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The Journal of Venomous Animals and Toxins Including Tropical Diseases logoLink to The Journal of Venomous Animals and Toxins Including Tropical Diseases
. 2021 Oct 18;27:e20210023. doi: 10.1590/1678-9199-JVATITD-2021-0023

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Maged M A Fouda 1,2, Mohammed Abdel-Wahab 2, Amal Mohammadien 3,4, Mousa O Germoush 1, Moustafa Sarhan 2,*
PMCID: PMC8525892  PMID: 34712278

Abstract

Background:

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species.

Methods:

In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail.

Results:

The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned.

Conclusion:

Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.

Keywords: Conus taeniatus, Conopeptides, Conotoxin, HPLC, Mass spectrometry, Cone snail venom

Background

Cone snails are venomous marine mollusks of the genus Conus that can produce small cysteine-rich peptides called conotoxins or conopeptides. These conopeptides display diverse pharmacological activities for prey capture, self-defense, competition, and other biological purposes [1,2]. According to their prey preference, cone snails are commonly classified into three main groups: vermivore, molluscivore or piscivore [3,4]. Conopeptides can modulate the nervous system of their targets by affecting ion channels [5-7]. Therefore, conopeptides have become a platform for discovering new drugs in these exceptionally potent venoms. Moreover, specific components in Conus venoms are used as therapeutics. For example, ω-MVIIA conotoxin is known commercially as ziconotide (Prialt®) and is utilized to cure chronic pain [8-12]. Several other conopeptides are being studied for the treatment of neuropathic pain, epilepsy, hypertension and myocardial infarction [13]. In addition to their contribution to neurobiological and therapeutic applications, conotoxins show high diversity. Conopeptides are stable, relatively small, and structurally diverse with various cysteine frameworks and numerous posttranslational modifications (PTMs) [14-16]. To date, over 800 species of cone snails have been described [17]. Assuming that the venom of each species contains 100 distinct peptides, a repertoire of more than 80,000 conopeptides could be obtained. However, currently only a restricted number of conopeptides (~3%) have been characterized [18,19]. Conopeptides are generated from mRNA-encoded conopeptide precursors that possess signal peptides followed by a variable region and a hypervariable mature peptide [20,21]. At present, conotoxins are classified based on three classification methods: (1) peptide precursor identity, (2) cysteine frameworks, and (3) pharmacological targets and activity. Thus far, twelve families of conotoxins have been identified [18,22].

The worm-hunting cone snail C. taeniatus is commonly distributed along the Egyptian Red Sea. However, there is no information regarding its venom composition. Thus, a proteomic analysis of C. taeniatus venom is of great interest and essential to uncover its various components. In the present study, high-performance liquid chromatography (HPLC) fractionation combined with LC/mass spectrometry (LC-MS) and offline matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-MS was used to assess the conopeptide content in the venom of C. taeniatus. This integrated approach provides an initial outline of C. taeniatus venom constituents and presents information about potential bioactive peptide candidates that may have pharmaceutical importance. To our knowledge, this is the first proteomic analysis of the venom of Red Sea endemic Conus species, and therefore, it provides information that complements and enriches the field of cone toxinology.

Methods

Crude venom extraction

Specimens of C. taeniatus (n = 40) were collected from several sites along the Red Sea coast of Egypt (Figure 1A and 1B). After carefully dissecting the snail venom apparatus, the venom ducts were sliced into small parts to extract the protein contents. For extraction, parts of the venom ducts were suspended in two percent acetic acid (AA) and then centrifuged at 500 × g for 5 minutes at 4°C. The venom was extracted three times, freeze-dried, and then saved at −80°C until use.

Figure 1. (A) General morphology of C. taeniatus shell (bar = 1 cm) and (B) map of the Red Sea in Egypt showing the collection sites of C. taeniatus.

Figure 1.

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LC/MS analysis

LC/MS measurements of C. taeniatus venom were analyzed using an electrospray ion source (ESI) equipped with an LCMS-IT-TOF (Shimadzu). A reversed-phase C18 HPLC (RP-HPLC) column (Cadenza CD-C18, 2.0 150 mm; Imtakt) was used for separation. The column was eluted with 0.1% formic acid (FA) in H2O (solvent A) and 0.1% formic acid in CH3CN (solvent B) at a flow rate of 0.2 mL/min with a linear gradient of 5%-60% solvent B in solvent A, over 55 minutes.

Reduction and carboxyamidomethylation of the venom

The reduction of crude venom (100 μg) was performed in a buffer containing 0.13 M NaHCO3 (pH 8.5), 2.7 M urea, and 35 mM dithiothreitol (DTT), and then the mixture was incubated at 50°C for one hour under argon gas. The combined reaction mixture was then mixed with iodoacetamide (IAA) at a final concentration of 125 mM and incubated for 1 h at 25°C for the alkylation process. The final mixture including the derivatized peptides was analyzed by LC/MS without purification.

MALDI-TOF/MS analysis

MALDI-TOF-TOF/MS analysis was performed on a TripleTOF™ 5600+ (AB Sciex, Canada). The venom samples were first desalted by using MonoSpin reversed-phase C18 columns (GlSciences, Cat. No. 5010-21701) prior to the measurement. The venom was dissolved in a matrix solution containing α‐cyano‐4‐hydroxy‐cinnamic acid (HCCA, 2.5 mg, Bruker Daltonics), dissolved in CH3CN (50%, 0.1% formic acid, Sigma‐Aldrich). One µl of the solution was spotted onto a target plate (Bruker Daltonics) and allowed to dry at room temperature. For high precision, external calibration of the sample batches was carried out to correct possible TOF deviation. Measurements were conducted in positive ion mode, and the MS and MS/MS ranges were 400‐1250 and 170‐1500 m/z, respectively. Mass spectra raw files from the TripleTOFTM 5600+ were converted into Mascot generic format (mgf) files using the script provided by AB Sciex and ProteoWizard. The MS/MS spectra were searched using X! Tandem in a Peptide-shaker (v1.16.38) against the UniProt Conus organism (Swiss-Prot and TrEMBL containing 10684 proteins) with reversed sequences. With initial mass tolerances of 20.0 and 10.0 ppm, the precursor and fragment masses were established, respectively. The carbamidomethylation of cysteine (mass 57.02 amu) was considered to be a static modification, and the oxidation at methionine (mass 15.99 amu), acetylation of the protein N‐terminus (mass 42.01 amu), deamidation of asparagine (mass 0.98 amu), and deamidation of glutamine (mass 0.98 amu) were considered to be variable modifications. Subsequently, the UniProtKB database (www.uniprot.org) and the Entrez PubMed database (www.ncbi.nih.gov) were used to determine the gene superfamilies found in the crude venom of C. taeniatus from known protein fragments.

