Toxicon 71 (2013) 11–17
Contents lists available at SciVerse ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Proteomic characterisation of toxins isolated from
nematocysts of the South Atlantic jellyfish Olindias
sambaquiensis
Andrew J. Weston a, Ray Chung a, Walter C. Dunlap b, André C. Morandini c,
Antonio C. Marques c, Ana M. Moura-da-Silva d, Malcolm Ward a,
Gabriel Padilla e, Luiziana Ferreira da Silva e, Nikos Andreakis f, Paul F. Long b, g, *
a
King’s College London Proteomics Facility, Institute of Psychiatry, London SE5 8AF, United Kingdom
Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH,
United Kingdom
c
Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, R. Matão, Tr. 14, 101, 05508-090 São Paulo, SP, Brazil
d
Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil
e
Departmento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-090 São Paulo, SP, Brazil
f
Centre for Marine Microbiology and Genetics, Australian Institute of Marine Science, PMB No. 3 Townsville MC, Townsville,
Queensland 4810, Australia
g
Department of Chemistry, King’s College London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 March 2013
Received in revised form 24 April 2013
Accepted 1 May 2013
Available online 18 May 2013
Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other
venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including conesnails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological
activities of these potential toxins include cytolysins, neurotoxins, phospholipases and
toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider
venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals
sequence orthologs to many representatives of the most important superfamilies of peptide
venoms suggesting that their origins in higher organisms arise from deep eumetazoan
innovations. Accordingly, cnidarian venoms may possess unique biological properties that
might generate new leads in the discovery of novel pharmacologically active drugs.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Keywords:
Cnidaria
Jellyfish
Nematocyst
Proteomics
Venom
1. Introduction
Cnidaria is a diverse phylum of basal metazoans
comprising over 10,000 species, predominately marine
organisms (Daly et al., 2007; Zhang, 2011). The phylum has
* Corresponding author. Institute of Pharmaceutical Science, King’s
College London, Franklin-Wilkins Building, Stamford Street, London SE1
9NH, United Kingdom. Tel./fax: þ44 207 848 4842.
E-mail address: [email protected] (P.F. Long).
two major lineages: Anthozoa (sea anemones and corals)
which live as sessile polyps, and the Medusozoa (jellyfishes
and Hydra), comprising the classes Cubozoa, Hydrozoa,
Scyphozoa and Staurozoa. Species of these four classes have
either a free-swimming or attached medusa stage and
many retain the ancestral stage of sessile polyps during
their life cycles. Cnidarians have external radial symmetry,
although many species are either asymmetric or bilateral in
their internal anatomy (Marques and Collins, 2004).
Cnidarian polyps and medusae have a single body opening
0041-0101/$ – see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxicon.2013.05.002
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A.J. Weston et al. / Toxicon 71 (2013) 11–17
that acts as both mouth and anus and is generally surrounded by tentacles bearing stinging cells. The nematocytes (stinging cells), sometimes called cnidocytes, are a
special type of cnidae and constitutes the defining synapomorphic trait of the phylum Cnidaria (Marques and
Collins, 2004). Cnidarians are toxin-producing animals
and, having existed since the Cambrian (Cartwright et al.,
2007) or even Pre-Cambrian (Van Iten et al., 2013), is
possibly the oldest lineage of animals to have evolved
means to inject toxins into prey. While they do not have the
macromorphological apparatus such as the fangs of snakes
to deliver its venomous substances, cnidarians have unique
secretory organelles (nematocysts) within their stinging
cells. There have been numerous studies characterising the
venoms and toxins of many poisonous animals such as
cone-snails, scorpions, snakes and spiders, but by comparison very few cnidarian venoms and toxins have been
examined in detail (Turk and Kem, 2009). This is surprising
because, although most cnidarians do not have harmful
nematocysts that are able to penetrate human skin, contact
with certain cubozoans, such as Chironex fleckeri (the Pacific Sea Wasp), Carukia barnesi and Malo kingi (the latter
two are both commonly called Irukandji Jellyfish) can be
fatal (Fenner and Harrisson, 2000). Other medusozoan
taxa, species of both planktonic (Haddad et al., 2002) and
benthic animals (Marques et al., 2002), are also involved in
human envenomation.
