Article
Cite This: Biochemistry XXXX, XXX, XXX-XXX
pubs.acs.org/biochemistry
Structure and Biological Activity of a Turripeptide from
Unedogemmula bisaya Venom
Carla A. Omaga,†,‡,§ Louie D. Carpio,† Julita S. Imperial,*,‡ Norelle L. Daly,∥ Joanna Gajewiak,‡
Malem S. Flores,† Samuel S. Espino,‡,⊥ Sean Christensen,‡ Olena M. Filchakova,‡,#
Estuardo López-Vera,∇,‡ Shrinivasan Raghuraman,‡ Baldomero M. Olivera,‡ and Gisela P. Concepcion†
†
Marine Science Institute, University of the Philippines, P. Velasquez Street, Diliman, Quezon City 1101, Philippines
Department of Biology, University of Utah, 257S 1400 E, Salt Lake City, Utah 84112, United States
§
Department of Chemistry, University of Utah, 315 1400 E, Salt Lake City, Utah 84112, United States
∥
Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Cairns, Queensland 4870, Australia
⊥
Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, United States
#
Biology Department, School of Science and Technology, Nazarbayev University, Qabanbay Batyr Avenue 53, Astana 010000,
Kazakhstan
∇
Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autonoma de Mexico, 04510 Coyoacan, DF, Mexico
‡
ABSTRACT: The turripeptide ubi3a was isolated from the
venom of the marine gastropod Unedogemmula bisaya, family
Turridae, by bioassay-guided purification; both native and
synthetic ubi3a elicited prolonged tremors when injected
intracranially into mice. The sequence of the peptide,
DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline)
follows the framework III pattern for cysteines (CC−C−C−
CC) in the M-superfamily of conopeptides. The threedimensional structure determined by NMR spectroscopy
indicated a disulfide connectivity that is not found in
conopeptides with the cysteine framework III: C1−C4, C2−C6, C3−C5. The peptide inhibited the activity of the α9α10
nicotinic acetylcholine receptor with relatively low affinity (IC50, 10.2 μM). Initial Constellation Pharmacology data revealed an
excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.
T
species of Unedogemmula, are shown in Figure 1B. The
taxonomy of this group needs revision, and the molluscan
literature has many errors with regard to species assignments; it
is likely that a significant number of species are undescribed.
Their venoms are uncharacterized, and this work and a
proteomic analysis of U. bisaya venom (B. Uberheide and coworkers, manuscript in preparation) are the first toxinological
characterization of any Unedogemmula species.
The venom of conoidean snails has been considered as a
bountiful resource of potential peptide drugs. The conotoxins
from cone snails are peptides that have been shown to
selectively affect the nervous system by binding to a specific
macromolecule such as an ion channel or receptor in the
targeted animal (prey, predator, or competitor).9 Because of
their high selectivity, several conopeptides have been used as
molecular tools to study ion channels and receptors;10,11 some
have been developed as therapeutic leads.12−15 The conopeptide, MVIIA,16 which is marketed as Prialt (generic name
he turrid snails (Turridae), along with the cone snails
(genus Conus in the family Conidae) and auger snails
(Terebridae), comprise the superfamily Conoidea within the
order Neogastropoda.1 Almost all species in Conoidea are
venomous, and the toxins produced by these animals are used
to capture prey, defend against predators, and deter
competitors.2 With almost 700 genera and over 10 000 species,
the turrids are considered to be one of the most diverse groups
among the marine molluscs.1,3,4 Morphologically, there is no
distinct turrid shell shape by which all members can be easily
identified, although one shell feature common to turrids is a slit
or aperture on the outer lip (Figure 1A), which is also referred
to as the “turrid notch”.1 Molecular phylogenetic data suggest
that the family Turridae, as defined by Powell, is polyphyletic.5
In most recent taxonomic work, the classical family has been
more narrowly circumscribed and restricted to forms in the
subfamily Turrinae, as defined by Powell.4
Unedogemmula bisaya was initially included in the genus
Lophiotoma but was reassigned to the genus Unedogemmula
based on molecular phylogenetic data.6,7 The genus Unedogemmula comprises a group of relatively large turrid species
that mostly live offshore in deeper water. Some of the species in
Unedogemmula, including U. bisaya8 and U. unedo, the type
© XXXX American Chemical Society
Received: May 19, 2017
Revised: September 11, 2017
A
DOI: 10.1021/acs.biochem.7b00485
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Figure 1. Shells of Unedogemmula species. (A) Unedogemmula bisaya. The average shell length of the mature turrid reaches up to 5 cm. The image on
the left shows the “turrid notch” on the shell aperture, a common feature of shells of all species of Turridae. The venom duct of U. bisaya is a thin,
mostly white tubular organ (inset, still attached to the bulb) with an average length of 1.5 cm. (B) Shells of eight species in the genus Unedogemmula.