Results

Molecular mass range and distribution of conopeptides detected by LC/MS

To study the total number of peptide profiles produced in the venom of C. taeniatus, an online LC/MS equipped with an ESI source (LCMS-IT-TOF; Shimadzu) was used to analyze quantified crude venom samples. The LC/MS spectra of the extracted crude venom from C. taeniatus demonstrate the remarkable complexity of conopeptides present in this species (Figure 2A and 2B). The LC/MS analysis revealed approximately 149 components from C. taeniatus venom. Those between 500 and 4,000 Da represented 84% of the conopeptides, and the large peptides (> 4,000 Da) constituted only 16% of all C. taeniatus components (Figure 3, Additional file 1). The molecular mass distribution of the components in C. taeniatus venom in relation to their total ion current intensity showed a bimodal distribution. The molecular mass can be observed with one major mode (500-3,000 Da) and one minor mode (3,000-7,000 Da). These results clearly show that C. taeniatus peptides between 1,000 and 2,000 Da are highly represented compared with those of other molecular masses.

Figure 2. LC/MS chromatograms of (A) native and (B) Cys-alkylated C. taeniatus venom.

Figure 2.

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Figure 3. Molecular mass distribution of the components in C. taeniatus venom detected by LC/MS.

Figure 3.

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Conopeptides with disulfide bridges and cysteine distribution

LC/MS analysis of the DTT-reduced venom component derivatives of C. taeniatus demonstrated an increase in molecular mass by 116.058 × n Da. Disulfide bond-containing components were detected in C. taeniatus venom (Additional file 2). Forty disulfide bond-containing components were confirmed and the cysteine distribution of those conopeptides is shown in Figure 4A and Additional file 3. The number of disulfide bonds ranged from one to five, and the 0-, 2-, and 3-disulfide frameworks were common in the C. taeniatus conopeptides. Peptides contained a 6-cysteine framework, which represents three disulfide bridges, were the most common in the venom. Conopeptides were also divided into “disulfide-poor” (containing two or no cysteines) and “disulfide-rich” (containing four to ten cysteines) groups. The results revealed that 68.75% of the identified peptides were disulfide-rich and the remaining 31.25% were mostly disulfide-poor (Figure 4B and Additional file 3).

Figure 4. Number of disulfide bridges determined based on the mass shift detected by LC/MS after reduction/alkylation of Cys residues.

Figure 4.

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Conotoxin diversity of C. taeniatus with respect to superfamily

A total of 290 peptide fragments (Additional file 4) were detected in the venom of C. taeniatus. A protein sequence similarity search in the database revealed that 170 peptides belonging to 153 conotoxin proteins were assigned to 23 conopeptide superfamilies: the A, B1, B2, E, F, H, I1, I2, M, O1, O2, O3, P, S, T, V, Conkunitzin, Con-ikot-ikot, Conodopin, Cerm, Pmag and two hormone families (Conopressin/Conophysin and prohormone-4). The sequences of these peptides are shown in Table 1. Notably, T, O1, M and O2 constitute the highest percentages (22.87%, 17.56%, 13.1% and 9.8%, respectively) of the known superfamilies. Furthermore, some rare superfamilies of conotoxins were found in the venom of C. taeniatus. Only one peptide fragment sequence was detected from each of the following conotoxin superfamilies: E, H, Con-ikot-ikot, Pmag and Prohormone-4 (Figure 5). Additionally, 48 non-conotoxin proteins were identified including conoporin, protein disulfide isomerase, arginine kinase and Kazal proteinase inhibitor (Table 2).

Table 1. List of identified conotoxin proteins in the venom of Egyptian C. taeniatus with their corresponding gene superfamilies, type of targets and possible applications.