Characterisation of the lethal components of Cubozoa
and Scyphozoa venoms has been elusive because it was
thought that these toxins are large polypeptides that are
unstable. Two haemolysin toxins designated cfTX-1 and
cfTX-2 from C. fleckeri has been examined by cDNA cloning,
protein expression and recent proteomic analysis
(Brinkman and Burnell, 2009; Brinkman et al., 2012).
Tentacle extracts of C. fleckeri have phospholipase A2
(PLA2) activity, as demonstrated in tissue extracts in species from four classes of Cnidaria that include hydrozoan
fire corals (Millepora spp.), the scleractinian coral Pocillopora damicornis and the sea anemones Adamsia carciniopados (Nevalainen et al., 2004) and Urticina crassicornis
(Razpotnik et al., 2010).
Most anthozoan toxins are neurotoxic, ion-channel
modulating peptides, for examples the potassium ion
channel blocking toxin AeK isolated from Actinia equina
(Minagawa et al., 1998) and the toxin ShK isolated from
Stichodactyla helianthus (Castañeda et al., 1995). Sea
anemone neurotoxins and cytolytic enzymes have also
been examined for cardiotonic properties (Suput et al.,
2001), which includes the peptide hK2a isolated from
Anthopleura sp. (Ouyang et al., 2005) and the actinoporins
such as equinatoxin isolated from A. equina (reviewed in
Kem, 1988; Macek et al., 1994). The paucity of data on unequivocal identification of cnidarian toxins stems from
early studies, which had been limited by the use of low
throughput, bioassay-guided chromatography (reviewed in
Suput, 2009). Nevertheless, several novel peptides with
ion-channel blocking activity have been identified in
venom released from the sea anemone Bunodosoma cangicum using liquid chromatography coupled to mass spectrometry (Zaharenko et al., 2008). The first comprehensive
proteome expression profile of toxins from a cnidarian
nematocyst using high-resolution, high throughput protein
analysis has been reported (Balasubramanian et al., 2012).
Although the primary objective of this study was to
examine the structural and mechanistic properties of
nematocysts of Hydra magnipapillata, the putative venoms
were described in Supplemental data. These toxins were
similar to the ion-channel blockers and pore-forming
toxins of sea anemones.
Using a similar high throughput proteomics technique,
we recently described the metaproteome of a symbiont
enriched fraction of the coral Stylophora pistillata (Weston
et al., 2012). Surprisingly, a more complex mixture of
toxins was revealed than was expected, and the presence of
non-dinoflagellate toxins was attributed to the venom
content of contaminating nematocysts. It is intriguing that
so many of the toxins from the venom of S. pistillata were
found to be related to such diverse toxins from such dissimilar organisms (e.g. bacteria, fungi, invertebrates and
vertebrates) compared to that reported from previous
cnidarian studies (Zaharenko et al., 2008; Brinkman et al.,
2012). Therefore, to further examine the provenance, biological complement and evolutionary significance of early
metazoan toxins, we now report a high throughput proteomic profile of putative toxins of the venom from the isolated nematocysts of Olindias sambaquiensis, a common
hydrozoan jellyfish endemic to the South Atlantic coastal
waters of Brazil, Uruguay and Argentina.
