Structure and Biological Activity of a Turripeptide from Unedogemmula

Nov 1, 2017 - Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Cairns, Queensland 4870, Australia. ⊥ Washin...
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Article Cite This: Biochemistry XXXX, XXX, XXX-XXX

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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 Biochemistry XXXX, XXX, XXX−XXX

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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.

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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 B

DOI: 10.1021/acs.biochem.7b00485 Biochemistry XXXX, XXX, XXX−XXX

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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 C

DOI: 10.1021/acs.biochem.7b00485 Biochemistry XXXX, XXX, XXX−XXX

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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.

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.

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 D

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Biochemistry 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.

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 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

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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 E

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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).

day-old ICR mice that lasted for at least 3 h after administration

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,

(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. G

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Article

Biochemistry 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.

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.

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

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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 H

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Biochemistry

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.

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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

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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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biological activities detected requires further investigation. The successful production of the bioactive synthetic peptide will allow us to pursue further structure and receptor binding studies. Insights on the biological role of turripeptide ubi3a would be dependent on obtaining additional information regarding the natural prey and feeding ecology of turrids; these studies are only in their infancy.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Tel: 801-581-8370 Fax: 801-585-2010. ORCID

Julita S. Imperial: 0000-0003-2794-0820 Notes

The authors declare no competing financial interest.

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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 (FT110100226). Electrophysiology experiments were partially supported by Consejo Nacional de Ciencia y Tecnologiá (CONACYT), Grant 153915 to E.L.V. The work of C.A.O. was done at the University of the Philippines Marine Science Institute and at the University of Utah Department of Biology in partial fulfillment of requirements for an M.S. in Chemistry with the University of the Philippines. J.S.I. thanks the DOST for visits to the University of the Philippines Marine Science Institute through the Balik Scientist Program. We thank Dr. Mehdi Mobli for providing the CYANA library file for the NMR structure determination, Dr. William Low for MS analyses, Dr. Robert Schackmann for peptide sequencing and 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 I

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