Protein superfamily Sequence Identified protein Protein accession no. No. of proteins Type of target Possible application
A QKGLVPSVITTCCGYDPGTMCPPCR α-Conotoxin S4.4 [C. striatus] AGK23185.1 9 Neuronal/neuromuscular nicotinic acetylcholine receptors (nAChR), K+ channels, Na+ channels Treatment of pain, neuronal disorder diseases such as epilepsy, schizophrenia, nicotinic addiction, Alzheimer’s and Parkinson’s disease
KTAQDNTDLNLITDLNAREDKPK α-Conotoxin [C. betulinus] AMP44778.1
LRECCGRVGPMCPK α-Conotoxin [C. bullatus] P0CY81.1
DERSDMYELKR α-Conotoxin [C. achatinus] ABD33864.1
EVSGSCSSR α-Conotoxin [C. stercusmuscarum] P0DPM2.1
MATLPSCPRHIVR α-Conotoxin [C. bullatus] P0CY88.1
YDKAGNGKYK α-Conotoxin [C. flavidus] ATF27517.1
DMEKKTVEALNTLEGELK α-Conotoxin [C. geographus] BAO65582.1
AAKFKAPALMELTVR α-Conotoxin [C. lividus] AFD18493.1
B1 EVAETVRELDAA Conantokin-Qu [C. quercinus] P0DOZ4.1 4 N-methyl-D-aspartate receptor (NMDA) antagonists Treatment of pain and epilepsy
SSEEEREHAEKLMTFQNQR
SSARSTDDNGNDR Conantokin-R [C. radiatus] P58806.2
AMAELEAKKAQEALK Conantokin-Oc [C. ochroleucus] P0DP00.1
TFEDVEELGKELDANLTK Conantokin-L2.2 [C. literatus] ADZ72981.1
B2 FNEGNKSPFDAEGGFGNFMNFMKENSN Conotoxin precursor superfamily B2 [C. ermineus] AXL95666.1 6 Unknown Unknown
FNEGNKSPFDAEGGFGNFMNFMKEN
DNLGGFMNFMK Conotoxin precursor superfamily B2 [C. ermineus] AXL95405.1
RDGAPADTANLQPFNQGMQAMPA Conotoxin precursor superfamily B2 [C. betulinus] AMP44597.1
DGAPADAANLQSFDPGMQAMPGMPNM
QHSQFNADENKA Conotoxin precursor superfamily B2 [C. magus] QFQ60977.1
EGAPADAANLQSFDPALMPMQGMQG
QMAGKASDQFLPFNPN Conotoxin precursor superfamily B2 [C. magus] DAC80549.1
NFDELVND Conotoxin precursor superfamily B2 [C. magus] QFQ60978.1
E TCVALSSLNECAVREK Conotoxin precursor superfamily E [C. ermineus] AXL95533.1 1 Unknown Unknown
F GQKLMHACSIANKYTYD Conotoxin precursor superfamily F [C. magus] QFQ60998.1 2 Unknown Unknown
LMHACSIANK
VYHSMMGDMVTCLNHFFRR
MNPYSPMNPVNSLYNPMK Conotoxin precursor superfamily F [C. magus] QFQ60999.1
H SLVYVNLKK Conotoxin precursor superfamily H [C. ermineus] AXL95408.1 1 Unknown Unknown
I1 MKLALTFLLILMILPLTTGGK MKLALTFLLILMILPLTTGGKK Conotoxin Im11.13 [C. imprialis] ADZ74137.1 3 Na+ channels activator Treatment of heart failures and pain
FQKTVPNKCAGDIEI Contoxin M11.2 [C. magus] P0C613.1
EDSLNCIETMATTATCMKSNK G115_VD_Superfamily_I1_precursor_conopeptide [C. geographus] BAO65648.1
I2 LSLASSAVLMLLLLFALGNFVGVQPGQITR Conotoxin Im9.12 [C. imprialis] ADZ99328.1 3 K+ channels Treatment of neuronal disorder diseases and cancer
SLNECAVR Conotoxin Sx11.2 [C. striolatus] P0C258.1
NEEDHLRLISMQKGGNLK Conotoxin Gla-TxX [C. textile] Q5I4E6.1
M QDLHPNERTGFILPAMR MLKM group conopeptide Eb3-H0 [C. ebraeus] AEX60108.1 20 K+ channels, Na+ channels and nAChR Useful for treatment of pain, stroke, epilepsy, neuronal disorder diseases and cancer
QDISPNERKR MLKM group conopeptide Vr3-DPP03 [C. varius] AEX60203.1
YAENKQDLNPAER MLKM group conopeptide [C. magus] DAC80581.1
YGWTCWLGCSPCGC Mu-conotoxin PnIVB [C. pennaceus] P58927.2
LTYHAGCPVLMGNKWIANKWIWHYGNMFR conotoxin superfamily M [C. eburneus] ACV87167.1
KYMYNIQR conotoxin superfamily M [C. magus] DAC80582.1
LATSLGDLR conotoxin precursor superfamily M [C. ermineus] AXL95471.1
QDLNLDERR MLKM group conopeptide Co3-S01 [C. coronatus] AEX60110.1
SLKCCSGR Conotoxin superfamily M [ C. magus] QFQ61028.1
VDGLNHPEPSFGED Conomarphin conotoxin precursor analog Bt2 [C. betulinus] AGE10520.1
NVENKQDLNLDKR MLKM group conopeptide Ec3-DA01 [C. emaciatus] AEX60080.1
QDLNLDKRR
RGIKLLAQR
DVKCIGSCDSTVWHRV MLKM group conopeptide [C. distans] AGE10511.1
QDLNPDERMKFK MLKM group conopeptide Cp3-I02 [C. capitaneus] AEX60051.1
MQDDISSEQNPLLEKR Mu-conotoxin BuIIIB [C. bullatus] C1J5M6.1
DQDLVEQYRNLK conotoxin superfamily M [C. magus] QFQ61035.1
RCCRVICSR Conotoxin TxMMSK-01 [C. textile] Q9BPJ1.1
EDGKSAALQPWFD Conotoxin Lt3.7 [C. literatus] ADZ99311.1
TLLRQWNK Conotoxin Reg3.17 [C. regius] A0A2I6EDN0.1
QSVTLSNDNRLTADHPNTFYLLIR Conotoxin precursor superfamily M [C. ermineus] AXL95450.1
SVGSSTADCLDNK Conotoxin precursor superfamily M [C. rattus] AEX60323.1
O1 LTCMMIVAVLSLTAWTFATADDPR Conotoxin precursor superfamily O1 [C. episcopatus] BAS22635.1 27 Ca+ channels, K+ channels, Na+ channels and nAChR Useful in pain, stroke, hypertension, arrhythmias, epilepsy, neuronal disorder diseases and cancer
FDNDCCDACMLREKQQPICAV Conotoxin precursor superfamily O1 [C. miles] Q3YEG3.1
TASKLLQGSQVAASPL Conotoxin precursor superfamily O1 [C. ermineus] AXL95342.1
NELENLFPKARHEMD Conotoxin precursor superfamily O1 [C. ermineus] AXL95353.1
NELESYAYSLKNQVNDKEK
KCLGFGEACLMFYSDCCSFCVRAVCL Conotoxin precursor superfamily O1 [C. episcopatus] BAS22577.1
GKGAPCRK Conotoxin precursor superfamily O1 [C. magus] 1OMN_A
NGLGNLFSNAHHEMK Conotoxin precursor superfamily O1 C. episcopatus] BAS22395.1
MKNPEASKLNNR Conotoxin precursor superfamily O1 C. episcopatus] BAS22442.1
QVYRAVGLTDKMR Conotoxin precursor superfamily O1 [C. magus] QFQ61065.1
ARNELQKLEASQLNER Conotoxin precursor superfamily O1 [C. virgo] Q3YED8.1
DKQEHPAVRGSDDMQDSEDLK Conotoxin precursor superfamily O1 [C. arenatus] Q9BP77.1
SHNCCGVCMIRKLPK Conotoxin precursor superfamily O1 [C. ermineus] AXL95510.1
ALMSTGTNYRLLK Conotoxin GeXXXIA [C. generalis] A0A2I4QAG8.1
NIDGREASGLRK Conotoxin precursor superfamily O1 [C. ermineus] AXL95529.1
RYYTCVALS Conotoxin precursor superfamily O1 [C. episcopatus] BAS22584.1
YYTCVALS
MLSMLAWTLMTAMVVMNA Conotoxin precursor superfamily O1 [C. episcopatus] BAS22670.1
CIVGTPCHVCRSQSKSCNGWLGK Conotoxin Bu6 [C. bullatus] P0CY65.1
LEKRDCQDK Conotoxin Tx6.6 [C. textile] P0DPM4.1
GLGYLTFCPSNLGTTLR Conotoxin precursor superfamily O1 [C. episcopatus] BAS22548.1
LDFGDLDPKNE Conotoxin precursor superfamily O1 [C. ermineus] AXL95735.1
CKSPGTPCSKGMR Conotoxin precursor superfamily O1 [C. geographus] BAO65621.1
VGTGLGEYMFDK Conotoxin ArMKLT1-02 [C. arenatus] Q9BP99.1
MLSMLAWTLMTAMVVMNA Conotoxin precursor superfamily O1 [C. episcopatus] BAS22670.1
LNHPEPDFGDLSKLGFGNLDPGG Conotoxin precursor superfamily O1 [C. episcopatus] BAS22556.1
LSATPGFKD Conotoxin precursor superfamily O1 [C. episcopatus] BAS22677.1
NLLKIGTRGQGGCVPPGGGR Conotoxin precursor superfamily O1 [C. geographus] BAO65614.1
MTKRCMHPEGGCR Conotoxin AbVIE [C. abbreviates] Q9UA85.1
O2 KEVGNPKASK Contoxin TxMEKL-P2 [C. textile] Q9BPA9.1 15 Neuronal pacemaker channels and Ca+ channels Useful in pain, hypertension, arrhythmias, epilepsy
