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Zuckerkandl
Prize Editorial (2004) Journal of Molecular Evolution 58:491-492
The Zuckerkandl Prize is awarded by the Journal of Molecular Evolution to the
first author of the paper judged to contain the most significant advance in the
field of molecular evolution published in JME each calendar year. Two papers
were cited for this award:
Fry
BG, Lumsden N, Wüster W, Wickramaratna J,
Hodgson WC and. Kini RM. (2003) "Isolation of a neurotoxin
(alpha-colubritoxin) from a 'non-venomous' colubrid: evidence for early origin
of venom in snakes. Journal of Molecular Evolution 57(4):446-452.
Fry
BG, Wuster W, Kini RM., Brusic V, Khan A,
Venkataraman D and Rooney AP. (2003) "Molecular evolution of elapid snake
venom three finger toxins" Journal of Molecular Evolution 57(1):110-129.
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Amongst
extant reptiles only two lineages are known to have evolved venom delivery
systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded
Lizard)1. Evolution of the venom system is thought to underlie the
impressive radiation of the advanced snakes (2,500 out of 3000 snake species)2,3,4,5.
In contrast, the lizard venom system is thought to be restricted to just two
species, and to have evolved independently from the snake venom system1.
Here we report the presence of venom toxins in two additional lizard lineages
(Monitor Lizards and Iguania) and show that all lineages possessing toxin
secreting oral glands form a clade, demonstrating a single early origin of the
venom systems in lizards and snakes. Construction of gland cDNA libraries and
phylogenetic analysis of transcripts revealed that nine toxin types are shared
between lizards and snakes. Toxinological analyses of venom components from the
Lace Monitor Varanus varius showed potent effects
upon blood pressure and clotting ability, bioactivities associated with a rapid
loss of consciousness and extensive bleeding in prey. The Iguanian lizard Pogona
barbata appears to retain characteristics of the
ancestral venom system, i.e. serial, lobular non-compound venom secreting
glands on both the upper and lower jaws, whereas the advanced snakes and
anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard)
have more derived venom systems characterised by the loss of the mandibular
(lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians,
and anguimorphs form a single clade provides overwhelming support for a single,
early origin of the venom system in lizards and snakes. These results provide
new insights into the evolution of the venom system in squamate reptiles and
open additional new avenues for biomedical research and drug design using
hitherto unexplored venom proteins
Pung YF, Kumar SV, Rajagopalan N, Fry BG, Kumar PP, Kini RM (2005) "Ohanin, a novel protein
from king cobra venom: Its cDNA and genomic organization" Biochemical
Journal (In press).
Ohanin, from king cobra venom, is a
novel protein which induces hypolocomotion and hyperalgesia in mice (Pung, Y.
F., Wong, P. T. H., Kumar, P. P., Hodgson, W. C., and Kini, R. M. (2005) J
Biol Chem. 280, 13137-
13147). It shows weak homology
to PRYSPRY domains (B30.2-like domain). Here we report the complete cDNA and
genomic organization of ohanin. There are two mRNA subtypes differing in their
5'-untranslated regions. Interestingly, cDNA sequence does not show sequence
similarity to any known sequences, including those of B30.2-like
domain-containing proteins. Its full-length cDNA sequence of 1558 bp encodes
for prepro-ohanin with a propeptide segment at the C-terminal. This is the
first reported snake venom protein with a C-terminal propeptide segment. Ohanin
is the first member of a new subfamily of proteins containing B30.2-like domain
with short N-terminal segment. We named this subfamily as vespryns. Southern
hybridization study shows that ohanin is encoded by a single gene. Its genomic
sequence is 7086 bp with five exons
and four introns, and the two types of mRNAs are generated by alternative
splicing of exon 2. Our results indicate that ohanin and vespryns may have
evolved from the same ancestral gene as B30.2 domain.
This study analyzed the origin and
evolution of snake venom proteome by means of phylogenetic analysis of the
amino acid sequences of the toxins and related nonvenom proteins. The snake
toxins were shown to have arisen from recruitment events of genes from within
the following protein families: acetylcholinesterase, ADAM
(disintegrin/metalloproteinase), AVIT, complement C3, crotasin/ defensin,
cystatin, endothelin, factor V, factor X, kallikrein, kunitz-type proteinase
inhibitor, LYNX/SLUR, L-amino oxidase, lectin, natriuretic peptide, nerve
growth factor, phospholipase A2, SPla/Ryanodine, vascular endothelial growth
factor, and whey acidic protein/secretory leukoproteinase inhibitor. Toxin
recruitment events were found to have occurred at least 24 times in the
evolution of snake venom. Two of these toxin derivations (CRISP and kallikrein
toxins) appear to have been actually the result of modifications of existing
salivary proteins rather than gene recruitment events. One snake toxin type,
the waglerin peptides from Tropidolaemus wagleri (Wagler's Viper), did not have a match with
known proteins and may be derived from a uniquely reptilian peptide. All of the
snake toxin types still possess the bioactivity of the ancestral proteins in at
least some of the toxin isoforms. However, this study revealed that the toxin
types, where the ancestral protein was extensively cysteine cross-linked, were
the ones that flourished into functionally diverse, novel toxin multigene
families.