2. Materials and methods
2.1. Nematocyst isolation
Animals were collected during a jellyfish monitoring
project along the central coast of São Paulo state (Guarujá
County). Animals were captured through bottom shrimp
trawls (2 cm mesh size) dragged at Enseada beach
(24 590 5200 S 46130 2600 W) at 10 m depth, with each trawl
lasting 10 min on May 7th 2012. The collected animals
measured 4–6 cm in bell diameter. After capture, the animals were transferred into plastic buckets containing
seawater and transported live to the marine laboratory at
the Instituto de Biociências, Universidade de São Paulo. The
animals were identified as O. sambaquiensis (Fig. 1) based
on general morphological characters (gonads on radial
canals; number of radial and centripetal canals per quadrant; tentacles of two types with and without adhesive
pads) according to the descriptions of Vannucci (1951)
and Bouillon (1999). After 2 days held in aquarium conditions (filtered seawater, natural light and at a controlled
temperature of 20 C), two animals had their tentacles
excised. Intact nematocysts were isolated by modification
of the method of Weber et al. (1987). The tentacles were
gently homogenized in a pestle and mortar in cold SuFi
solution (300 mM sucrose containing 50% v/v Ficoll-Paque
Plus, GE Healthcare). This material was kept at 4 C for
30 min and then passed through a 2 mm diameter sieve.
The sample was centrifuged for 10 min at 3000 g at 4 C.
The supernatant containing debris and cell fragments was
removed. The pellet containing intact nematocysts was
carefully suspended and washed three times in cold SuFi
A.J. Weston et al. / Toxicon 71 (2013) 11–17
13
then de-stained. The stacked gel was then divided into
bands and each band excised. In-gel reduction, alkylation,
and proteolytic digestion with trypsin were performed
for each band prior to liquid chromatographic separation
and mass spectrometric analysis (Schevchenko et al., 1996)
as follows. Briefly, cysteine residues were reduced with
10 mM dithiothreitol and alkylated with 55 mM iodoacetamide in 100 mM ammonium bicarbonate to form stable
carbamidomethyl derivatives. Trypsin digestion of the
section was carried out overnight at 37 C in 50 mM
ammonium carbonate buffer and the supernatant was
retained. Peptides were extracted from the excised gel
portion by two washes with 50 mM ammonium bicarbonate and acetonitrile. Each wash involved shaking gel
sections for 15 min before collecting the peptide-containing
extract. The extract was pooled with the initial digestion
supernatant and then lyophilised. The extract residue was
reconstituted in 50 ml of 50 mM ammonium bicarbonate
prior to LC-MS/MS analysis with 5 ml of the sample injected.
2.4. LC-MS/MS
Fig. 1. The South Atlantic jellyfish Olindias sambaquensis F. Müller, 1861
(courtesy of Dr. Alvaro Migotto from the University of São Paulo).
solution. The final material was submitted, after microscopic inspection, for lyophilisation.
2.2. Protein extraction
A volume of 200 ml of 50 mM TEAB was added to two
centrifuge tubes containing approximately 25 mg of the
freeze dried nematocysts. The reconstituted material was
disrupted in a sonic bath (VWR, Lutterworth, UK) for
15 min. The tubes were then centrifuged for 5 min at
10,000 g and 4 C. The supernatants were decanted and
lyophilised before reconstituting in 50 ml TEAB. Both protein extract solutions were pooled and quantified by
Nanodrop (Thermo Scientific, Wilmington, DE, USA)
spectrophotometry.
2.3. Protein preparation
One microlitre of protein extract (equivalent to 88.65 mg
of protein determined by Nanodrop analysis) was amended
to 20 ml in TEAB before adding 20 ml of Laemmli buffer,
heated for 10 min at 90 C and loaded onto a 4–20% polyacrylamide gel for separation by SDS-PAGE electrophoresis.