IMEKLTIMLLVAAILMLT Contoxin [C. ermineus] AXL95503.1
QEDPVVRSSDKVQR Contoxin Bt6.2 [C. betulinus] AGE10507.1
EKLTVLILVATVLLAIQVLVQSDREKPLK Contoxin Tx15a [C. textile] AGK23206.1
EMINVLSKGKTNAER Contoxin MaI51 [C. marmoreus] QFQ61084.1
LIILLLVAAVLMSTQALFQEKRPMKK Contoxin Vc6.12 [C. victoriae] G1AS78.1
EKLTVLILVAIVLLTIQVLGQSDRDK Contoxin Ml15b [C. miles] C8CK75.1
SSVDEKIKNK Conotoxin precursor superfamily O2 [C. episcopatus] BAS22689.1
MLSGNNEKR Conotoxin precursor superfamily O2 [C. magus] QFQ61085.1
MEKLTILLLVAALLVLTQALIQGGVEK Conotoxin Fla6.7 [C. flavidus] AFU50755.1
AEINFLSK Conotoxin precursor superfamily O2 [C. terebra] AGK23197.1
MEKLTILLLVAAVLMSTQALIQEKRPK Conotoxin VnMEKL-0222 [C. ventricosus] AXL95751.1
YYTCVALSSLNECAVR Conotoxin VnMEKL-0111 [C. ventricosus] Q9BPC4.1
AKIDFSNR Conotoxin Vc6.10 [C. victoriae] G1AS76.1
LMSAQALMQEK Conotoxin precursor superfamily O2 [C. ermineus] AXL95556.1
O3 AKPEFMAAAAK Conotoxin precursor superfamily O3 [C. ermineus] AXL95644.1 3 Unknown Induce sleep in mice
GEKQAMQR Conotoxin precursor superfamily O3 [C. ermineus] BAO65632.1
EQNKTCCGLTNGRPRCVGVCFG Conotoxin VnMSGL-0123 [C. ventricosus] Q9BP59.1
P LSLASSAVLMLLLLFALGNFVGVQPGQITR Conotoxin Im9.12 [C. imprialis] ADZ99328.1 2 May target a glycine receptor Induce hyperactivity and spasticity in mice
QHSSDAVDLQTGQIK Conotoxin Fla9.1 [C. flavidus] AFU50766.1
S KTHLKSGFYR Conotoxin Tx8.1 [C. textile] AGK23266.1 6 Serotonin receptor or nAChR Treatment of pathologies including neuropathic pain.
CYCKNGGR Conotoxin precursor superfamily S [C. ermineus] AXL95481.1
YDNNLCGK Conotoxin precursor superfamily S [C. ermineus] BAS22723.1
QLKCHRNFSVDK Conotoxin precursor superfamily S [C. ermineus] BAS22797.1
CFGESNCR Conotoxin precursor superfamily S [C. ermineus] BAS22751.1
NKIQRSDYLK Conotoxin precursor superfamily S [C. ermineus] BAS22859.1
T DDMSPASFHDNAKRTQHVFWSK Conotoxin precursor superfamily T [C. episcopatus] BAS25056.1 35 Noradrenaline transporter, somatostatin-3 receptor and possibly Ca2+ channels and Na+ channels. Treatment of pain, stroke, hypertension, arrhythmias, epilepsy
CLPVLIILLLLTASGPSIEARPR Conotoxin precursor superfamily T [C. episcopatus] BAS25321.1
ETDKNLDAVR Conotoxin Ts5.7 [C. tessulatus] AGK23242.1
ECCSDGWCCPQNLK Conotoxin precursor superfamily T [C. episcopatus] BAS23229.1
RCLPVFVILLLLIAFAPSVDVRPKAK Conotoxin precursor superfamily T [C. episcopatus] BAS23428.1
RCLPVLVILLLLIASAPSVDVRPKAK Conotoxin precursor superfamily T [C. episcopatus] BAS25341.1
DDMPLASFHANVK Conotoxin Mr5.2 [C. marmoreus] Q6PN84.1
IQMEKTTVDALNTL Conotoxin precursor superfamily T [C. episcopatus] BAS25067.1
MRCLPVFVILLLLIASAPSVDVLLKAK Conotoxin precursor superfamily T [C. episcopatus] BAS23994.1
TLQTPLNK Conotoxin precursor superfamily T [C. episcopatus] BAS24922.1
LCLPVFIILLLLVSPAATLRVQSKLER Conotoxin Qc5.4 [C. quercinus] AGK23244.1
MRCLPVLIILLLLTASGPSVDAKVHLK Conotoxin precursor superfamily T [C. episcopatus] BAS25470.1
EPYFGEDKLDFGDLDPK Conotoxin precursor superfamily T [C. episcopatus] BAS24907.1
CLPVFVILLLLIASTPNVDALPKTK Conotoxin precursor superfamily T [C. episcopatus] BAS24300.1
FGYRNMTLDETPAKCPWM Conotoxin precursor superfamily T [C. episcopatus] BAS24141.1
DDVPLASFHEDANGILQMLWK Conotoxin Pu5.1 [C. pulicarius] BAS24327.1
NLQTLLNK Conotoxin precursor superfamily T [C. episcopatus] BAS24637.1
LCLPVFIIPLLLVSPAATLRVQSKLER Conotoxin Lv5.5 [C. lividus] AGK23255.1
CGKNCCPKGWGCIR Conotoxin Im5.1 [C. imprialis] Q9U6Z5.1
MRCLPVFVILLLLIASTPIVDALLKTK Conotoxin precursor superfamily T [C. episcopatus] BAS24288.1
CSEIKENDFG Conotoxin precursor superfamily T [C. episcopatus] BAS24381.1
CLPVFIILLLLIPSALSLIAKPK Conotoxin precursor superfamily T [C. magus QFQ61106.1
QKTKDDIPQASFQDNAK Conotoxin precursor superfamily T [C. episcopatus] BAS23977.1
QLSVELDLQR Conotoxin Vx5.2 [C. vexillum] AGK23237.1
RILQVLENK Conotoxin precursor superfamily T [C. episcopatus] BAS23633.1
GGGPLSSFRDNAK Conotoxin precursor superfamily T [C. episcopatus] BAS25155.1
KGVEAVIK Conotoxin Ts5.5 [C. tessulatus] Q9BP46.1
RCLPVFVILLLLIASAPSVDALPR Conotoxin precursor superfamily T [C. episcopatus] BAS23533.1
CFPVFVILLLLIATAPSVDVRPKAK Conotoxin precursor superfamily T [C. episcopatus] BAS23041.1
MGEVPLNTCPEL Conotoxin precursor superfamily T [C. episcopatus] BAS24903.1
DMPLASSQANVK Conotoxin mr5.3 [C. marmoreus] Q6PN83.1
MTDSYTGENECFYDNNLCGK Conotoxin precursor superfamily T [C. episcopatus] BAS25019.1
TGENECFYDNNLCGK
YTGENECFYDNNLCGK
KPAQFNSPQEVKEYVRK Conotoxin precursor superfamily T [C. ermineus] BAS23732.1
TVPDDVNAER Conotoxin Ts5.5 [C. tessulatus] Q9BPF7.1
ESKAKLDSLGR Conotoxin precursor superfamily T [C. episcopatus] AXL95513.1
V SDPPVSLVKVDCTAETK Conotoxin Vi15a [C. virgo] B3FIA5.1 2 Unknown Unknown
LGLTEFEAIQEMR Conotoxin Fla15.3 [C. flavidus] AFU50801.1
Con-ikot-ikot SDVERALNIEIRR Con-ikot-ikot [C. magus] QFQ60982.1 1 α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors Inhibiting channel desensitization
Conkunitzin LEPDAGLCR Conkunitzin [C. ermineus] AXL95648.1 3 K+ channels Neuronal disorder diseases and cancer
ISMQKGGNLK Conkunitzin [C. magus] DAC80559.1
SMQKGGNLK
RMGEVPLNTCPELFE Conkunitzin [C. ermineus] AXL95589.1
Conodipin HFLAACDR Conodipin [C. magus] DAC80618.1 4 Conotoxin with PLA2 activity. Potent neurotoxicity, Neurologic application for pain reduction
QVASDRATSIAR Conodipin [C. purpurascens] QEO32927.1
LISMQMGGNLK Conodipin [C. buxeus loroisii] ATJ04131.1
ACFIRNCPK Conodipin [C. ermineus] AXL95508.1
Cerm CSTDSDHTITVVQSYINGYPEKR Cerm-13 [Pionoconus magus] QFQ61140.1 2 Unknown Unknown
QVCPTMTDSYTGENECFYDNNLCGK
CVALSSLNECAVR
CSSRCYCKNGGR
LTPDKVEMATLTR Cerm-18 [C. ermineus] AXL95455.1
Conopressin/Conophysin RATKECMYCSLGQCVGPR Conopressin/ Conophysin [C. ermineus] AXL95508.1 2 Vasopressin receptors Antidiuresis, stimulation of liver glycogenolysis, and central regulation of somatic functions
ACFIRNCPK
DPISVKVLCR Conopressin/Conophysin [C. magus] DAC80606.1
Prohrmone-4 YALRLATSLGDLRWSLALTDENINNTK hormone superfamily prohormone-4 DAC80609.1 1 Unknown Unknown
Pmag QVALGLEEGWR Pmag295 ferritin [C. magus] AXL95451.1 1 Iron receptor Unknown
Total 170 153
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Figure 5. Percentage composition of conotoxin superfamilies and non-conotoxin proteins in C. taeniatus venom proteome. The relative abundance of conopeptide superfamilies in C. taeniatus venom is expressed as the percent relative abundance of total identified proteins by LC-MS/MS.

