J Mol Evol
DOI 10.1007/s00239-016-9764-6
LETTER TO THE EDITOR
Viper Venom Botox: The Molecular Origin and Evolution
of the Waglerin Peptides Used in Anti-Wrinkle Skin Cream
Jordan Debono1 • Bing Xie2 • Aude Violette3 • Rudy Fourmy3 • Marc Jaeger4
Bryan G. Fry1
Received: 2 November 2016 / Accepted: 9 November 2016
Ó Springer Science+Business Media New York 2016
Abstract The molecular origin of waglerin peptides has
remained enigmatic despite their industrial application in
skin cream products to paralyse facial muscles and thus
reduce the incidence of wrinkles. Here we show that these
neurotoxic peptides are the result of de novo evolution
within the prepro region of the C-type natriuretic peptide
gene in Tropidolaemus venoms, at a site distinct from the
domain encoding for the natriuretic peptide. It is the same
region that yielded the azemiopsin peptides from Azemiops
feae, indicative of a close relationship of this toxin gene
between these two genera. The precursor region for the
molecular evolution is a biodiversity hotspot that has
yielded other novel bioactive peptides with novel activities.
We detail the diversity of components in this and other
species in order to explore what characteristics enable it to
be such a biodiscovery treasure trove. The unusual func-
tion of Tropidolaemus venoms may have been selected for
due to evolutionary pressures brought about by a high
likelihood of prey escape.
& Bryan G. Fry
bgfry@uq.edu.au
1
Venom Evolution Lab, School of Biological Sciences,
University of Queensland, St Lucia, QLD 4072, Australia
2
BGI-Shenzhen, Shenzhen 518083, China
3
Alphabiotoxine Laboratory sprl, Barberie 15,
7911 Montroeul-au-bois, Belgium
4
Planet Exotica, 5, Avenue des Fleurs de la Paix,
17204 Royan Cedex, France
•
Introduction
Tropidolaemus (temple vipers) are arboreal snakes with
venom shaped by prey escape pressures to be unusually neu-
rotoxic for pitvipers, selectively blocking the post-synaptic
nicotinic acetylcholine receptors (epsilon subunit) to produce
rapid collapse, spasms, ocular proptosis and tachypnea in prey,
with death resulting from respiratory failure (Fry et al. 2015a
Lin et al. 1995; McArdle et al. 1999; Schmidt and Weinstein
1995; Schmidt et al. 1992). These effects are produced by
short (21 amino acid) peptides, which do not display any
homology to any other known peptide type and thus the gene
precursor identity is unknown (Fry 2005; Fry et al. 2015b).
A skin cream with the trade name SynÒ-Ake has been
developed using the tripeptide synthetic compound dipeptide
diaminobutyroyl benzylamide diacetate (molecular formula of
C23H37N5O7 and a molecular weight of 495.57 g/m) to mimic
the action of the full-length peptide (Balaev et al. 2014;
Trookman et al. 2009). Similar to botox, it blocks signal
transmission and relaxes the muscles at the area of application,
in order to reduce the formation and appearance of wrinkles.
Despite the industrial usefulness of these peptides, their
molecular origin and evolution has remained unelucidated.
It has previously been hypothesised that based on the pro-
line-rich nature, they share evolutionary relationships with
the neurotoxic peptides from Azemiops feae (Brust et al.
2013; Fry 2005; Fry et al. 2003). However, in the absence of
a gene sequence, this link has remained speculative.
Methods
Sequencing, transcriptomics and phylogenetic analysis of
Bicol, Philippines Tropidolaemus subannulatus were
undertaken as per our previously described methods (c.f.
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Fry et al. 2008; Yang et al. 2016). mRNA was extracted
using the Trizol method, and venom gland transcriptomics
were conducted by the IMB Sequencing Facility (Institute
for Molecular Bioscience, The University of Queensland,
St Lucia, Qld, Australia). Libraries were prepared with the
TruSeq Stranded mRNA kit (Illumina, San Diego, CA,
USA), and were sequenced on the Illumina NextSeq (Il-
lumina) 500 using 2 9 150 bp reads and V2 chemistry. To
identify the full sequence of waglerin-Ts-1, forward and
reverse sequences were merged using MacQIIME (Werner
Lab, SUNY Cortland, NY, USA) join_paired_end.py and
matched to the sequence determined by Edman degradation
using standalone BLAST.
Results and Discussion
Sequence blasting revealed cDNA library ORFs containing
regions with homology to waglerin peptides (Fig. 1). Like
waglerins 2 and 4 from T. wagleri, the version from T.
subannulatus Waglerin-Ts-1 contained a -CYPPC- motif
instead of CHPPC as seen in waglerins from T. wagleri
Fig. 1 1 P24335 (waglerin-1/3) Tropidolaemus wagleri, 2 P58930
(waglerin-2/4) Tropidolaemus wagleri, 3 S9L004TR1742 Tropidolae-
mus subannulatus, 4 K4IT20 Azemiops feae, 5 Q9PW56 Bothrops
jararaca, 6 A0A0B4SX88 Philodryas chamisso, 7 A8YPR6 Echis
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J Mol Evol
(see waglerin domain in Fig. 1). This is a change previ-
ously shown to slightly decrease activity (Schmidt et al.
1992). As toxins evolve under selection pressure towards a
more potent form (Fry 2005; Fry et al. 2003; 2010), this
indicates that the -CYPPC- motif is the plesiotypic form,
with the change from Y to H as a structural derivation that
impacts upon functionality. Conversely, the region down-
stream from the second of the two cysteines is CHHR in T.
subannulatus, while it was CHYI in T. wagleri, with the
switch from H to Y being likely to impact upon function-
ality (Fig. 1). Histidine is an amino acids with an electri-
cally charged side chain (basic), while tyrosine is an amino
acid with a hydrophobic side chain (aromatic). While both
amino acid types usually participate in hydrogen bonds as
proton donors or acceptors, the significant variation in
electrochemical characteristics has the potential to impact
upon activity. Thus, future work will examine the struc-
ture–function relationships of these peptides.
Blasting of the full-length precursor sequence revealed
that waglerin peptides not only evolved in the prepro
region of the C-type natriuretic peptide, but that they
specifically evolved at the same site as did the similarly
ocellatus, 8 P23582 Homo sapiens. Post-translationally cleaved
peptides are highlighted in grey, including the ancestral natriuretic
gene, and cysteines are underlined
J Mol Evol