Top row, from left to right: Unedogemmula deshayesii, Kagoshima, Japan; Unedogemmula kilburni, South Mozambique; Unedogemmula capricornica,
Lady Musgrave Island, Queensland Australia; Unedogemmula tayabasensis, Sogod, Cebu, Central Philippines; bottom row, from left to right:
Unedogemmula unedo, Philippines (type species of genus); Unedogemmula bisaya, Vietnam (the focus of this article); Unedogemmula panglaoensis,
Panglao Island, Central Philippines; Unedogemmula f riedrichbonhoef feri, Aliguay Island, Central Philippines.
Ziconotide),17 was approved by the Federal Drug Administration for the treatment of chronic pain. Conopeptides have
been extensively studied to a much greater extent than
augerpeptides (or teretoxins), which are produced by auger
snails (family Terebridae), and turripeptides, which are
produced by turrid snails. Considering the number of turrid
species (>10 000) and assuming that the absence of molecular
overlap in the sets of 50−200 conopeptides in the venom of
each Conus species18 also applies to turrids, the projected
molecular diversity in turripeptides is ≥0.5 × 106.
Little is known about the feeding ecology of turrid species.
Documented observations on their feeding behavior suggest
that they prey on marine worms that belong to the class
Polychaeta in the phylum Annelida.7 Considering the much
higher total molecular diversity in turripeptides compared to
conopeptides, studying the mechanisms of turripeptide action
enlarges the resource pool of potential peptide drug candidates.
In this report, we describe the purification and characterization of the first peptide to be directly isolated and
characterized from the venom of Unedogemmula bisaya8 in
the family Turridae. The peptide sequence of the turripeptide,
currently assigned the temporary name ubi3a, shows a cysteine
pattern that is similar to Framework III (CC−C−C−CC),
which is found in conopeptides belonging to the Msuperfamily.19 The molecular structure of turripeptide ubi3a
was determined by NMR spectroscopy, and its biological
activity was assessed. The amino acid sequence of this
turripeptide indicated a similarity to some alpha conotoxins
that act on neuronal subtypes of the nicotinic acetylcholine
receptor (nAChR);11 thus, its bioactivity was screened on
several nAChR subtypes. ubi3a was found to be active on two
nAChR subtypes, but with relatively low affinity. Therefore,
subsequent tests on DRG neurons were carried out to get a
lead on other possible molecular targets. The newly developed
technique of Constellation Pharmacology20 is a screening
platform for assessing molecular targeting profiles of new
compounds on DRG neurons. The results from an initial
constellation pharmacology experiment using ubi3a and their
significance are discussed.
MATERIALS AND METHODS
Snail Collection and Dissection. Live specimens of
Unedogemmula bisaya were collected using trawl nets set up
in the waters of Cavite, Luzon Island, Philippines. The snails
were identified based on shell morphology (Figure 1), and the
adults with an average shell length of 5 cm were transported to
the laboratory in seawater enriched with oxygen. Dissection was
carried out immediately on a cold block. The venom ducts were
excised, placed in microcentrifuge tubes, and stored at −20 °C
until peptide extraction.
Extraction of Venom Ducts and Peptide Purification
by High Performance Liquid Chromatography (HPLC).
Venom ducts from 200 U. bisaya snails were suspended in 1 mL
of 10% v/v aqueous acetonitrile (CH3CN) containing 0.1% v/v
trifluoroacetic acid (TFA), cut into small pieces, and
homogenized in an Eppendorf tube with a disposable plastic
pestle. The mixture was allowed to stand at 4 °C for 1 h with
occasional vortex mixing and then centrifuged at 12000g for 10
min. The pellet was reextracted using 1 mL of 40% v/v aqueous
CH3CN containing 0.1% v/v TFA. The supernatants were
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supernatant with cold methyl-tert-butyl ether (MTBE) at −20
°C for 20 min. Following centrifugation, the crude peptide
pellet was washed twice with cold MTBE, dissolved in 10% of
solution B and subsequently purified by C18 semipreparative
HPLC (Vydac 218TP510, 250 × 10 mm, 5 μm particle
diameter) over a linear gradient ranging from 10% to 40% of
solution B in 30 min at 4 mL min−1 flow rate. The purity of the
linear ubi3a was assessed with an analytical C18 HPLC run
using a linear gradient ranging from 15% to 45% of solution B
in 30 min at a flow rate of 1 mL min−1.