Figure 5.

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Table 2. List of non-conotoxin proteins identified in Egyptian C. taeniatus venom by using MALDI/TOF/MS.

Protein family Sequence Identified protein Protein accession no. No. of proteins Biological process
Conoporin VSCIIQVENWTR Conoporin [C. lividus] ATG85040.1 8 Punching Holes in Membranes. Osmotic stress and cell death of microorganisms
LVASEVVTPG
AEGAMTNGNHAQVK Conoporin [C. ebraeus] ASF90529.1
VIVRPTRNNWK
YSNWMGLGMTR
VQVENWTRYPLMTPR
GKREAFAVR Conoporin [C. magus] DAC80623.1
LQTIYAKDK Conoporin [C. consors] P0DKQ8.1
EAFAVQMPSSGR Conoporin [C. ermineus] AXL95502.1
RFVLMWSAPFDFN Conoporin [C. lividus] ATG85040.1
LGLTEFEAIQEMR Conoporin [C. monile] ANC48005.1
ALQQKRSLQR Conoporin [C. magus] QFQ61164.1
Protein disulfide isomerase TFIDSDEVIVMGFFKDQEGKGA Protein disulfide isomerase [C. ebraeus] ASF90532.1 27 Oxidative folding of conopeptides
VLFIYLDTAKT
EDVVFGITSEDSVFKEHK Protein disulfide isomerase [C.geographus] AMM62652.1
GKVLFIYLDTAKEENEHI Protein disulfide isomerase [C. ermineus] AXL95726.1
ADSPAMRLIQLGEDLAK Protein disulfide isomerase [C. magus] QFQ61177.1
LFIYLDTAKEESEHIMGFFGLKAADAPTMR Protein disulfide isomerase [C. lividus] ATG85035.1
TENFDKFIK
FFMNGQSVDYTGGRQ Protein disulfide isomerase [C. bullatus] AMM62658.1
GSNIKLAKVDATVEK Protein disulfide isomerase [C. ermineus] AXL95393.1
LAKVDIIAEMD Protein disulfide isomerase [C.geographus] AMM62650.1
LAKVDIIAEM
QLAPQYSAAA Protein disulfide isomerase [C. literatus] ARS01447.1
TVETDLAGKFEVK Protein disulfide isomerase [C. magus] DAC80628.1
TAQKIFAGDIQNH
Protein disulfide isomerase EDWDAQPVKVLVR Protein disulfide isomerase [C. frigidus] ARU12136.1 27 Oxidative folding of conopeptides
EGAEDILDTF
QLAPQYSAAAG Protein disulfide isomerase [C. literatus] ARS01447.1
DATIEKDLAGK
AVLNGEVEAYLK Protein disulfide isomerase [C. magus] QFQ61181.1
QTSDFITWLKKK Protein disulfide isomerase [C. monile] ANC47993.1
MDSMANELEEIQ Protein disulfide isomerase [C.geographus] AMM62651.1
APMYSKAAGK Protein disulfide isomerase [C. magus] QFQ61177.1
DLASKFEVKGFPT Protein disulfide isomerase [C. bullatus] AMM62660.1
GITSEDSVFEEHKMK Protein disulfide isomerase [C. magus] DAC80628.1
STMTKFVQDF Protein disulfide isomerase [C. araneosus] AQM52452.1
DTPAMRLIQLGK
KAADTPAMRLIQLGK
YKPESDSLDKSTMTKF
VAAEIDNIAFGI
VQNYLMLFVK Protein disulfide isomerase [C.geographus] AMM62653.1
ITSEDSIFK Protein disulfide isomerase [C. ebraeus] ASF90532.1
NDFSGDFEEAAMSKFVKD Protein disulfide isomerase [C. ermineus] AXL95421.1
GKLMDEGSSIK Protein disulfide isomerase [C. miles] AQQ10870.1
KLATVFSLTLLAFVACEEVKQEEK
FIKDNYLPLINEFTQETSQKL Protein disulfide isomerase [C. ermineus] AXL95599.1
TCDQAKTFIDSDEVIVMGFFKDQEGK Protein disulfide isomerase [C. textile] AMM62657.1
TGDVQSYLMLFIK Protein disulfide isomerase [C. bullatus] AMM62659.1
DLAGKFNVTSYPTIKF
VEYKGEQK Protein disulfide isomerase [C. magus] DAC80629.1
Arginine kinase LAATPEFK Arginine kinase [C. anemone novaehollandiae] ADK73590.1 3 Neurotoxicity, leading to paralysis and subsequent death of prey
KGVEAVIK Arginine kinase [C. ebraeus] ASF90538.1
IQMEKTTVDALNTLEGELAGTYYPLLG Arginine kinase [C. miles] AQQ10876.1
ATP synthase F0 subunit 8 VMFSGKGLDCADLK ATP synthase F0 subunit 8 [C. betulinus] YP_009538431.1 3 important enzyme that provides energy to be used by the cell through the synthesis of ATP
IMFSSKSLTYINLGKENK ATP synthase F0 subunit 8 [C. iosephinae] ATZ70391.1
KIMFSSKSSTYTNLSK ATP synthase F0 subunit 8 [C. borgesi] YP_003204749.1
Kazal protease inhibitor CAGDIEICK Kazal protease inhibitor [C. ermineus] AXL95542.1 1 Inhibits serine proteases, including trypsin, chymotrypsin, and elastase.
NADH dehydrogenase subunits NNSSEVDIIMSK NADH dehydrogenase subunits [C. pseudonivifer] ATZ70266.1 6 Mitochondrial membrane respiratory
ELFVLFCVMSGGSALIGGMGGLNQTQVR NADH dehydrogenase subunits [C. (Lautoconus) sp] ATZ70968.1
SIVVMISMLNVVGSVLILLSNFAEGM NADH dehydrogenase subunits [C. geographus] ATZ70864.1
LGILLANFLILVIFPLAGK NADH dehydrogenase subunits [C. venulatus] APH08616.1
MLGILLANFLILVILPLISKKWSWYLK NADH dehydrogenase subunits [C. trochulus] ATZ69913.1
ETSKPASQLILN NADH dehydrogenase subunits [C. borgesi] YP_003204755.1
Total 64 48
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Discussion

