating a
fully functional vascular endothelium and lack of effect of
venom on responses to this endothelium-dependent agonist.
Pre-treatment with the muscarinic antagonist atropine or the
cyclooxygenase inhibitor indomethacin did not cause a significant rightward shift of the overall relaxation curve to venom, but
did attenuate the maximum relaxation (to 270 g/mL) to 34 5
(p = 0.011) and 33 5% KPSS (p = 0.0005), respectively (n = 3
each; Fig. 6A). In separate treatment groups, pre-treatment with
the nitric oxide synthase inhibitor L-NAME (n = 4) or, anecdotally, the KATP channel inhibitor glibenclamide (n = 1) did not affect
the vasorelaxant response curve to venom (p > 0.05; data not
shown). The initial pre-tone was the same in all treatment
groups (p > 0.05, 1-way ANOVA).
To examine the effects of M. ikaheka venom on sympathetic
nerve-induced vascular contractions, electrical field stimulation
was applied (6.2525 Hz) to rat mesenteric arteries. In the absence
of venom, this caused frequency-dependent arterial contractions
to a maximum of 48 5% KPSS at 25 Hz (n = 6; p < 0.0001; Fig. 6B).
Venom at 90 and 270 g/mL did not affect contractile responses
to electrical stimulation (data not shown), however at 360 g/mL
225
Fig. 6 Effects of M. ikaheka venom on arterial vascular tone and nerve-induced contraction. A: Rat isolated mesenteric arteries
were pre-treated for 30 min with vehicle (PSS; venom control group), the cyclooxygenase inhibitor indomethacin (3 M) or
muscarinic receptor antagonist atropine (3 M) before endothelin-1 pre-contraction (ET-1 tone) and M. ikaheka venom (0.9
270 g/mL) application. Initial pre-tone was compared between groups using one-way ANOVA with Dunnett's post hoc test for
multiple comparisons (P > 0.05). Relaxation within each group was analysed with RM ANOVA; a Dunnett post hoc test was used
to compare each venom concentration with its respective baseline (*P < 0.05). B: Electrical nerve stimulation (6.25
25 Hz)-induced contraction of rat isolated mesenteric arteries in the absence (no venom control group) or presence (30 min
pre-treatment) of M. ikaheka venom (360 or 450 g/mL). The highest venom concentration (450 g/mL) attenuated the
contractile responses to stimulation at 25 Hz (*P = 0.0025; RM ANOVA and Dunnett post hoc test). Vascular tone is expressed as
% KPSS maximum contraction. n, Number of arteries per group (each taken from separate rats). Error bars are average SEM
from RM ANOVA (see Materials and methods).
responses tended to be decreased. With the highest concentration of venom tested, 450 g/mL, the maximum contraction to
25 Hz was attenuated to 26 7% KPSS (n = 6; p = 0.0025; Fig. 6B).
Following venom washout, the frequencycontractile response
curve to electrical stimulation returned to (no venom) control
levels (data not shown), indicating that the effects of this high
concentration of venom were reversible.
In rat isolated left and right atria, M. ikaheka venom (0.009

90 g/mL) caused concentration-dependent positive ionotropy
(EC50 2.2 0.5 g/mL; n = 6) (i.e., strengthens the force of the
heartbeat) and tachycardia (EC50 1.6 0.9 g/mL; n = 4), respectively (p < 0.0001; Fig. 7A and B). Pre-treatment with the adrenoceptor antagonist propranolol to inhibit sympathetic
nerve-mediated ionotropic and chronotropic responses had
Fig. 7 Effects of M. ikaheka venom and pre-treatment with antagonists or abrogation of PLA2 activity on contractile force of left
atria and rate of spontaneously beating right atria isolated from the rat. A: Change () in left atrial contractile force (g). B: Change
() in right atrial rate (beats/min). Atria were incubated for 30 min with the b-adrenoceptor antagonist propranolol (1 M) or
calcitonin gene-related peptide (CGRP) antagonist CGRP8-37 (1 M) prior to M. ikaheka venom (0.00990 g/mL) application. In
the anti-PLA2 groups, M. ikaheka venom was incubated with pBPB to abrogate PLA2 activity prior to application to the atria. n,
Number of atria. Vertical error bars are average SEM from RM ANOVA and horizontal error bars represent the EC50 (where
applicable) 1 SEM.
226
no effect on responses to the venom in either left or right