Electrophoresis was performed using Tris-Glycine buffer
(Sigma, Dorset, UK) at 200 V for approximately 30 min. The
gel was fixed, stained with Coomassie Blue, visualised with
ImageQuant (GE Healthcare, Buckinghamshire, UK) and
Samples were analysed on a Thermo Scientific Orbitrap
Velos Pro mass spectrometer coupled to an EASY-nLC II
(Proxeon) nano LC system. Samples were trapped on a
0.1 20 mm Easy-column (Fisher Scientific, Loughborough,
UK) packed with ReproSil-Pur C18, 5 mm. After loading and
washing of the adsorbed samples the separation was run on
a 0.075 100 mm Easy-column packed with ReproSil-Pur
C18, 3 mm (Fisher Scientific) for 60 min using a gradient
ranging from 5% to 40% of 99.9% acetonitrile: 0.1% formic
acid (buffer B) against 0.1 % formic acid in 99 % H20 (buffer
A) at a flow rate of 300 nL/min for 40 min. Then the
gradient was changed from 40% to 80% of buffer B for
10 min followed by 10 min at 80% B. Mass spectra ranging
from 400 to 1800 Da (m/z) were acquired from proteins in
the Orbitrap at a resolution of 30,000 and the 20 most
intense ions were subjected to MS/MS by CID fragmentation in the ion trap using a threshold of 5000 counts. The
isolation width of precursor selection was 2 units and the
normalized collision energy for peptides was 35. Automatic
gain control settings for FTMS survey scans were 106 counts
and for FT MS/MS scans 104 counts. Maximum acquisition
time was 500 ms for survey scans and 250 ms for MS/MS
scans. Charge-unassigned and þ1 charged ions were
excluded for MS/MS.
2.5. Data analysis
Rawfile data from mass spectrometry analysis were
processed for database spectral matching using Proteome
Discoverer (Thermo Scientific) software. Mascot was
used as the search algorithm with the following variable
modifications: methionine oxidation, phosphorylation on
S/T/Y, deamidation on N/D. Carbamidomethyl cysteine was
selected as a fixed modification. A digestion enzyme of
trypsin was set allowing up to two missed cleavages. The
data were searched with a parent ion tolerance of 10 ppm
and a fragment ion tolerance of 0.8 Da. The numerous MS/
MS spectra relating to these peptide mixtures were analysed using a two-step process. Firstly, a profile was created
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A.J. Weston et al. / Toxicon 71 (2013) 11–17
by searching against three databases 1/ against all entries in
the uniprot_sprot_110118 database (524420 entries), 2/ a
custom database created from Uniprot entries retrieved
from separate keyword searches for ‘toxin’ and ‘venom’,
and 3/ a custom database created from entries retrieved
from the annotated UniProtKB/Swiss-Prot Tox-Prot program (Jungo et al., 2012). All database search results were
reviewed by loading the MASCOT result files into Scaffold
3.5.1 (Proteome Software). Peptides where two or more
spectal counts were obtained were retained for further
analysis. When single spectra where matched to regions of
identical sequence within species homologues contained in
the database(s), a 95% confidence score was required for
the spectra to be retained for further analysis. Secondly, the
three profiles were merged, duplicates removed and the
peptides manually annotated to select only peptides
thought to originate from known toxins or venoms. Spectra
for these toxins and venoms were manually validated and
only those that contained an unbroken series of overlapping b-type and y-type sequence-specific fragment ions
with neutral losses consistent with the sequence were
considered as valid. Proteins that contained similar peptides that could not be differentiated by MS/MS analysis
alone were grouped to satisfy the principles of parsimony.
3. Results
Olindias sambaquiensis has three types of tentacles and
all contain the same three nematocysts (microbasic mastigophore, pseudostenole, and microbasic euryteles). The
use of bottom shrimp trawls inevitably leads to the
amputation of tentacles of any great length. For this reason,
all remaining tentacles were cut from around the bell
margin, ensuring that all three tentacle types were
sampled. Light microscopy revealed that greater than 90%
of nematocyst capsules were not discharged after processing. The size and shape of the nematocysts indicated
that these were microbasic mastigophores, the most common and most often associated with envenomation (AC
Marques pers. comm.). Total soluble proteins extracted
from the nematocyst preparation were first separated by
1D-gel electrophoresis and then 15 gel slices covering
molecular masses up to 200 kDa were analysed by LC-MS/
MS following in-gel tryptic digestion. Since LC/MS/MS experiments are capable of generating MS/MS spectra for
large numbers of peptides within complex mixtures of
proteins, we needed a way to extract information for
numerous novel jellyfish proteins in a timely manner. We
avoided extensive de novo sequencing by adopting an
approach which involved the matching of peptides representing identical regions of sequence to related homologues already contained with the database. Hence, in this
way only a few matching peptides were required to identify, with good confidence, a number of species specific
homologues for particular toxic proteins or venoms.