The venom components of marine cone snails have evolved bioactive peptides targeting various biological activities to quickly paralyze their preferred prey. Studies have focused on both fish- and mollusk-hunting cone snail venoms because of the biomedical interest of their conopeptides [23]. Information on the peptide profile of worm-hunting species remains limited, despite their significance as a source of pharmacological compounds [24-26]. Thus, vermivore snails might also be promising pharmacological sources [27,28].

It is technically difficult to determine the precise number of components in the venom using biological activity methods [29]. In contrast, LC/MS supplied with an ESI source (LCMS-IT-TOF) is an effective way to provide an abundance of valuable data. This approach revealed a high degree of conopeptide diversity and increased the predicted number from 200 to >1100 distinct toxins per Conus species. In the present study, we observed diverse components in the venom of C. taeniatus. After mass deconvolution and filtering, a total of more than one hundred different molecular masses were detected from the venom of C. taeniatus. Previous studies reported between 50 and 1,000 conopeptides for a Conus species [14,30,31]. This variability may enable C. taeniatus to modify the composition of the injected venom according to the predatory or defensive stimuli. A total of 276, 298 and 488 different molecular masses were identified in C. imperialis, C. fulgetrum and C. crotchii venoms, respectively [14,32]. Furthermore, more than 500 different compounds were detected in the venom of C. consors by MALDI-MS alone and more than 700 by ESI-MS [33]. In our proteomic study, LCMS-IT-TOF and MS/MS were used to discover the peptide profile and predict putative conotoxin gene superfamilies in the neglected worm-hunting snail C. taeniatus. The number of distinct peptides previously reported in different species varies considerably. For example, 290 peptides were detected in C. taeniatus venom (this study), 1,746 peptides in the venom of C. textile [14], and 8,000 peptides in the venom of C. marmoreus [34]. Significant differences in peptide numbers in the proteomic analysis of Conus species may be due to the difference in methods of venom collection, total number of collected specimens and pooled data, or different conditions used for peptide authentication [35,36].

In the present study, we reported that the majority (84%) of C. taeniatus components were 500-4,000 Da, whereas only 16% of all components were large peptides (>4,000 Da). In addition, over 50% of the conopeptides detected in the venom of the studied species were smaller than 2,500 Da. [37]. Similarly, low molecular weight peptides were the most abundant in C. fulgetrum venom [37], C. marmoreus and C. bandanus venoms [2]. Although these species share worm-like prey, they evolved different strategies to produce diverse conopeptides. Low molecular weight peptides in venom specifically alter Na+, Ca2+, K+, and Cl- ion channels [38,39]. Because these low molecular weight peptides have the ability to block voltage-gated channels, they can be employed in tumor growth impairment [40,41]. Therefore, the discovered low molecular weight peptides in C. taeniatus and other Conus venoms could be employed in tumor treatment because they can most likely control the signal transduction pathways in malignant tumor cells.

Peptide toxins are usually highly bridged proteins with multiple pairs of intrachain disulfide bonds. The analysis of disulfide connectivity is important in protein structure determination [42]. The disulfide pattern in the venom peptides of C. taeniatus was estimated directly by LCMS-IT-TOF without venom fractionation. We reported herein that most C. taeniatus peptides were disulfide-rich, with the highest possibility of 3 disulfide bridges. Disulfide-rich peptides were also abundant in the venom of C. consors [43], C. bandanus and C. marmoreus [2] and C. fulgetrum [37]. It is well known that disulfide bonds confer conformational stability to folded proteins [44]. Therefore, an understanding of disulfide linkage patterns is necessary for further studies relating the structure to the function of Conus venom peptides.