Oxidative folding of ubi3a was carried out in a buffered
solution consisting of 0.1 M Tris-HCl, pH 7.5, 1 mM EDTA,
and 1 mM each of reduced and oxidized glutathione. Linear
ubi3a was resuspended in 0.01% v/v aqueous TFA solution and
added to the folding solution to a final peptide concentration of
20 μM. The progress of the folding reaction was monitored by
analytical C18 HPLC using the gradient ranging from 15% to
45% of solution B at 1 mL min−1 flow rate. The folding reaction
reached equilibrium after 30 min at room temperature and was
quenched with 8% v/v aqueous formic acid. The folded peptide
mixture was fractionated by HPLC using a semipreparative C18
column over a linear gradient ranging from 5% to 35% of
solution B in 30 min at a flow rate of 4 mL min−1. Quantitation
of the folded ubi3a was done by amino acid analysis at the
University of Utah Health Sciences Center Core Research
Facility.
In order to establish whether the synthetic ubi3a and native
ubi3a were identical, their HPLC retention times were
compared by loading the peptides separately on the analytical
C18 column using a linear gradient of 10−40% of solution B in
60 min at a flow rate of 1 mL min−1. A coelution experiment
was also conducted where the native and synthetic ubi3a
peptides were mixed in a 1:2 ratio and then applied on the C18
analytical column using the same gradient.
NMR Spectroscopy and Structure Calculation. The
spectra of a purified sample of ubi3a (2.5 mg) in 90% H2O/
10%D2O, pH 5.5 were recorded on a Bruker 600 MHz
AVANCE III spectrometer equipped with a cryoprobe. The
spectra included TOCSY, NOESY, COSY, and HSQC and
were recorded at 290 K. The TOCSY and NOESY spectra23
were recorded with mixing times of 80 and 250 ms,
respectively. Sequence specific assignments were made using
the TOCSY and NOESY spectra and angle restraints were
derived using TALOS+.24 Preliminary three-dimensional (3D)
structures were calculated using automated NOE assignment
within CYANA25 without disulfide bond restraints, and analysis
of the distances between the sulfur atoms was carried out to
provide an indication of the likely connectivity.
A final set of 100 structures was calculated with the Cys 2−
12, Cys 3−17, and Cys5−16 disulfide connectivity and the 20
lowest energy structures selected to represent the structure of
ubi3a. Structures were analyzed using Promotif, Procheck
(www.ebi.ac.uk/thornton-srv/databases/pdbsum) and MolMol.26
Nicotinic Acetylcholine Receptor (nAChR) Assay in
Oocytes. Capped cRNAs of the clones for human α9 and
human α10 nAChRs in pSGEM vector were transcribed in vitro
using the mMessage mMachine T7 kit (Ambion, TX, USA) and
purified using the Qiagen RNeasy kit (Qiagen, CA, USA). The
RNA concentration was determined by spectrophotometry at
260 nm. Each Xenopus laevis oocyte was injected with 23 nL of
cRNA (11 ng/oocyte of each subunit). The injected oocytes
were kept at 17 °C in ND96 (96 mM NaCl, 2.0 mM KCl, 1.8
pooled and fractionated by HPLC in a C18 analytical column
(Phenomenex Jupiter, 250 × 4.60 mm, 5 μm particle diameter,
300 Å pore size) with a C18 guard column (Phenomenex, 10 ×
4.60 mm, 5 μm particle diameter). Elution was accomplished
using solution A (0.1% v/v aqueous TFA) and solution B (90%
v/v aqueous CH3CN with 0.1% v/v TFA) in a linear gradient
of 6% to 60% of solution B in 60 min followed by 60% to 100%
of solution B in 20 min at 1 mL min−1 flow rate. The
absorbance of the eluate was measured at 220 and 280 nm
using a diode array detector. The individual peaks were
collected21 and bioassayed by intracranial injection in mice.
Purification of ubi3a from the bioactive fraction was achieved
by HPLC, as described above, but using a shallow linear
gradient of 15−20% of solution B in 25 min.
Intracranial (ic) Mouse Bioassay. Fourteen-day-old and
22-day-old ICR (Institute of Cancer Research) mice (n = 2 per
dose, male and female) were intracranially injected with dried
native fractions or synthetic ubi3a samples; each sample was
resuspended in 20 μL of 0.9% NaCl. Behavior after injection
was observed in the treated mice for at least 3 h simultaneous
with that in control mice injected with pure saline solution.22
The use of mice followed protocols that conform to the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals and approved by the University of Utah
Institutional Animal Care and Use Committee.
Peptide Characterization. The purified peptide was
analyzed by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS) using a
Voyager DE-STR mass spectrometer at The Vincent J. Coates
Foundation Mass Spectrometry Center, Salk Institute.
The amino acid sequence of ubi3a was obtained by Nterminal sequencing at the Health Sciences Center Core
Research Facilities of the University of Utah. The sequence was
analyzed using CLUSTALW2 multiple sequences alignment
program (www.ebi.ac.uk/Tools/msa/clustalw2/) in pairwise
mode to identify conserved sequence regions and to determine
whether the purified peptide has any overlap with known
conotoxins.