Manual validation of ms/ms spectra leading to the annotation of the putative toxins given in Table 1 is shown in
Supplemental File 1. Our two step analysis of the soluble
nematocyst proteome of O. sambaquiensis revealed 29
probable toxins similar to venom proteins from diverse
phyla (Table 1). Biological activities of these venoms
include hypothetical cytolysins, neurotoxins, phospholipases and toxic peptidases. Several major families of lytic
enzymes are represented with metalloproteases being
the most prominent. Other enzyme classes include lipid
hydrolytic enzymes that cause membrane dysfunction.
4. Discussion
Identification of toxic peptides in cnidarians is limited to
a small number of toxins (mainly from sea anemones)
which have been identified by traditional protein analytical
methods, and the identification of these toxins is still
under-represented taxonomically. 29 Putative toxins were
identified in this study which used a high throughput
proteomics platform to characterise the nematocyst proteome of a jellyfish, O. sambaquiensis (Table 1). Representatives from all of the important protein superfamilies of
toxins were identified. The most ancient toxic device of
venom elaboration is the ability to interfere with ionchannels by acting as blockers or activators. Ion-channels
are effective targets to subdue prey and predators, and
most toxins that act on these channels result in neurotoxic
or paralysing effects on the target organism. Toxins that
interfere with ion-channels are found in diverse venomous
animals that include molluscs, spiders and scorpions, and
perhaps the best studied examples are from the very large
family of Conus peptide toxins. More than a 100 different
conotoxins have been described, and a great majority of
these peptides selectively target a specific-type ion-channel receptor (Terlau and Olivera, 2004). Because conotoxins
discriminate between closely related subtypes of ionchannels they are widely used as molecular probes (agonists or antagonists) in ion-channel research, several have
direct diagnostic and therapeutic potential, and the
synthetic derivative of the u-conotoxin peptide SNX-111
(Ziconotide marketed as PrialtÒ) was the first to be
approved in 2004 for clinical use by the USA Food and Drug
Administration as a non-opioid analgesic (McGivern, 2007).
Only two of the hits revealed the presence of conotoxin
homologs (Table 1) that likely have neurotoxic activity
(Zhangsun et al., 2006; Biggs et al., 2010). Ion-channel
toxins from Cnidaria (Uniprot Q3C258, Beadlet Anemone),
spiders (Uniprot Q9XZC0, Mediterranean Black Widow
Spider; the theraphotoxins (Uniprot B1P1E7 and B1P1A0,
Chinese Earth Tiger Tarantula) and snakes (Uniprot P85061,
Australian Death Adder) were also identified. In addition to
postsynaptic polypeptide neurotoxins common to venoms
of elapid and hydrophid snakes, are selective antagonists of
nicotinic acetylcholine receptors (Uniprot O12961, Chinese
Krait Snake), revealing the great potential of this proteome
for screening of pharmacologically active compounds.
Hydrolytic enzymes have also been recruited into the
toxic milieu of venoms. For example, phospholipases are
commonly found in the venoms of wasps (Pinto et al.,
2012), scorpions (Diego-Garcıa et al., 2012), sea anemones
(Landucci et al., 2012), spiders (Chaim et al., 2011) and
snakes (Zychar et al., 2010) where they accelerate the toxic
effects of venoms as allergens, inflammatory agents or
as tissue and blood cytotoxins. In snake venoms, phospholipases such as phospholipase A2 (PLA2) are often
abundant with many acting on the pre-synaptic nerve
A.J. Weston et al. / Toxicon 71 (2013) 11–17
15
Table 1
Potential toxin components of venoms identified through similarity searching of ms/ms events relating to peptides isolated following the proteomic analysis
of nematocysts from the hydrozoan jellyfish Olindias sambaquensis.