Classical peptide identification methods, including Sanger sequencing and isolation, are generally considered laborious with limited efficiency and are sometimes limited by sample availability. The advance of high-throughput sequencing combined with bioinformatics analysis has allowed for more precise identification of conopeptides to predict and discover novel conotoxins from a variety of Conus species [34,45-49]. Here, the majority of conotoxins identified in C. taeniatus belonged to the T-superfamily, suggesting an important function for C. taeniatus. The T-superfamily peptides in Conus venom target different types of ion channels or neurotransmitters [50,51]. Similarly, the T-superfamily is predominant in C. victoriae venom [52]. Evidently, the T-superfamily is abundant in C. taeniatus and other Conus species; however, little is known about this group of conotoxins. Variations in conotoxin targets enable them to be included in the treatment of several diseases, such as pain, cancers and depression [1,53,54]. For example, M-superfamily peptides, which are ubiquitous in Conus venom [55], are blockers of voltage-gated sodium and potassium channels or nicotinic acetylcholine receptors. Conopeptides from the O-superfamily, which have O1, O2, and O3 variations, can block voltage-gated calcium and potassium channels [56,57]. Currently, ziconotide from the O1 superfamily is commercially available and works as an analgesic that relieves pain by selectively inhibiting the N-type voltage-gated Ca++ channel, and thus inhibiting the release of pro-nociceptive neurochemicals in the spinal cord [58,59]. The M- and O-superfamilies are the predominant superfamilies in C. tribblei, C. bullatus, C. marmoreus, and C. pulicarius [52]. Additionally, A-superfamily conopeptides are the most abundant in C. consors, C. geographus, and C. bullatus [52], and together with the O-superfamilies, can block potassium channels and affect nicotinic acetylcholine receptors [32,60]. As conopeptides in C. taeniatus can target different ion channels and receptors, they are promising candidate compounds for biomedical applications and drug development.

In addition to conopeptides, different non-conopeptide proteins and enzymes were detected. Conoporin, which is known as a potent cytolytic and hemolytic protein, was detected in C. taeniatus venom. Conoporins exert toxicity by forming pores in membranes, leading to cell death [61]. Interestingly, different peptide fragments of conoporins were identified, indicating the potential antimicrobial activity of C. taeniatus venom. The enzyme family protein disulfide-isomerase (PDI) was detected in the venom of C. taniatus and can catalyze the oxidation, isomerization, and reduction of disulfide bonds to ensure the proper folding of proteins. PDI confers stability to proteins by covalently linking specific cysteine residues [53,62]. This enzyme family has also been identified in the venom glands of several insects, including Aphidius ervi [63] andPsytalliaspecies [64], and in the crude venom extract ofPteromalus puparum [65],Diversinervus elegans [66] andCotesia chilonis [67]. In venomous cone snails, PDIs are only located in the venom glands directing the folding of conotoxins but not in the secreted venom [68,69]. PDIs rarely exist in the extracellular space and are principally localized in the endoplasmic reticulum [70]. Therefore, the presence of PDI in the extracted venom ofC. taeniatus is probably due to the rupture of venom-producing cells during venom collection. In this study, several of the detected protein fragments could not be attributed to conopeptides. One possible explanation is that the extracted venom may contain other untreated peptides and cellular debris. In addition, whole conotoxin sequences are not described and available in the database.

Conclusion

The data described herein contribute to addressing the gap of knowledge regarding the venom composition of the neglected vermivore cone snail C. taeniatus at the proteomic level. We used different proteomic approaches to characterize various peptide compositions of C. taeniatus venom. We successfully identified 170 out of 234 peptide fragments and classified them into 23 known gene superfamilies. Many conopeptide superfamilies targeting various types of ion channels and receptors were identified in the venom composition of the worm-hunting C. taeniatus, making them valuable lead compounds for drug development and biomedical applications. Therefore, further research with more sensitive methods are required to determine the peptide composition of untapped cone snail venoms.

Abbreviations

AA: acetic acid; DTT: dithiothreitol; ESI: electrospray ion source; FA: formic acid; HPLC: high-performance liquid chromatography; MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight; MM: Monoisotopic molecular masses; MS: mass spectrometry; PDI: disulfide-isomerase; PTM: posttranslational modifications; RP-HPLC: reversd-phase HPLC.

Acknowledgments

The authors are grateful to the Deanship of Scientific Research at Jouf University, Saudi Arabia, for funding this work (research grant number 40/284). We are grateful to Dr. M. Miyashita, associate professor of Kyoto University, for his valuable collaboration in the preliminary experiments. The authors extend their appreciation to the proteomics and metabolomics unit at Children Cancer Hospital (CCHE-57357)

Supplementary material.

The following online material is available for this article:

Additional file 1. Monoisotopic molecular masses (MM) of native components in Conus taeniatus venom detected by LC/MS analysis.
Click here for additional data file. (131KB, pdf)
Additional file 2. Monoisotopic molecular masses (MM) of reduced and alkylated components in Conus taeniatus venom detected by LC/MS analysis.
Click here for additional data file. (137.5KB, pdf)
Additional file 3. Estimation of the number of disulfide bridges included in each component in Conus taeniatus venom.
Click here for additional data file. (142.8KB, pdf)
Additional file 4. List of peptide sequences detected in C. taeniatus venom by using MALDI/TOF/MS.
Click here for additional data file. (214.5KB, pdf)

Footnotes

Availability of data and materials: All data generated or analyzed during this study are included in this article.

Funding: This work was supported by grants from the Deanship of Scientific Research at Jouf University, Saudi Arabia (research grant number 40/284).

Ethics approval: Not applicable

Consent for publication: Not applicable.