Accelerated evolution of toxins is a
unique feature of venoms, with the toxins evolving via the birth-and-death mode
of molecular evolution. The venoms of sea snakes, however, are remarkably
simple in comparison to those of land snakes, which contain highly complex
venoms. Aipysurus eydouxii (Marbled sea snake) is a particularly unique sea
snake, feeding exclusively upon fish eggs. Secondary to this ecological change,
the fangs have been lost and the venom glands greatly atrophied. We recently
showed that the only neurotoxin (a three-finger toxin) gene found in the sea
snake A. eydouxii has a dinucleotide deletion, resulting in the loss of
neurotoxic activity. During these studies, we isolated and identified a number
of cDNA clones encoding isozymes of phospholipase A2 (PLA2) toxins from its
venom gland. Sixteen unique PLA2 clones were sequenced from the cDNA library
and TA cloning of reverse transcription-polymerase chain reaction products.
Phylogenetic analysis of these clones revealed that less diversification of the
PLA2 toxins has occurred in the A. eydouxii venom gland in comparison to
equivalent terrestrial and other marine snakes. As there is no longer a
positive selection pressure acting upon the venom, mutations have accumulated
in the toxin-coding regions that would have otherwise had a deleterious effect
upon the ability to use the venom for prey capture. Such mutations include
substitutions of highly conserved residues; in one clone, the active site His48
is replaced by Arg, and in two other clones, highly conserved cysteine residues
are replaced. These mutations significantly affect the functional and
structural properties of these PLA2 enzymes, respectively. Thus, in A.
eydouxii, the loss of the main neurotoxin is accompanied by a much slower rate
of molecular evolution of the PLA2 toxins as a consequence of the snake's shift
in ecological niche. This is the first case of decelerated evolution of toxins
in snake venom.
1. Pseudechis species (black snakes)
are among the most widespread venomous snakes in Australia. Despite this, very
little is known about the potency of their venoms or the efficacy of the
antivenoms used to treat systemic envenomation by these snakes. The present
study investigated the in vitro neurotoxicity of venoms from seven Australasian
Pseudechis species and determined the efficacy of black and tiger snake
antivenoms against this activity. 2. All venoms (10 g/mL) significantly
inhibited indirect twitches of the chick biventer cervicis nervemuscle
preparation and responses to exogenous acetylcholine (ACh; 1 mmol/L), but not
to KCl (40 mmol/L), indicating activity at post-synaptic nicotinic receptors on
the skeletal muscle. 3. Prior administration of either black or tiger snake antivenom
(5 U/mL) prevented the inhibitory effects of all Pseudechis venoms. 4. Black
snake antivenom (5 U/mL) added at t 90 (i.e. the time-point at which the
original twitch height was reduced by 90%) significantly reversed the effects
of P. butleri (28 +/- 5%), P. guttatus (25 +/- 8%) and P. porphyriacus (28 +/-
10%) venoms. Tiger snake antivenom (5 U/mL) added at the t 90 time-point
significantly reversed the neurotoxic effects of P. guttatus (51 +/- 4%), P.
papuanus (47 +/- 5%) and P. porphyriacus (20 +/- 7%) venoms. 5. We show, for
the first time, the presence of neurotoxins in the venom of these related snake
species and that this activity is differentially affected by either black snake
or tiger snake antivenoms.
Studies so far have correlated the
variation in the composition of snake venoms with the target prey population
and snakeÅfs diet. Here we present the first example of an alternative
evolutionary link between venom composition and dietary adaptation of snakes.
We describe a dinucleotide deletion in the only three finger toxin gene
expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how
it may have been the result of a significant change in dietary habits. The
deletion leads to a frame shift and truncation with an accompanying loss of
neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a
mutation of a single toxin can have dramatic effects on the whole venom, in
this case likely explaining the 50- to 100-fold decrease in venom toxicity in
comparison to that of other species in the same genus. This is a secondary
result of the adaptation of A. eydouxii to a new dietary habit Å\ feeding
exclusively on fish eggs and, thus, the snake no longer using its venom for
prey capture. This was parallel to greatly atrophied venom glands and loss of
effective fangs. It is interesting to note that a potent venom was not
maintained for use in defense, thus reinforcing that the primary use of snake
venom is for prey capture.