Peptide Synthesis by Fmoc Chemistry. The peptide
ubi3a was synthesized using an Apex 396 automated peptide
synthesizer (AAPPTec, KY, USA) applying standard solidphase Fmoc (9-fluorenylmethyloxycarbonyl) protocols. The
peptide was constructed on preloaded Fmoc-Rink amide
MBHA resin (substitution: 0.4 mmol g−1, Peptides International Inc., KY, USA). All standard amino acids were purchased
from AAPPTec, and side-chain protection for each of the
following amino acids was Asp: O-tert-butyl; Arg: 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl; Cys: trityl. N-αFmoc-O-t-butyl-L-trans-4-hydroxyproline was purchased from
NovaBiochem/EMD Chemicals (NJ, USA). The peptide was
synthesized at a 50-μmol scale, using 10-fold excess of amino
acids. Coupling activation was achieved with 0.4 M
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2 M N,N-diisopropylethyl amine
(DIPEA) in N-methyl-2-pyrrolidone (NMP) following the
1:1:2 molar ratio of amino acid/PyBOP/DIPEA. The coupling
reaction was conducted for 60 min and Fmoc deprotection
reaction was carried out for 20 min with 20% (v/v) piperidine
in N,N-dimethylformamide (DMF).
The linear ubi3a was cleaved from the resin by treatment
with Reagent K (82.5/5/5/5/2.5 v/v TFA/water/phenol/
thioanisole/1,2-ethanedithiol) for 3.5 h with constant stirring.
After filtration, the peptide was precipitated from the
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Figure 2. Purification of ubi3a by HPLC using a C18 analytical column as described in Materials and Methods. The elution gradients used are
indicated (blue line). (A) The elution profiles at 220 nm (dark line) and at 280 nm (light line) of the crude venom extract are shown, and the
bioactive fraction is indicated by an arrow. (B) The elution profile of the bioactive fraction from A is shown, and the subfraction with ubi3a is
indicated by an arrow. (C) The purified peptide ubi3a.
Voltage-clamp recordings were done with the membrane
potential kept at −70 mV using a two-electrode voltageclamp amplifier (model OC-725B, Warner Instrument Corp.,
CT, USA). Acetylcholine (ACh)-gated currents were elicited by
a 1 s pulse of 10 μM ACh at a frequency of 1 pulse min−1.
Three ACh responses preceding the toxin application were
averaged in order to establish control response.
For the test response, ubi3a was resuspended in ND96 and
applied to the oocytes expressing α9α10 nAChR subtype for 5
mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES at pH 7.1−7.5)
supplemented with 100 U mL−1 penicillin, 100 μg mL−1
streptomycin, 100 μg mL−1 amikacin sulfate, 160 μg mL−1
sulfamethoxazole, and 32 μg mL−1 trimethoprim.27 Recordings
were made 1−7 days after injection.
The injected oocytes were placed in 30 μL of ND96 and
gravity-perfused with the same solution at a rate of ∼2 mL
min−1. All solutions contained 0.1 mg mL−1 of bovine serum
albumin in order to reduce nonspecific adsorption of toxins.
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major component (Figure 2C) was established to induce
tremors when injected (ic) in mice.
The HPLC elution profile of the crude extract from the U.
bisaya venom ducts revealed a complex mixture of peptidic
components (Figure 2A). MALDI-TOF-MS analysis of the
HPLC fractions showed that the masses of most of the peptides
were within 1000−4000 Da. The peptide, ubi3a, is the major
component of one of the major peaks in the HPLC profile of
the U. bisaya crude venom duct extract, suggesting that it is one
of the highly expressed peptides in the U. bisaya venom.
The sequence of the peptide was determined to be
DCCOCOAGAVRCRFACC-NH2, with O representing the
posttranslationally modified amino acid residue, 4-hydroxyproline. The calculated monoisotopic mass, 1798.65 Da, agrees
with the measured monoisotopic mass of 1798.52 Da, which
was obtained by MALDI-MS in the reflector mode. The
cysteine arrangement follows the framework III pattern
(C1C2−C3−C4−C5C6)) in the M-superfamily conopeptides.19,31 This turripeptide was designated the name of ubi3a,
prior to the determination of its actual molecular target; the
first three letters being derived from the species name, the
Arabic numeral 3 designating the cysteine framework and the
final letter “a” denoting that ubi3a is the first peptide from the
turrid snail, U. bisaya, with the cysteine framework III.