atria. Further, inhibition of the sensory nerve neurotransmitter, calcitonin gene-related peptide, with the antagonist
CGRP837 did not affect venom-induced increases in contractility or rate (Fig. 7A and B). However, when venom was
treated overnight with pBPB to abrogate PLA2 activity, the
positive ionotropic and chronotropic atrial responses were
completely abolished (n = 3 each; Fig. 7). The PLA2 activity of
M. ikaheka venom pre- and post-pBPB treatment was 4593 and
0.4 mol/min/mg, respectively. Separate untreated aliquots
of venom were left at room temperature overnight and then
tested in left and right atria (n = 2 each); this venom retained
its full potency in both sets of tissues (data not shown).
In summary, these in vitro pharmacological studies suggest
that M. ikaheka venom is cardiotoxic inducing tachycardia and
positive ionotropy, together with significant vascular smooth
muscle relaxation (which has been correlatated with systemic
hypotension [11]) and, at high concentrations, reversible inhibition of sympathetic nerve-induced vascular contraction. In
arteries, arachidonic acid metabolites and muscarinic receptor
activation contributed only in small part to the M. ikaheka venom
vasorelaxation; further, NO synthase and ATP-sensitive K+
channels did not appear to be involved. Additional studies are
required to determine the mechanism of action of M. ikaheka
venom in vascular smooth muscle. In cardiac tissues, much
lower concentrations (approximately 10-fold less) of venom
were required to elicit major increases in atrial rate and force
of contraction compared with relaxant effects in vascular
smooth muscle. Inhibition of the sympathetic nervous system
(1-adrenoceptor signalling) or the sensory neurotransmitter
CGRP had no effect on either the tachycardia or positive
inotropy. However, when PLA2 activity was abrogated, the
right and left atrial effects of the venom were completely
prevented, indicating that PLA2s are responsible for these
cardiotoxic effects.
4. Concluding remarks
Our joint venom gland cDNA sequencing and proteomics
approach revealed peptide evidence for ~50 venom proteins
including 25 of the 27 full-length toxin sequences identified in
the novel sequences of M. ikaheka venom gland cDNA clones.
Several toxins sequenced in this study shared close identity to
proteins in Asian elapid venoms, notably Naja spp. and
Bungarus spp., an observation in broad agreement with the
widely accepted concept of an Asian (perhaps Bungarus
Laticauda) origin leading to colonization by rapidly diversifying ancestral species that split from a Laticauda-like predecessor between 13 and 19 MYBP and then diversified into the
present day Australo-Papuan snake fauna [2,6971]. At the
same time, the remarkable homology of the venom toxin
sequences of M. ikaheka to venom proteins from terrestrial
and marine Australian Hydrophiinae and viviparous sea snakes
also supports the hypothesis of a sister-group relationship
between the monotypic Melanesian Micropechis genus and the
subfamily Oxyuraninae within the family Hydrophiidae [4]. In
concordance with this view, an initial investigation of the
venoms of some small Australian elapids has shown them to
be equally as complex as those of their larger, better-investigated
cousins [72], suggesting that their overall venom composition

was derived at the base of the Australian elapid snake radiation.
The split between Laticauda and Oxyuraninae snake lineages is
estimated at 12.6 MYBP, and the last common ancestor of
Micropechis and all living oxyuranines is dated at 11.5 MYBP
[1,2]. Bayesian relaxed clock analyses indicated that the ~160
species of the core Australian radiation arose recently, within
the last 10 MYBP, with most inter-generic splits dating to
between 10 and 6 MYBP. Analysis of the rapid evolutionary
radiation of Australasian elapids and sea snakes from a
venomics perspective may shed light on the lethal mechanisms of elapid snake venoms. In this respect, the recent
report of the sequencing of the first genome of a venomous
snake, the king cobra, O. hannah, has revealed that toxin
genes important for prey capture have massively expanded
by gene duplications and evolved under positive selection,
resulting in protein neofunctionalisation [73]. Gene duplicates exhibit varying degrees of structural resemblance to
their progenitor loci. The rate at which a new functional gene
copy appears in a population depends on both the time since
duplication (genetic drift) and the adaptive potential of the
duplicated gene [74,75]. The high structural conservation
exhibited by M. ikaheka and other Australo-Papuan elapid
venom toxins suggests a high conservation of protein classspecific immunologic epitopes across this diverse radiation
of species.
M. ikaheka is a large, powerfully built elapid that averages
1.21.7 m in length [5] but very large specimens of 2.12.3 m
have been recorded [76]. This species often holds onto tightly
when it bites, and envenoming may lead to significant illness or
to death [8,76,77]. Although there is no known specific antivenom available for the treatment of M. Ikaheka envenoming in
Papua New Guinea, positive clinical outcomes and recovery have
been documented through the use of bioCSL Australo-Papuan
polyvalent antivenom and bioCSL monovalent tiger snake
antivenom, if given early [8,77,78]. These methods of treatment,
however, have been largely empirical, leading us to undertake
further studies [22] to elucidate which toxins harbour immunologic epitopes for neutralizing bioCSL antivenom immunoglobulins, and while these and other bioCSL products demonstrated
good neutralisation of lethality, as well as dose-dependent attenuation of myotoxicity, they were poorly efficacious in the
neutralisation of PLA2 and anticoagulant activity [22]. Formal
studies of envenoming by this species should in future enable us
to evaluate the practical effectiveness of these antivenoms, and
determine whether a need for an improved antivenom against
this species is required in order to achieve the best possible
clinical outcomes for all patients.
Acknowledgements
Thanks are due to Daniela Solano, Instituto Clodomiro Picado, for
her support in the laboratory work. Funding for the research
described in this paper was provided by grants BFU2010-17373
from the Ministerio de Ciencia e Innovacin (currently, Ministerio
de Economa y Competitividad), Madrid; PROMETEO/2010/005
from the Generalitat Valenciana; CYTED project BIOTOX P211RT
0412; project 741-B2-652 (Vicerrectora de Investigacin, UCR);
and FEES-CONARE (Costa Rica). Research in PNG was supported
by AVRU from a grant by the Australian Government's Department of Health & Aging (DoHA) and a grant [APSF 07/1] from the
Australia-Pacific Science Foundation (APSF), as well as the
International Cooperative Biodiversity Group (Professor Teatulohi
Matainaho & Professor Louis Barrows). We are particularly
grateful to Prof. Martin Lavin, Dr. Geoff W. Birrell, and Dr. Liam
St Pierre (Queensland Institute of Medical Research, Brisbane,
Queensland, Australia) who made it possible for Owen Paiva to
carry out much of his work in their laboratories, using primers
and protocols developed by them.
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J O U RN A L OF P ROT EO M IC S 1 1 0 ( 2 01 4 ) 2 0 9 22 9