In this study, we have
pharmacologically characterised boigatoxin-A, a three finger toxin isolated
from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the
chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 mM) displayed
poorly reversible postsynaptic blockade as evidenced by the inhibition of
indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous
acetylcholine (1 mM) and carbachol (20 mM). Boigatoxin-A (0.3 0.5 mM) caused a
concentration-dependent depression of the maximum response of cumulative
concentration response curves to CCh (0.680 mM). Boigatoxin-A (1 mM) induced
readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70 100
V) twitches of the prostatic segment of the rat vas deferens. This inhibition
was not significantly attenuated by 8- phenyltheophylline (20 mM) or idazoxan
(1 mM). Boigatoxin-A (1 mM) did not affect a,b-mATP (10 mM) or noradrenaline
(25 mM) responses in unstimulated epididymal segments of the rat vas deferens.
Our data suggests that this toxin has weak postsynaptic neurotoxicity in
skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle
of the rat vas deferens to inhibit the release of neurotransmitter(s), but not
via prejunctional purinergic or adrenergic receptors. This is the first report
of such activity for a toxin isolated from snake venom and reinforces the
largely untapped potential of colubrid venoms.
The brown-headed snake (Glyphodon
tristis) inhabits the forest regions of Papua New Guinea, Torres Strait
Islands, and far northern Queensland, Australia. Although bites by Glyphodon
dunmalli have been reported, G. tristis was regarded as innocuous until 1989
when a healthy 20 year old man was bitten (Sutherland, S.K., Tibballs, J.,
2001. Australian Animal Toxins, the Creatures, their Toxins and Care of the
Poisoned Patient. University Press, Oxford). Treatment of envenomation by this
species is empirical with no specific antivenom available. While no published
studies on the venom of G. tristis are available, unpublished studies suggest
neurotoxicity as being the main symptom of envenomation. In this study, the in
vitro effects of G. tristis venom were examined using the chick biventer
cervicis nerve muscle (CBCNM) preparation. Venom (10 mg/ml) inhibited indirect
(0.2 ms, 0.1 Hz, supramaximal V) twitches of the CBCNM. This inhibition
appeared to be presynaptic in origin as evidenced by the lack of effect of
venom on responses to exogenous acetylcholine (1 mM), carbacho (20 mM) and KCl
(40 mM) in the non-stimulated CBCNM. Prior addition (10 min) of polyvalent
snake antivenom (5 U/ml; CSL Ltd) attenuated twitch inhibition. The venom (1050
mg/ml) also appears to be myotoxic as indicated by a slowly developing
contracture and inhibition of direct (2 ms, 0.1 Hz, supramaximal V, in the
presence of tubocurarine 10 mM) twitches. Myotoxicity was confirmed by
subsequent histological examination of tissues. This myotoxicity was prevented
by the prior addition of polyvalent snake antivenom (30 U/ml). The
phospholipase A inhibitor 4-BPB (1.8 mM) significantly attenuated the
inhibition of indirect and direct twitches of the CBCNM preparation, indicating
the involvement of a PLA2 component in the toxic action of the venom.
Three natriuretic-like peptides (TNP-a,
TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus
(inland taipan) and were also present in the venoms of Oxyuranus
scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus
scutellatus (coastal taipan). They were isolated by HPLC, characterised by
mass spectrometry and Edman analysis, and consist of 35–39 amino acid residues.
These molecules differ from ANP/BNP through replacement of invariant residues
within the 17-membered ring structure and by inclusion of proline residues in
the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or
aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A
over-expressing cells and endothelium-denuded aortic rings) or weakly active
(endothelium-intact aortic rings). TNP-a and TNP-b were also unable to
competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A)
or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms
provide a new platform for further investigation of structure–function
relationships of natriuretic peptides.
Summary
1. The great taxonomic and prey base diversity of colubrids (non-front-fanged
snakes) suggests that their venoms may represent a literal gold mine for
scientists eager to find novel pharmacological probes.
2. While pharmacological characterization is lacking for most of these venoms,
this is even more so with regard to activity of colubrid venoms on the
mammalian autonomic nervous system. This study characterizes the activity of
venom from the colubrid, Boiga dendrophila using in vitro smooth
muscle preparations and the anaesthetized rat.