Following the further classification of conopeptides with the
framework III cysteine pattern based on the number of amino
acid residues between the fourth and fifth cysteine residues, the
turripeptide, ubi3a, is similar to the conopeptides belonging to
the M-3 branch.19,32 The M-3 conopeptides are shown with
ubi3a in Table 1. The sizes of the intercysteine regions
min in a static bath before pulsing with 200 μM ACh. The test
response was normalized to control response in order to get “%
response”. Each relevant toxin concentration was tested on
three different oocytes expressing the human neuronal nAChR
in order to establish a concentration−response curve and
IC50.28 The equation: % response = 100/(1 + ([toxin]/
IC50)nH), where nH is the Hill coefficient, was fit to the
concentration−response data using Prism software (Graph Pad
Software Inc., CA, USA).
Calcium Imaging of Native Dorsal Root Ganglion
(DRG) Neurons. Mice lumbar DRG neurons were isolated and
cultured following a previously established protocol.29 Briefly,
dorsal root ganglia (DRG) were harvested from the lumbar
region of three-week-old ICR mice. The DRG cells were
cultured in MEM (Invitrogen) supplemented with 10% fetal
bovine serum, 2.4% glucose, 1% glutamax, and 1% penicillin/
streptomycin. After 18 h of incubation at 37 °C, the DRG cells
were loaded with Fura-2-AM dye at one h before calcium
imaging. The experiments were performed at room temperature (25 °C) in a 24-well plate format using fluorescence
microscopy. Typically, > 300 neurons were imaged per
experiment with individual cells treated as single samples, so
that the individual responses of diverse neuronal subtypes from
the DRG could be examined. Changes in the cytosolic Ca2+
concentration upon depolarization by the application of 20 mM
KCl were measured by taking the ratio of the emissions
resulting from excitation of the dye at 340 and 380 nm. The
cells were depolarized by three 10-s applications of 20 mM KCl.
After the third depolarization, 1 μM turripeptide ubi3a was
applied for a duration of 6 min. After the 6 min incubation, a
depolarizing pulse consisting of 20 mM KCl and 1 μM ubi3a
was applied to determine the effect of ubi3a on the responses of
the neurons to depolarization. Two additional depolarizations
using 20 mM KCl were performed after the application of ubi3a
to determine the reversibility of the responses of the cells to
depolarization in the presence of ubi3a.
To identify specific cellular targets of ubi3a, calcium imaging
experiments were performed on a specific strain of transgenic
mice (Tg(Calca-EGFP)FG104Gsat/Mmucd;
RRID:MMRRC_011187-UCD).30 These mice were provided
by David Ginty (Harvard University). In these mice, neurons
expressing the calcitonin-gene-related peptide (CGRP) were
genetically labeled with green fluorescent protein (GFP) to
identify peptidergic nociceptive neurons. To subclassify these
neurons, the pharmacological agonists (400 μM Menthol, Me;
100 μM allyl isothiocyanate, AITC and 300 nM capsaicin, C)
were applied during calcium-imaging experiments.20 The cells
were labeled with Alexa Fluor 568 Isolectin-B4 (IB4)
(Thermofischer scientific, catalog no. I21412) at the end of
experiments to identify nonpeptidergic neurons.
Table 1. Comparison of ubi3a to Conopeptides within the
M-3 Branch of the M-Superfamily (Jacob and McDougal,
2010)
name
ubi3a
reg12a
Vn3.4
Tx3.5
species
Unedogemmula
bisaya
Conus regius
Conus ventricosus
Conus textile
sequence
reference
DCCOCOAGAVRCRFACC
this study
GCCOOQWCGODCTSOCC
GCCEPDWCDSGCDDGCC
RCCKFPCPDSCRYLCC
33
34
19
differentiate ubi3a from the M-3 conopeptides. While ubi3a has
only one amino acid residue in the first loop (between C2 and
C3), the conopeptides in the M-3 branch have three or four
residues; likewise, the size of the second loop (between C3 and
C4) also diverges from the known M-3 conotoxins (six amino
acid residues for ubi3a and three residues for the Conus
peptides).
Chemical Synthesis and Structure of ubi3a. ubi3a was
chemically synthesized using the standard solid-phase Fmoc
protocol. Oxidative folding in the presence of glutathione
provided a good yield (20−25 nmol for every 100 nmol of
linear form) of the peptide isoform that coeluted with the
native peptide. The mass of the synthetic ubi3a showed that the
synthetic sample was identical to the native ubi3a as
determined by MALDI-MS in the reflector mode: calculated
monoisotopic [MH]+1: 1799.635; determined monoisotopic
[MH]+1: 1799.654.
NMR spectroscopy was used to determine the 3D structure
of ubi3a (Figure 3). Analysis of the sulfur−sulfur distances in
structures calculated without disulfide bond restraints revealed
that in all 15 structures the distance between the sulfur atoms of
RESULTS AND DISCUSSION
Purification and Characterization of Native Turripeptide ubi3a. Turripeptide ubi3a was directly isolated from U.
bisaya venom using mouse-bioassay-directed fractionation.