Peptide
Mode of action
Uniprot
accession
number
Organism with closest homology
Acanmyotoxin-3
Phospholipase A2 lipid degradation
Possible ion channel inhibitor
Zinc metalloprotease
Zinc metalloprotease
Neurotoxin - inhibits nicotinic
acetylcholine receptors
Unknown
Pre-synaptic neurotoxin
P85061
Acanthophis seram (Australian Death adder)
Q3C258
Q9W7S2
P0C7A9
O12961
Actinia equina (Beadlet Anemone)
Deinagkistrodon acutus (Chinese Sharp Nose Viper)
Bothrops jararaca (Brazilian Golden Lancehead Viper)
Bungarus multicinctus (Chinese Krait Snake)
H2CYR5
Q9XZC0
Acrorhagin-1
Aculysin-1
Bothrojaractivase
k-4-Bungarotoxin
Cytotoxic linear peptide
a-Latrocrustotoxin-Lt1a
Metalloprotease
Metalloproteinase
Natriuretic peptide Oh-NP
Proteolysis
Endopeptidase
Hypotensive and vasodepressor
activity
Unknown
Potassium ion channel inhibitor
Lipid degradation
F1CJ78
E9JG55
D9IX98
Pandinus cavimanus (Tanzanian Redclaw Scorpion)
Latrodectus tredecimguttatus (Mediterranean
black Widow Spider)
Buthotus judaicus (Israeli Black Scorpion)
Echis coloratus (Burton’s Carpet Viper)
Ophiophagus hannah (King Cobra)
Q3SAY4
P0C185
Q6H3D0
Oxyuranus scutellatus (Coastal Taipan)
Tityus costatus (Brazilian Scorpion)
Viridovipera stejnegeri (Chinese Green Tree Viper)
Lipid degradation
Zinc metalloendopeptidase
Lipid degradation
A8CG84
Q90220
C5J8C9
Daboia russelli (Eastern Russell’s Viper)
Gloydius halys (Siberian Pit Viper)
Opisthacanthus cayaporum (South American Scorpion)
Neublin-like protein
Butantoxin-like peptide
Phospholipase A2 isozyme
Ts-A1
Phospholipase A2
Prepro-halystatin
Phospholipase A2
isozyme 1
Serine proteinase 8
Serine protease HS112
Sphingomyelin
phosphodiesterase D
U14-Theraphotoxin-Cj1b
k -Theraphotoxin-Cj1b
Toxin AvTX-60A
Toxin Gkn9.1
Toxin PsTX-60A
Turritoxin UID-02
Venom allergen 5
Endopeptidase
Endopeptidase
Lipid degradation
F8S120
Q5WVSP
C0JB55
Crotalus adamanteus (Eastern Diamondback Rattlesnake)
B. jararaca (Brazilian Golden Lancehead Viper)
Sicarius damarensis (South American Assassin Spider)
Possible ion channel inhibitor
Possible ion channel inhibitor
Hemolysis
Unknown
Hemolysis
Possible ion channel inhibitor
Unknown
B1P1E7
B1P1A0
Q76DT2
P0C848
P58911
D5KXG9
A9YME1
Venom allergen 5.01
Venom dipeptidylpeptidase IV
Venom serine protease 34
Unknown
Endopeptidase
Endopeptidase
G7Y9P2
A6MJH9
Q8MQS8
Chilobrachys jingzhao (Chinese Earth Tiger Tarantula)
Chilobrachys jingzhao (Chinese Earth Tiger Tarantula)
Actineria villosa (Okinawan Sea Anemone)
Gemmula kieneri (Kiener’s Turrid Sea Cone)
Phyllodiscus semoni (Night Anemone)
Gemmula speciosa (Splendid Turris Sea Cone)
Microctonus hyperodae (South American
Parasitoid Wasp)
Clonorchis sinensis (Chinese Liver Fluke)
Notechis scutatus (Eastern Tiger Snake)
Apis mellifera (European Honey Bee)
terminal where they hydrolyse plasma membrane phospholipids into lysophospholipids and fatty acids. Such
plasma membrane alterations can trigger synaptic vesicle