3. In the prostatic segment of the rat vas deferens, cumulative additions of
venom (1–150 lg ml)1) induced concentration-dependent inhibition of
electrically evoked (0.2 Hz, 0.3 ms, 70–100 V) twitches. The inhibitory effect
of venom (100 lg ml)1) was attenuated by 8-phenyltheophylline (8-PT) (20 lM)
and 8-cyclopentyl-1, 3-dipropylxanthine (20 lM) but not idazoxan (1 lM), or a
combination of ranitidine (0.2 lM) and thioperamide (10 lM). The inhibitory
effect of venom (100 lg ml)1) was augmented by dipyridamole (10 lM) but
abolished by pretreatment with adenosine deaminase (7.5 units/100 ll)
suggesting that it contains components with adenosine A1 receptor activity,
most likely adenosine.
4. In isolated segments of guinea-pig ileum, venom (10–100 lg ml)1) caused
concentration dependent contractions which were inhibited by the muscarinic
receptor antagonist atropine (0.1 lM) but not by the histamine receptor
antagonist mepyramine (0.5 lM).
5. In the anaesthetized rat, venom (5–7.5 mg kg)1, i.v.) caused a hypotensive
effect.
6. Our data suggest that the venom contains components with purinergic and
muscarinic receptor activity.
Lumsden
NG, Fry BG, Kini RM and Hodgson WC
(2004) "In vitro neuromuscular activity of 'colubrid' snake
venoms: clinical and evolutionary implications." Toxicon 43(7):819-827.
In this study, venoms from species
in the Colubrinae, Homalopsinae, Natricinae, Pseudoxyrhophiinae and
Psammophiinae snake families were assayed for activity in the chick biventer
cervicis skeletal nerve muscle preparation. Boiga dendrophila, Boiga cynodon,
Boiga dendrophila gemincincta, Boiga drapiezii, Boiga irregularis, Boiga
nigriceps and Telescopus dhara venoms (10 mg/ml) displayed postsynaptic
neuromuscular activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms,
supramaximal V) twitches. Neostigmine (5 mM) reversed the inhibition caused by
B. cynodon venom (10 mg/ml) while the inhibitory effects of Psammophis
mossambicus venom (10 mg/ml) spontaneously reversed, indicating a reversible
mode of action for both venoms. Trimorphodon biscutatus (10 mg/ml) displayed
irreversible presynaptic neurotoxic activity. Detectable levels of phospholipase
A2 activity were found only in T. biscutatus, T. dhara and P. mossambicus
venoms. The results demonstrate a hitherto unsuspected diversity of
pharmacological actions in all lineages which may have implications ranging
from clinical management of envenomings to venom evolution.
Chetty N, Du
A, Hodgson WC, Winkel K and Fry BG (2004)
"A pharmacological examination of Indo-Pacific sea-snake venoms: efficacy
of antivenom." Toxicon 44(2):193-200
We examined the neurotoxicity of
the following sea snake venoms: Enhydrina schistosa (geographical variants from Weipa and
Malaysia), Lapemis curtus
(Weipa and Malaysia), Laticauda colubrina, Aipysurus laevis, A. fuscus and A. foliosquamatus. Venom from a terrestrial snake, Notechis scutatus (tiger snake), was used as a reference. All
venoms (1 & 3 mg/ml) abolished indirect twitches of the chick biventer
cervicis muscle and significantly inhibited responses to ACh (1 mM) and CCh (20
mM), but not KCl (40mM), indicating the presence of post-synaptic toxins. Prior
administration (10 min) of beaked sea snake antivenom (1 unit/ml) attenuated
the twitch blockade produced by N. scutatus venom and all sea snake venoms (1 mg/ml). Prior
administration (10 min) of tiger snake antivenom (1 unit/ml) attenuated the
twitch blockade of all venoms except those produced E. schistosa (Malaysia and Weipa) and A. foliosquamatus. Administration of beaked sea snake antivenom
(1 unit/ml) at t90 (ie. time at which 90% inhibition of initial twitch height
occurred) reversed the inhibition of twitches (20-50%) produced by the sea
snakes venoms (1 mg/ml) but not by N. scutatus venom (1 mg/ml). Tiger snake antivenom (1 unit/ml)
administered at t90, produced only minor reversal (ie. 15-25%) of the twitch
blockade caused by L. curtus (Weipa),
A. foliosquamatus, L.
colubrina and A. laevis venoms (1 mg/ml). Differences in the rate of
reversal of the neurotoxicity produced by the two geographical variants of E.
schistosa venom, after addition
of beaked sea snake antivenom, indicate possible differences in venom
components. This study shows that sea snake venoms contain potent postsynaptic
activity that, despited the significant genetic distances between the lineages,
can be neutralised with the monovalent beaked sea snake antivenom. However, the
effects of tiger snake antivenom are more variable.