Intracranial (ic) injection of the crude venom duct extract in
14-day-old mice resulted in apparent paralysis of the limbs
within 20 s followed by death in 20 min. Fractionation of the
crude venom duct extract gave the HPLC profile in Figure 2A;
the fraction indicated with an arrow caused tremors in the
injected mice. Further fractionation indicated the presence of
one major component and a few minor ones (Figure 2B). The
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Figure 3. Structure determination of ubi3a and comparison with RgIA. (A) Superposition of the 20 lowest energy structures of ubi3a determined
using NMR spectroscopy. Structures are superimposed over the backbone atoms and the disulfide bonds are shown in magenta. (B) Ribbon
representation of the lowest energy structure of ubi3a. (C) Superposition of the structural ensemble of RgIA (PDB ID code 2JUT). (D)
Superposition of ubi3a and RgIA over the backbone atoms of residues 9−13. The structures superimposed with an RMSD of 0.34 Å. ubi3a is shown
in blue and RgIA in green.
Cys 3 and Cys 17 was less than 6 Å. Similarly, the distance
between the sulfur atoms of Cys 5 and Cys 16 was also less
than 6 Å in all structures. The sulfur atoms of Cys 2 and Cys 12
were within 6 Å in 11 of the 15 structures. On the basis of this
result it is likely that Cys 3−17 and Cys 5−16 are disulfide
bonded, and consequently, the remaining disulfide bond
involves Cys 2−12. Thus, the disulfide connectivity in ubi3a
is C1−C4, C2−C6, C3−C5.
The structure statistics of structures calculated with the
Cys2−12, Cys3−17, and Cys5−16 connectivity are given in
Table 2. The major element of secondary structure in ubi3a is a
310 helix from residues 14 to 16. Four type IV β-turns are
present between residues 5−8, 7−10, 8−11, and 9−12 and an
inverse gamma turn is present between residues 11−13. The
disulfide bond between residues 2 and 12 conforms to a righthanded hook conformation.35 By contrast, the other two
disulifde bonds do not conform to a regular conformation.
RgIA is a potent inhibitor of the α9α10 subtype of nicotinic
acetylcholine receptor and turripeptide ubi3a shares a sequence
similarity with RgIA (Table 4). Arg7 in loop 1 of RgIA has been
demonstrated to play a critical role in the block of the α9α10
subtype, whereas Arg9 in loop 2 of RgIA is crucial for the
specificity of binding to the α9α10 subtype.36 Ubi3a contains a
three-amino-acid RCR sequence motif (residues 11−13), which
is analogous to RgIA residues 7−9, but the two sets of
structures do not align well when superimposed over these
Table 2. Structural Statistics for the ubi3a Ensemble
Experimental restraints
interproton distance restraints
intraresidue
sequential
medium range (i − j < 5)
long range (i − j ≥ 5)
hydrogen-bond restraints1
disulfide-bond restraints
dihedral-angle restraints
R.m.s. deviations from mean coordinate structure (Å)
backbone atoms (1−17)
all heavy atoms (1−17)
Molprobity Statistics
Molprobity score
Ramachandran (%)
CA Geometry outliers (%)
174
54
53
27
40
6
9
19
0.27 ± 0.11
0.83 ± 0.21
3.44 ± 0.08
90
0
residues. There is some structural similarity in the C-terminal
regions (Figure 3D), which might account for the weak activity
of ubi3a at the α9α10 (Figure 4), but analysis of ubi3a bound to
the receptor is likely required to determine if this structural
similarity plays a role in activity.
Biological Activity of ubi3a in Mice. Intracranial
injection of synthetic ubi3a induced strong tremors in 14F
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Figure 4. Activity of ubi3a on the α9α10 subtype of nAChR. (A) The peptide was applied at 14.3 μM to oocytes expressing the nAChR subtype. The
arrow indicates the first current elicited after equilibration with the peptide for 5 min. C is the control trace right before application of ubi3a. (B)
Concentration−response curve. Each data point is the average of responses obtained from three oocytes, and the curve was generated using Prism;
the IC50 is 10.2 μM (8.5−12.1 μM, 95% CI).
nmol) were also observed in mice treated with the native
peptide (∼3 nmol). In all cases, the tremors were accompanied
by apparent difficulty in walking. All control mice exhibited
normal behavior.
The difference in the mouse bioassay symptoms of the crude
venom extract (partial paralysis followed by death in 20 min),\
and those resulting from the injection of pure ubi3a (tremors
that lasted for at least a few hours) clearly indicate the presence
of other bioactive components in U. bisaya venom that affect
the mammalian CNS.