exocytosis causing depletion of synaptic vesicles and
increase membrane permeability to calcium ions resulting
in degradation of cell organelles such as mitochondria,
which are essential for the function of nerve terminals
leading to rapid paralysis from permanent membrane
dysfunction of the neuromuscular junction (Montecucco
and Rossetto, 2000; Montecucco et al., 2009; Tedesco
et al., 2009). Proteases are also constituents of the toxic
armament of venomous animals and are well represented
(Table 1) by serine protease and metalloprotease enzymes
in this dataset (for example, Uniprot Q9W7S2, Chinese
Sharp Nose Viper; Uniprot P0C7A9, Brazilian Golden Lancehead Viper; Uniprot E9JG55, Burton’s Carpet Viper; Uniprot F8S120, Eastern Diamondback Rattlesnake). These
proteases are essentially digestive enzymes that in venom
may elicit cytotoxic, hemotoxic, hemorrhagic and myotoxic
effects to subdue and kill prey, and these enzymes may
additionally assist in subsequent prey digestion (reviewed
in Marques et al., 2002). Notable are the pore-forming
toxins characterised in this study (Table 1) with similarity
to other cnidarian toxins that cause disruption to normal
transmembrane ion concentration gradients, some with
haemolytic activity, that include Toxin AvTX-60A (Okinawan Sea Anemone) and Toxin PsTX (Night Anemone).
Previously, toxins from jellyfish venoms, such as the Box
Jellyfish, have only been characterised by use of cDNA
cloning and proteomics (Brinkman and Burnell, 2009;
Brinkman et al., 2012). Detection of Cubozoa and Scyphozoa toxins by direct proteome analysis coupled with
advanced bioinformatic homology searching now offers a
new and powerful technique to characterise venoms from
these potentially lethal animals, which were thought previously to be too labile to characterise by traditional
bioassay-guided chromatographic methods. Of those jellyfish venoms related to spider toxins, the most distinguishable is sphingomyelin phosphodiesterase B (Smase
D), which is a highly destructive component in the venom
of brown spiders that causes dermonecrosis in mammals
and, up to now, Smase D has been described only in
Loxosceles, Sicarius and Drimusa spider venoms (Binford
et al., 2009). The presence of these enzymes in a jellyfish
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A.J. Weston et al. / Toxicon 71 (2013) 11–17
proteome is therefore intriguing, since tissue necrosis has
not been reported in any case of envenomation attributed
to O. sambaquiensis (Kokelj et al., 1993; Haddad et al., 2002).
The most predominant of the jellyfish toxins are family
members of snake venom metalloproteinase (SVMP) disintegrins, with homologs identified in the peptide search.
SVMPs share ancestral genes with matrix metalloproteinase endogenous enzymes (Moura da Silva et al.,
1996) that in the pathology of viper envenomation cause
the disruption of capillary vessels and tissues that is manifest by severe inflammation (Moura da Silva et al., 2007).