Wickramaratna
J, Fry BG, Loiacono RE, Aguilar MI and
Hodgson WC (2004) "Isolation and characterization at cholinergic nicotinic
receptors of a neurotoxin from the venom of the Acanthophis sp.
seram death adder." Biochemical Pharmacology 68:383-394.
The present study describes the
isolation of the first neurotoxin (acantoxin IVa) from Acanthophis sp. Seram
death adder venom and an examination of its activity at nicotinic acetylcholine
receptor (nAChR) subtypes. Acantoxin IVa (MW 6815; 0.11.0 mM) caused
concentration-dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms,
supramaximal V) and inhibited contractile responses to exogenous nicotinic
agonists in the chick biventer cervicis nerve-muscle, confirming that this
toxin is a postsynaptic neurotoxin. Acantoxin IVa (110 nM) caused
pseudo-irreversible antagonism at skeletal muscle nAChR with an estimated pA2
of 8:36 0:17. Acantoxin IVa was approximately two-fold less potent than the
long-chain (Type II) neurotoxin, a-bungarotoxin.With
a pKi value of 4.48, acantoxin IVa was approximately 25,000 times less potent
than a-bungarotoxin at a7-type neuronal nAChR.
However, in contrast to a-bungarotoxin,
acantoxin IVa completely inhibited specific [3H]-methyllycaconitine (MLA)
binding in rat hippocampus homogenate. Acantoxin IVa had no activity at
ganglionic nAChR, a4b2 subtype neuronal nAChR or cytisine-resistant
[3H]-epibatidine binding sites. While long-chain neurotoxin resistant [3H]-MLA
binding in hippocampus homogenate requires further investigation, we have shown
that a short-chain (Type I) neurotoxin is capable of fully inhibiting specific
[3H]-MLA binding.
Fry
BG and Wüster W (2004) "Assembling an
arsenal: Origin and evolution of the snake venom proteome inferred from
phylogenetic analysis of toxin sequences". Molecular Biology and
Evolution 21(5): 870-883.
We analyzed the origin and
evolution of snake venom toxin families represented in both viperid and elapid
snakes by means of phylogenetic analysis of the amino acid sequences of the
toxins and related non-venom proteins. Out of eight toxin families analyzed,
five provided clear evidence of recruitment into the snake venom proteome prior
to the diversification of the advanced snakes (Kunitz-type protease inhibitors,
CRISP toxins, galactose binding lectins, M12B peptidases, nerve growth factor
toxins), and one was equivocal (cystatin toxins). In two others (phospholipase
A2 and natriuretic toxins), the non-monophyly of venom toxins demonstrates that
presence of these proteins in elapids and viperids results from independent
recruitment events. The ANP/BNP natriuretic toxins are likely to be basal while
the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited
twice. In contrast to the basal recruitment of the galactose binding lectins,
the C-type lectins were shown to be Viperidae only, with the alpha- and
beta-chains resulting from an early duplication event. These results provide
strong additional evidence that venom evolved once, at the base of the advanced
snake radiation, rather than evolving multiple times in different lineages,
with these toxins also present in the venoms of the 'colubrid' snake families.
Moreover, they provide a first insight into the composition of the earliest
ophidian venoms, and point the way towards a research program that could
elucidate the functional context of the evolution of the snake venom proteome.
Fry
BG, Wüster W, Ramjan SFR, Jackson T,
Martelli P and Kini RM. (2003) "LC/MS (liquid chromatography, mass
spectrometry) analysis of Colubroidea snake venoms: evolutionary and
toxinological implications." Rapid Communications in Mass Spectrometry 17: 2047-2062.
The evolution of the
venomous function of snakes and the diversification of the toxins has been of
tremendous research interest and considerable debate. It has become recently
evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated
the evolution of the advanced, front-fanged delivery mechanisms. Historically,
the venoms of snakes lacking front-fanged venom delivery systems
(conventionally grouped into the paraphyletic family Colubridae) have been
largely neglected. In this study we used LC/MS (liquid chromatography with mass
spectrometry) to analyze a large number of venoms from a wide array of species
representing the major advanced snake clades Atractaspididae, Colubrinae,
Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and
Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the
Colubrinae. The large body of data on molecular masses and retention times thus
assembled demonstrates a hitherto unsuspected diversity of toxins in all
lineages, having implications ranging from clinical management of envenomings
to venom evolution to the use of isolated toxins as leads for drug design and
development. Although definitive assignment of a toxin to a protein family can
only be done through demonstrated structural studies such as N-terminal
sequencing, the molecular mass data complemented by LC retention information,
presented here, do permit formulation of reasonable hypotheses concerning snake
venom evolution and potential clinical effects to a degree not possible till
now. The data will also be useful in biodiscovery.