Fig. 2 Bayesian reconstruction

of venom CNP molecular
evolution. Non-venom
outgroups P23582 Homo
sapiens and P55207 Rattus
norvegicus are not shown

123

post-synaptic neurotoxic peptides from Azemiops feae
(Fig. 1) (Brust et al. 2013; Fry et al. 2015b; Utkin et al.
2012a, b). Phylogenetic analysis showed the affinity of the
waglerin peptides with the azemiopsin peptides (Fig. 2),
confirming that the two types of post-synaptic nicotinic
acetylcholine receptor binding peptides share a common
molecular evolutionary history, with the cysteines of
waglerin peptides representing a derived state.
De novo evolution of novel proline-rich bioactive pep-
tides within the propeptide region of natriuretic peptides
has not only occurred in the snake venoms (Fry et al.
2015b), but also independently in the natriuretic peptide
form (B-type) convergently evolved within lizard venoms
(Fry et al. 2015c). This reinforces the dynamic nature of
this region, which is distinct from that encoding the ple-
siotypic natriuretic peptide. Genes coding for the normal
body form do not have such proline-rich regions (Fig. 1).
Another example of de novo evolution of bioactive pep-
tides within a propeptide region has occurred in Psam-
mophis mossambicus which has a myriad of bioactive
peptides (Brust et al. 2013; Fry et al. 2008). These include
novel neurotoxins which have explosively evolved within
the propeptide region of the snake venom metalloprotease
gene, accompanied by stop codons, preventing expression
of the metalloprotease enzyme that was the original func-
tion of this gene.
These variants underscore the dynamic nature of venom
evolution, which not only accelerates the molecular evo-
lution of the plesiotypic proteins, but may result in neo-
functionalisation in gene regions not normally associated
with a secreted product. This study also reinforces the
value of studying evolutionarily diverse lineages as sources
of novel lead compounds for drug design and develop-
ment. The value of using evolution as a biodiscovery is
further borne out by the apparent link of the evolution of
these fast acting neurotoxins with the selection pressure of
prey escape by avian prey of these arboreal tropical
pitvipers.
Acknowledgements BGF was funded by an Australian Research
Council and by the University of Queensland. JD was funded by an
Australian Postgraduate Award.
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