Snake and scorpion venom phospholipases A2 were also
identified in the jellyfish proteome (for example, Uniprot
Q6H3D0, Chinese Green Tree Viper; Uniprot A8CG84,
Eastern Russell’s Viper and Uniprot C5J8C9, South American Scorpion). These enzymes disturb blood clotting and
cause local tissue damage that frequently occurs in envenomation by viper snakes. These enzymes are not present in the basal milieu of the Toxicofera reptile venom
system and were recruited most likely during early differentiation of venomous snakes (Fry et al., 2008). Hemotoxic
serine proteases can have both fibrinolytic (coagulant) and
fibrinogenolytic (anticoagulant) activities to disrupt hemostasis, and those that elaborate only thrombin-like activity (blood coagulant factor II) cause fibrinogen
coagulation. In addition, several hemotoxic serine proteases specifically stimulate protein C inactivation of
coagulation factor V to inhibit blood clotting plasminogen
to dissolve fibrin blood clots as a necessary cofactor of the
mammalian thrombinase complex (reviewed in Crammer
and Gale, 2012). Like most serine proteases, venom metalloproteases are also activators of coagulation factor II, and
a few have a unique substrate specificity to activate coagulation factor X (Kini, 2006) or act by disrupting the
integrity of blood vessels to cause fatal hemorrhagic lesions
(Moura da Silva et al., 2007). Moreover, some group II PLA2s
or class P-II SVMPs enzymes detected in the jellyfish proteome appear in Toxicofera reptiles only after differentiation between Elapid and Viperid snakes (Fry et al., 2008;
Juarez et al., 2008). This evidence raises important questions concerning their identification in the jellyfish proteome suggesting independent evolution of toxic enzymes in
these distinct organisms. However, in this case, it is unclear
what would be the evolutionary pressure to select such
toxins with specific action to mammalian blood clotting or
damage to vascular systems, although mature jellyfish are
known to prey on juvenile fish (Underwood and Seymour,
2007).
A number of different phospholipases and metalloproteinases were identified by MS/MS peptide sequences
corresponding to signal-peptides. The most intriguing was
the detection prepro-halystatin from the Siberian Pit Viper
Gloydius halys (Uniprot Q90220). This is a RVV-X peptide
comprising the signal peptide and the N-terminus of the
pro-domain (Takeda et al., 2007) and is a P-IIId representative of SVMPs that activates coagulation factor X and is
very rare, even in snake venoms.
Our results demonstrates the effective utility of a high
throughput proteomics platform for toxin identification,
and for potential bio-discovery these results highlight the
urgency of future biochemical assessments for testing
in vivo the physiological effects of marine toxins. It is estimated that there are up to 700 species of Conus snails, each
possessing around 200 conopeptides in their venom, thus
theoretically there are over 50,000 pharmacologically
active components to be found in the genus Conus venoms
alone (Adams et al., 1999). If the 29 potential toxins presented in Table 1 of our data were extrapolated to all 10,000
species of Cnidaria, 29,000 toxins have yet to be explored as
potential therapeutics, novel templates for drug design or
diagnostic tools. This predicted magnitude of unique toxins
may be realistic given that 55 putative toxins have been
described solely in the proteome of H. magnipapillata
nematocysts (Balasubramanian et al., 2012) and 23 putative
toxins were detected from contaminating nematocysts in
the proteome of a symbiont enriched fraction of the coral S.
pistillata (Weston et al., 2012). From an evolutionary
perspective, the sequence similarities between toxic peptides of the jellyfish O. sambaquiensis and that of phylogenetically unrelated metazoans represent an intriguing
result. Both the amount of toxins and venoms gathered
from a proteome preparation of a single cnidarian species,
together with the degree of variation recovered, are
remarkable. In the view of these new data, the molecular
evolution of super gene families coding for toxins and
venoms arising in the Cnidaria should be addressed.
Acknowledgements
The authors thank Prof Robert Hider for his useful
comments. This work was funded by a co-operation grant
between King’s College London and Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP). Additional
funding to support animal collection under SISBIO license
15031-2 was provided from Brazil by FAPESP (grant 2010/
50174-7), Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Appendix A. Supplementary material
Supplementary material related to this article can be
found at http://dx.doi.org/10.1016/j.toxicon.2013.05.002.
Conflict of interest statement
There are no competing interests.
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