Fry
BG, Lumsden N, Wüster W, Wickramaratna J,
HodgsonWC and. Kini RM. (2003) "Isolation of a neurotoxin
(alpha-colubritoxin) from a 'non-venomous' colubrid: evidence for early origin
of venom in snakes. Journal of Molecular Evolution 57(4):446-452
The evolution of venom in advanced
snakes has been a focus of long-standing interest. Here we provide the first
complete amino acid sequence of a colubrid toxin, which we have called
'_-colubritoxin', isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal non-venomous snake as sold in
pet stores. This potent postsynaptic neurotoxin displays readily reversible,
competitive antagonism at the nicotinic receptor. The toxin is homologous with,
and phylogenetically rooted within, the three-finger toxins, previously thought
unique to elapids, suggesting that this toxin family was recruited into the
chemical arsenal of advanced snakes early in their evolutionary history. LC-MS
analysis of venoms from most other advanced snake lineages revealed the
widespread presence of components of the same molecular weight class,
suggesting the ubiquity of three-finger toxins across advanced snakes, with the
exclusion of Viperidae. These results support the role of venom as a key
evolutionary innovation in the early diversification of advanced snakes and
provide evidence that forces a fundamental rethink of the very concept of
non-venomous snake.
Fry
BG, Wuster W, Kini RM., Brusic V, Khan A,
Venkataraman D and Rooney AP. (2003) "Molecular evolution of elapid snake
venom three finger toxins" Journal of Molecular Evolution
57(1):110-129.
Animal venom components are of
considerable interest to researchers across a wide variety of disciplines,
including molecular biology, biochemistry, medicine and evolutionary genetics.
The three-finger family of snake venom peptides is a particularly interesting
and biochemically complex group of venom peptides, because they are encoded by
a large multigene family and display a diverse array of functional activities.
In addition, understanding how this complex and highly varied multigene family
evolved is an interesting question to researchers investigating the biochemical
diversity of these peptides and their impact on human health. Therefore, the
purpose of our study was to investigate the long-term evolutionary patterns
exhibited by these snake venom toxins in order to understand the mechanisms by
which they diversified into a large, biochemically diverse, multigene family.
Our results show a much greater diversity of family members than what was
previously known, including a number of subfamilies that did not fall within
any previously identified groups with characterised activities. In addition, we
found that the long-term evolutionary processes that gave rise to the diversity
of three-finger toxins are consistent with the birth-and-death model of
multigene family evolution. It is anticipated that this 'Three Finger Toxin
Toolkit' will prove to be useful in providing a clearer picture of the
diversity of investigational ligands or potential therapeutics available within
this important family.
Wickramaratna
JC, Fry BG and Hodgson WC. (2003)
"Species dependent variations in the in vitro myotoxicity of
death adder (Acanthophis) venoms." Toxicological
Science. 74(2): 352-360.
Based on early studies on Acanthophis antarcticus (common death adder) venom it has long been thought that
death adder snake venoms are devoid of myotoxicity. However, a recent clinical
study reported rhabdomyolysis in patients following death adder envenomations,
in Papua New Guinea, by a species thought to be different to A. antarcticus. Subsequently, a myotoxic phospholipase A2 component was
isolated from A. rugosus (Irian Jayan death adder) venom. The present
study examined the venoms of A.
praelongus (northern), A. pyrrhus (desert), A. hawkei
(Barkly Tableland), A. wellsi (black head), A. rugosus,
A. sp. Seram and the
regional variants of A. antarcticus for in vitro myotoxicity. Venoms (10-50 mg/ml) were examined for
myotoxicity using the chick directly (0.1Hz, 2ms, supramaximal V) stimulated
biventer cervicis nerve-muscle preparation. A significant contracture of skeletal
muscle and/or inhibition of direct twitches were considered signs of
myotoxicity. This was confirmed by histological examination. All venoms
displayed high phospholipase A2 activity. The venoms (10-50 mg/ml) of A. sp. Seram, A. praelongus, A. rugosus and A. wellsi caused
a significant inhibition of direct twitches and an increase in baseline tension
compared to the vehicle (n =
4-6; two-way ANOVA, p <
0.05). Furthermore, these venoms caused dose-dependent morphological changes in
skeletal muscle. In contrast, the venoms (10-50 mg/ml; n = 3-6) of A. hawkei,
A. pyrrhus and regional variants of A. antarcticus were devoid of myotoxicity. Prior incubation
(10 min) of CSL death adder antivenom (5 units/ml) prevented the myotoxicity
caused by A. sp. Seram, A. praelongus, A. rugosus and A. wellsi
venoms (50 mg/ml; n = 4-7).