Available online at www.sciencedirect.com

Combined venom gland cDNA sequencing and

Received 1 June 2014

Accepted 14 July 2014

Available online 8 August 2014

Venom gland cDNA

role for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed by

wide variety of small ground dwelling animals, particularly

2. Materials and methods

2.2. Venom and venom glands

2.3. RNA isolation and complementary DNA (cDNA) template

was then synthesized from 1 g of total RNA with an oligo(dT)

2.4. Amplification of toxin sequences from M. ikaheka venom

2.5. Reverse-phase and SDS-PAGE analyses

For venomics characterisation, 22.5 mg of crude, lyophilized

2.6. Characterization of the venom proteome

calculated from the relationship of the sum of the areas of the

2.7. Fractionation of M. ikaheka venom into 3FTx- and PLA2rich fractions

2.8. Estimation of the median lethal dose (LD50) of venom

2.9. Myotoxic activity of the PLA2 fraction

in 0.1 M Tris, 0.7 M EDTA, pH 8.0, buffer) was incubated with

2.11. Pharmacological effects in rat isolated cardiac and

2.12. Mesenteric arteries

inhibition protocol to enhance the contractile responses to

2.13. Rat right and left atria

2.14. Statistical analysis

(RM) ANOVA; a Dunnett post hoc test was used to compare

3. Results and discussion

(Hydrophis hardwickii) venom. Similarly while PLA2 primers

[AHZ08813] represents a new acidic PLA2 composed of 119

3.2. The venom proteome of M. ikaheka

of the most toxic venoms in the world [5]. Neurotoxicity

cells. In addition, L-type Ca2+ channel antagonists of the 3FTx

snake venoms (reviewed by Yamazaki and Morita [55]; but

3FTx (long -neurotoxin)

(continued on next page)

3FTx (long -neurotoxin)

(continued on next page)

3.3. Lethal activity of M. ikaheka venom fractions

prolonged (more than 12 h) when compared to mice injected

myotoxic activities of M. ikaheka venom, but exhibited poor

3.4. Myotoxic activity is associated with the PLA2 venom

Mice injected with PBS, 30 g PLA2 and 60 g PLA2 showed CK

3.5. Pharmacological effects in rat isolated cardiac and

Fig. 5 Inhibition of the myotoxic activity of M. ikaheka venom

In rat isolated left and right atria, M. ikaheka venom (0.009

no effect on responses to the venom in either left or right

cousins [72], suggesting that their overall venom composition

[15] Gao R, Kini RM, Li G, Luo R, Selvanayagam ZE,

[33] Angus J, Broughton A, Mulvany M. Role of -adrenoceptors in

[50] Aird SD. Ophidian envenomation strategies and the role of

[71] Scanlon JD, Lee MSY, Archer M. Mid-Tertiary elapid snakes

[75] Katju V, Bergthorsson U. Copy-number changes in evolution:

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