Effects of Turripeptide ubi3a on the Activity of
Nicotinic Acetylcholine Receptors Expressed in Oocytes.
The bioactivity of ubi3a was tested using two-electrode voltage
clamping on nAChRs expressed in X. laevis oocytes. The
peptide was demonstrated to be a low-affinity inhibitor of the
α9α10 subtype of human neuronal nAChR with an IC50 of 10.2
μM (Figure 4). At 14.3 μM, it was active (22% inhibition of
response) on the rat α3β4 subtype and inactive on the α3β2,
α4β2, α6α3β2β3, α6α3β4, and α7 subtypes
To date, there are three A-superfamily conotoxins that have
been reported to be antagonists of the α9α10 subtype of
nAChR (Table 4): α-conotoxin PeIA from the venom of Conus
pergrandis, α-conotoxin RgIA isolated from Conus regius, and αconotoxin Vc1.1, also known as ACV1, from Conus victoriae.
With values that were obtained from tests that utilized the rat
α9α10 nAChR subtype, RgIA is the most potent with an IC50
value of 1.5 nM. However, a comparison of the activity of RgIA
on the human α9α10 nAChR revealed a 2-order of magnitude
lowered potency (IC50 value of 494 nM), which was accounted
for by a single point mutation (Thr56 to Ile56) in the α9
subunit.37 Thus, in human α9α10 nAChR, the IC50 of ubi3a is
approximately 20 times higher than that of RgIA.
Effects of Turripeptide ubi3a on Mouse Dorsal Root
Ganglion (DRG) Neurons. Turripeptide ubi3a at 1 μM
affected an average of 5% ± 2% of the total neurons. These
effects of ubi3a were observed in the depolarizing pulse
following the incubation of 1 μM ubi3a. As shown in Figure 5B,
day-old ICR mice that lasted for at least 3 h after administration
(Table 3). The same effects induced by the synthetic ubi3a (3.6
Table 3. Effects of ubi3a on Micea
nmol of
ubi3a in
NSS
age
(days)
weight (g)
14
6.82 ± 0.25
0
14
6.78 ± 0.40
1.4
14
6.90 ± 0.23
3.6
14
7.01 ± 0.1 6
7.1
14
6.98 ± 0.30
14.3
22
8.10 ± 0.11
0
22
8.60 ± 0.28
1.4
22
8.90 ± 0.05
3.6
22
9.28 ± 0.37
7.1
22
8.74 ± 0.84
14.3
observations, post-injection (IC)
No adverse effect or unusual behavior
was observed.
Palpable tremors were evident within
3 min and lasted for >3 h.
Tremors started within 3 min and lasted
for >3 h.
These were more intense than those
observed with 1.4 nmol.
Tremors started within 3 min and lasted
for >3 h.
These were more intense than those
observed with 3.6 nmol.
Tremors started within 3 min and lasted
for >3 h.
These were more intense than those
observed with 7.1 nmol.
No adverse effect or unusual behavior
was observed.
The behavior was similar to that of the
control group; if trembling occurred, it
was imperceptible.
Tremors started within 3 min and lasted
for >2.5 h.
Tremors started within 3 min and lasted
for >2.5 h.
These were more intense than those
observed with 3.6 nmol.
Tremors started within 3 min and lasted
for >2.5 h.
These were more intense than those
observed with 7.1 nmol.
a
(ICR mice, n = 2 per dose, male and female). The symptoms
observed in 14-day-old mice are similar to those shown by 22-day-old
mice.
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Table 4. Comparison of ubi3a to Conopeptides That Are Active on the α9α10 nAChR Subtype
name
species
sequence
α9α10 nAChR
IC50 (ref)
ubi3a
α-RgIA
Unedogemmula bisaya
Conus regius
DCCOCOAGAVRCRFACC-NH2
GCCSDPRCRYRCR-OH
α-PeIA
α-Vc1.1
Conus pergrandis
Conus victoriae
GCCSHPACSVNHPELC-NH2
GCCSDPRCNYDHPEIC-NH2
human
human
rat
rat
rat
10.2 μM (this study)
494 nM37
1.49 nM37
6.9 nM38
19 nM11
Figure 5. Selected calcium-imaging traces from dissociated DRG neurons show the effects of 1 μM ubi3a on a subset of neurons. The ratio 340/380
nm is described in MATERIALS AND METHODS. Each arrow represents a 15-s application of 20 mM extracellular potassium (KCl), to depolarize
the neurons. In addition, other pharmacological ligands were applied toward the end of the experiment to identify different subclasses of DRG
neurons; ME: 400 μM menthol, AITC: 100 μM allyl isothiocyanate, C: 300 nM capsaicin. The horizontal bar indicates when ubi3a (1 μM) was
present in the bath. (A) Example of calcium imaging trace responses from 4 neurons that were unaffected by ubi3a. (B) Representative traces from
four neurons that were affected by ubi3a, typically with amplified responses to depolarization (K+). Notably, ubi3a elicited amplified responses in two
subsets of DRG neurons: medium-diameter isolectin B4+ neurons and a subset of small-diameter capsaicin-sensitive CGRP expressing DRG
neurons.
comprise extremely small forms, Unedogemmula may be one of
the few groups of Turridae where bioassay-guided characterization is at all feasible.