In conclusion, clinicians may need to be mindful of possible myotoxicity
following envenomations by A.
praelongus, A. rugosus, A. sp.
Seram and A. wellsi species.
Fry
BG, Wickramaratna JC, Hodgson WC, Winkel
K, and Wuster W. (2003) "Effectiveness of snake antivenom: species and
regional venom variation and its clinical impact" Journal of
Toxicology-Toxin Reviews. 22, No. 1, pp. 2334,
The ubiquity of venom variation in
snakes poses special problems for the manufacture of antivenom and has
undermined the commercial attractiveness of this class of therapeutic agent. In
particular, it has been amply documented that both interspecific and
intraspecific variation in venom composition can affect the neutralisation
capacity of antivenoms. This may be exacerbated by the selective use of tests
of venom toxicity and antivenom efficacy, such as the lethal dose and ED50,
resulting in inadequate neutralisation of time, rather than dose, dependent
toxins, particularly enzymes involved in defibrinogenating, haemorrhagic and
necrotising venom activities. The clinical consequences can be reduced efficacy
against some important venom activities or even complete treatment failure in
critical envenomations. All these factors, combined with the ongoing reduction
in the number of antivenom manufacturers world-wide, and concomitant contraction
in the range of available antivenoms, present significant challenges for the
treatment of snakebite in the 21st century.
Hodgson WC,
Eriksson CO, Alewood PF and Fry BG.
(2003) A comparison of the in vitro neuromuscular activity of venom from three
Australian snakes, Hoplocephalus stephensi, Austrelaps superbus and
Notechis scutatus: efficacy of tiger snake
antivenom. Clinical and Experimental Pharmacology and Physiology
.30(3):127-132.
Tiger snake antivenom, raised
against Notechis scutatus venom,
is indicated not only for the treatment of envenomation by this snake but also
that of the copperhead (Austrelaps superbus) and Stephen's banded snake (Hoplocephalus stephensi). This study compared the neuromuscular
pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. In the chick
biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all
venoms (3-10 _g/ml) produced inhibition of indirect twitches. In the biventer
muscle, venoms (10 _g/ml) inhibited responses to ACh (1 mM) and carbachol (20
mM), but not KCl (40 mM). The prior (10 min) administration of 1 unit/ml
antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 _g/ml) but was less effective against H.
stephensi venom (10 _g/ml).
While 5 units/ml antivenom attenuated the neurotoxic activity of all venoms.
Administration of 5 units/ml antivenom at t90 partially reversed, over a period
of 3 hours, the inhibition of twitches produced by N. scutatus (10 _g/ml, 41 % recovery), A. superbus (10 _g/ml, 25 % recovery) and H. stephensi (10 _g/ml, 50 % recovery) venoms. All venoms
(10-100 _g/ml) also displayed signs of in vitro myotoxicity. The results of this study indicate that all
three venoms contain neurotoxic activity which is effectively attenuated by
tiger snake antivenom.
Wickramaratna
JC, Fry BG, Aguilar MI, Kini RM and
Hodgson WC (2003) "Isolation and pharmacological characterisation of a
phospholipase A2 myotoxin from the venom of the Irian Jayan death adder (Acanthophis
rugosus)" British Journal of Pharmacology 138, 333 342
It has long been thought that death
adder venoms are devoid of myotoxic activity based on studies done only on Acanthophis antarcticus (Common death adder) venom. However, a recent clinical
study reported rhabdomyolysis in patients following death adder envenomations,
in Papua New Guinea, by a species thought to be different to A. antarcticus (Lalloo et al., 1996: QJM, 89, 25-35). Consequently, the present study examined A. rugosus (Irian Jayan death adder) venom for myotoxicity, and
isolated the first myotoxin (acanmyotoxin-1) from a death adder venom. A. rugosus (10-50 mg/ml) and acanmyotoxin-1 (MW 13811; 0.1 1 mM)
were screened for myotoxicity using the chick directly (0.1Hz, 2ms,
supramaximal V) stimulated biventer cervicis nerve-muscle (CBCNM) preparation.
A significant contracture of skeletal muscle and/or inhibition of direct
twitches were considered signs of myotoxicity. This was confirmed by
histological examination. High phospholipase (PL) A2 activity was detected in
both A. rugosus venom (140.2 ± 10.4 mmol/min/mg; n=6) and
acanmyotoxin-1 (153.4 ± 11 mmol/min/mg; n=6). Both A. rugosus venom (10-50 mg/ml) and acanmyotoxin-1 (0.1 1 mM) caused
dose-dependent inhibition of direct twitches and increase in baseline tension
(n=4-6). In addition, dose-dependent morphological changes in skeletal muscle
were observed. Prior incubation (10 min) of CSL death adder antivenom (5
units/ml; n=4) or inactivation of PLA2 activity with 4-bromophenacyl bromide
(1.8 mM; n=4) prevented the myotoxicity caused by acanmyotoxin-1 (1 m M).
Acanmyotoxin-1 (0.1 mM; n=4) displayed no significant neurotoxicity when it was
examined using the indirectly (0.1Hz, 0.2ms, supramaximal V) stimulated CBCNM
preparation. In conclusion, the myotoxic effects of A. rugosus venom may be of important clinical relevance given the use
of anticholinesterases to reduce the amount of death adder antivenom required
to treat systemic envenomation.
Fry
BG, Wickramaratna JC, Hodgson WC, Alewood
PF,Kini RM, Ho H and Wuster W. (2002) "Electrospray liquid
chromatography/mass spectrometry fingerprinting of Acanthophis (death adder)
venoms: taxonomic and toxinological implications" Rapid Communications
in Mass Spectrometry 16:600-608.
Death adders (genus Acanthophis) are unique among elapid snakes in both
morphology and venom composition. Despite this genus being among the most
divergent of all elapids, the venom has been historically regarded as
relatively quite simple. In this study, LC-MS analysis has revealed a much
greater diversity in venom composition, including the presence of molecules of
novel molecular weights which may represent a new class of venom component.
Furthermore, there exists significant variation between species and populations,
which allow for the LC-MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness
of this technique which underpins fundamental studies ranging from
chemotaxonomy to drug design.
Winkel KD, Fry BG, Hawdon GM and
Carroll T. (2001) "Black snake, brown snake or mistake? A cautionary
tale." Australian Family Physician. 2001 30(9):832.
The snake venom detection kit (SVDK), manufactured by CSL Limited and first
introduced in 19791, has made a unique and valuable contribution to snake bite
management in Australia by facilitating the choice of the correct monovalent
antivenom when indicated. However, despite its robustness 'in the field' the
SVDK has limitations that should always be borne in mind when interpreting the
assay results2. We present a case that reinforces those limitations and
provides important lessons for clinical practice.
Fry
BG, Wickramaratna JC, Jones A, Alewood P,
and Hodgson WC (2001) "Species and regional variations in the
effectiveness of antivenom against the in vitro neurotoxicity of
death adder (Acanthophis) venoms." Toxicology
and Applied Pharmacology 175, 140148.
Although viper-like in appearance
and habit, death adders belong to the Elapidae family of snakes. Systemic
envenomation represents a serious medical problem with antivenom, which is
raised against Acanthophis antarcticus venom, representing the primary treatment. This study
focused upon the major Acanthophis variants from Australia and islands in the Indo-Pacific
region. Venoms were profiled using liquid chromatography - mass spectrometry,
and analysed for in vitro
neurotoxicity (0.310 mg/ml), as well as the effectiveness of antivenom (1-5
units/ml; 10 min prior to the addition of 10 mg/ml venom). The following death
adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland,
South Australia & Western Australia), A. hawkei,
A. praelongus, A. pyrrhus,
A. rugosus, A. wellsi
and venom from an unnamed species from the Indonesian island of Seram. All
venoms abolished indirect twitches of the chick isolated biventer cervicis
nerve-muscle preparation in a dose dependent manner. In addition, all venoms
blocked responses to exogenous acetylcholine (1 mM) and carbachol (20 mM), but
not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom
(1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus and A. hawkei venoms, however,
was markedly less effective against venoms from A. antarcticus (NSW; SA; WA), A. rugosus, A. wellsi and A.
sp. Seram. However at 5 units/ml, antivenom was
effective against all venoms tested. Death adder venoms, including those from A.
antarcticus geographic
variants, differed not in only in their venom composition, but also in their
neurotoxic activity and susceptibility to antivenom. For the first time
toxicological aspects of A. hawkei, A. wellsi, A. rugosus and A. sp. Seram
venoms were studied.
Fry
BG (1999) "Structure-function
properties of venom from Australian elapids." Toxicon
37(1):11-32.
A comprehensive review of venom
components isolated thus far from Australian elapids. Illustrated is that a
tremendous structural homology exists among the components but this homology is
not representative of the functional diversity. Further, the review illuminates
the overlooked species and areas of research.