Turripeptide ubi3a shares the cysteine framework of the Msuperfamily conopeptides in the M-3 branch; however, due to
size differences in two intercysteine regions in turripeptide
ubi3a and those of the conopeptides belonging to the M-3
branch of the M-superfamily, the disulfide connectivity
indicated in ubi3a is probably different from those present in
Conus peptides within the M-3 branch.
The relatively low affinity of the inhibitory activity of
turripeptide ubi3a on the α9α10 neuronal subtype of human
nicotinic acetylcholine receptor (IC50 = 10.2 μM) indicates that
the effects of ubi3a may not be physiologically significant and
that the true molecular target of the peptide is different. The
initial results using the constellation pharmacology platform
suggest that the excitatory effects of the peptide may be more
physiologically relevant.
The peptide is clearly active in a variety of bioassays using
mammalian systems. Providing a rationale for the spectrum of
1 μM ubi3a amplified the responses from the depolarizing
stimulus (20 mM KCl).
The quantitative analysis revealed that two small categories
of neurons displayed this effect. In Figure 6, a subset of
medium-diameter IB4+ DRG neurons (13 out of 57 neurons,
average area = 393 um2) were affected by ubi3a. Similarly, the
other subset of DRG neurons that were affected by ubi3a were
found to be small-diameter capsaicin-sensitive DRG neurons
that predominantly expressed CGRP (8 out of 62 CGRP-GFP+
neurons, average area = 250 um2). Data were obtained from a
total of 1110 neurons collected from three mice and an average
of 370 neurons per mouse.
CONCLUSIONS
The exploration of the >10 000 species of turrids is clearly a
vast and formidable endeavor, and this study is the first one to
be carried out on a lineage that may be particularly suitable for
systematic investigation. The species in the genus Unedogemmula are among the largest of turrids (U. bisaya is one of the
smaller forms of Unedogemmula). Since many lineages of turrids
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Biochemistry XXXX, XXX, XXX−XXX
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appear in the Uniprot Knowledgebase under the accession
number C0HKK6. The structures are deposited in the protein
data bank (PDB code 5VR1), and the chemical shifts were
deposited in the Biological Magnetic Resonance Bank with the
code 30291.
ABBREVIATIONS
TFA, trifluoroacetic acid; ic, intracranial; IC, inhibitory
concentration; ICR, Institute for Cancer Research; DRG,
dorsal root ganglion; Ach, acetylcholine; nAChR, nicotinic
acetylcholine receptor; NSS, normal saline solution; MEM,
minimal essential medium; CGRP, calcitonin-gene related
peptide; GFP, green fluorescent protein; AITC, allyl
isothiocyanate; Me, menthol; C, capsaicin; IB4, isolectin-B4
■
Figure 6. Effects of ubi3a on subsets of DRG neurons. 1 μM ubi3a
affected two subsets of DRG neurons. An average of 370 ± 70 neurons
per mouse were analyzed. There were 62 small-diameter neurons that
expressed CGRP and 8 of these neurons were affected by ubi3a.
Similarly, there were 57 medium-diameter IB4+ neurons per
experiment and out of these, 13 neurons were affected by ubi3a. In
contrast, large-diameter DRG neurons were unaffected by ubi3a. Data
were obtained from three mice.
■
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successful production of the bioactive synthetic peptide will
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■
REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail: imperial@biology.utah.edu Tel: 801-581-8370 Fax:
801-585-2010.
ORCID
Julita S. Imperial: 0000-0003-2794-0820
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was funded by grants to G.P.C. from the
Department of Science and Technology (DOST) Philippine
Council for Aquatic and Marine Research and Development
through the Philippine PharmaSeas Drug Discovery Program
and to B.M.O. from the National Institute of General Medical
Sciences (GM 48677 and GM103362). N.L.D. was supported
by an Australian Research Council Future Fellowship
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supported by Consejo Nacional de Ciencia y Tecnologiá
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NMR structure determination, Dr. William Low for MS
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My Huynh, Iris Bea Ramiro, and Terry Merritt for assistance in
preparing the figures and manuscript. The protein sequence
data reported in this paper (ubi3a in Unedogemmula bisaya) will
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