References

  1. Lewis RJ, Dutertre S, Vetter I, Christie MJ. Conus venom peptide pharmacology. Pharmacol Rev. 2012;64(2):259–298. doi: 10.1124/pr.111.005322. [DOI] [PubMed] [Google Scholar]
  2. Nguyen B, Molgó J, Lamthanh H, Benoit E, Khuc TA, Ngo DN, Nguyen NT, Millares P, Le Caer JP. High accuracy mass spectrometry comparison of Conus bandanus and Conus marmoreus venoms from the South Central Coast of Vietnam. Toxicon. 2013;75:148–159. doi: 10.1016/j.toxicon.2013.06.005. [DOI] [PubMed] [Google Scholar]
  3. Röckel D, Korn W, Kohn AJ. Manual of the living Conidae. Vol. 1, Indo-Pacific Region. Wiesbaden: Verlag Christa Hemmen; 1995. [Google Scholar]
  4. Olivera BM. E.E. Just Lecture, 1996. Conus venom peptides, receptor and ion channel targets, and drug design: 50 million years of neuropharmacology. Mol Biol Cell. 1997;8(11):2101–2109. doi: 10.1091/mbc.8.11.2101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Favreau P, Stöcklin R. Marine snail venoms: use and trends in receptor and channel neuropharmacology. Curr Opin Pharmacol. 2009;9(5):594–601. doi: 10.1016/j.coph.2009.05.006. [DOI] [PubMed] [Google Scholar]
  6. Lewis RJ, Garcia ML. Therapeutic potential of venom peptides. Nat Rev Drug Discov. 2003;2(10):790–802. doi: 10.1038/nrd1197. [DOI] [PubMed] [Google Scholar]
  7. Terlau H, Olivera BM. Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol Rev. 2004;84(1):41–68. doi: 10.1152/physrev.00020.2003. [DOI] [PubMed] [Google Scholar]
  8. Olivera BM. In: Drugs from the Sea. Fusetani N, editor. Switzerland: Karger; 2000. ω-Conotoxin MVIIA: from marine snail venom to analgesic drug; pp. 74–85. [Google Scholar]
  9. Miljanich GP. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem. 2004;11(23):3029–3040. doi: 10.2174/0929867043363884. [DOI] [PubMed] [Google Scholar]
  10. Bingham JP, Mitsunaga E, Bergeron ZL. Drugs from slugs - past, present and future perspectives of omega-conotoxin research. Chem Biol Interact. 2010;183(1):1–18. doi: 10.1016/j.cbi.2009.09.021. [DOI] [PubMed] [Google Scholar]
  11. Pennington MW, Czerwinski A, Norton RS. Peptide therapeutics from venom: current status and potential. Bioorg Med Chem. 2018;26(10):2738–2758. doi: 10.1016/j.bmc.2017.09.029. [DOI] [PubMed] [Google Scholar]
  12. Brinzeu A, Berthiller J, Caillet JB, Staquet H, Mertens P. Ziconotide for spinal cord injury-related pain. Eur J Pain. 2019;23(9):1688–1700. doi: 10.1002/ejp.1445. [DOI] [PubMed] [Google Scholar]
  13. King GF. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin Biol Ther. 2011;11(11):1469–1484. doi: 10.1517/14712598.2011.621940. [DOI] [PubMed] [Google Scholar]
  14. Davis J, Jones A, Lewis RJ. Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS. Peptides. 2009;30(7):1222–1227. doi: 10.1016/j.peptides.2009.03.019. [DOI] [PubMed] [Google Scholar]
  15. Espiritu DJ, Watkins M, Dia-Monje V, Cartier GE, Cruz LJ, Olivera BM. Venomous cone snails: molecular phylogeny and the generation of toxin diversity. Toxicon. 2001;39(12):1899–1916. doi: 10.1016/s0041-0101(01)00175-1. [DOI] [PubMed] [Google Scholar]
  16. Rodriguez AM, Dutertre S, Lewis RJ, Marí F. Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS. Anal Bioanal Chem. 2015;407(20):6105–6116. doi: 10.1007/s00216-015-8787-y. [DOI] [PubMed] [Google Scholar]
  17. Puillandre N, Bouchet P, Duda TF, Jr, Kauferstein S, Kohn AJ, Olivera BM, Watkins M, Meyer C. Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea) Mol Phylogenet Evol. 2014;78:290–303. doi: 10.1016/j.ympev.2014.05.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaas Q, Yu R, Jin AH, Dutertre S, Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res. 2012;40(D1):D325–D330. doi: 10.1093/nar/gkr886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lu A, Yang L, Xu S, Wang C. Various conotoxin diversifications revealed by a venomic study of Conus flavidus. Mol Cell Proteomics. 2014;13(1):105–118. doi: 10.1074/mcp.M113.028647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Duda TF, Jr, Palumbi SR. Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc Natl Acad Sci U S A. 1999;96(12):6820–6823. doi: 10.1073/pnas.96.12.6820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Conticello SG, Gilad Y, Avidan N, Ben-Asher E, Levy Z, Fainzilber M. Mechanisms for evolving hypervariability: the case of conopeptides. Mol Biol Evol. 2001;18(2):120–131. doi: 10.1093/oxfordjournals.molbev.a003786. [DOI] [PubMed] [Google Scholar]
  22. Kaas Q, Westermann JC, Halai R, Wang CKL, Craik DJ. ConoServer, a database for conopeptide sequences and structures. Bioinformatics. 2008;24(3):445–446. doi: 10.1093/bioinformatics/btm596. [DOI] [PubMed] [Google Scholar]
  23. Norton RS, Olivera BM. Conotoxins down under. Toxicon. 2006;48(7):780–798. doi: 10.1016/j.toxicon.2006.07.022. [DOI] [PubMed] [Google Scholar]
  24. Aguilar MB, López-Vera E, Imperial JS, Falcón A, Olivera BM, Heimer de la Cotera EP. Putative γ-conotoxins in vermivorous cone snails: the case of Conus delessertii. Peptides. 2005;26(1):23–27. doi: 10.1016/j.peptides.2004.10.012. [DOI] [PubMed] [Google Scholar]
  25. Loughnan M, Nicke A, Jones A, Schroeder CI, Nevin ST, Adams DJ, Alewood PF, Lewis RJ. Identification of a novel class of nicotinic receptor antagonists: dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum. J Biol Chem. 2006;281(34):24745–24755. doi: 10.1074/jbc.M603703200. [DOI] [PubMed] [Google Scholar]
  26. Zugasti-Cruz A, Maillo M, López-Vera E, Falcón A, Heimer de la Cotera EP, Olivera BM, Aguilar MB. Amino acid sequence and biological activity of a γ-conotoxin-like peptide from the worm-hunting snail Conus austini. Peptides. 2006;27(3):506–511. doi: 10.1016/j.peptides.2005.07.021. [DOI] [PubMed] [Google Scholar]
  27. Romeo C, Di Francesco L, Oliverio M, Palazzo P, Massilia GR, Ascenzi P, Polticelli F, Schininà ME. Conus ventricosus venom peptides profiling by HPLC-MS: a new insight in the intraspecific variation. J Sep Sci. 2008;31(3):488–498. doi: 10.1002/jssc.200700448. [DOI] [PubMed] [Google Scholar]
  28. Abdel-Rahman MA, Abdel-Nabi IM, El-Naggar MS, Abbas OA, Strong PN. Conus vexillum venom induces oxidative stress in Ehrlich’s ascites carcinoma cells: an insight into the mechanism of induction. J Venom Anim Toxins incl Trop Dis. 2013;19(1):10. doi: 10.1186/1678-9199-19-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Favreau P, Menin L, Michalet S, Perret F, Cheneval O, Stöcklin M, Bulet P, Stöcklin R. Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. Toxicon. 2006;47(6):676–687. doi: 10.1016/j.toxicon.2006.01.020. [DOI] [PubMed] [Google Scholar]
  30. Jones A, Bingham JP, Gehrmann J, Bond T, Loughnan M, Atkins A, Lewis RJ, Alewood PF. Isolation and characterization of conopeptides by high-performance liquid chromatography combined with mass spectrometry and tandem mass spectrometry. Rapid Commun Mass Spectrom. 1996;10(1):138–143. doi: 10.1002/(SICI)1097-0231(19960115)10:1<138::AID-RCM442>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  31. Abdel-Rahman MA, Abdel-Nabi IM, El-Naggar MS, Abbas OA, Strong PN. Intraspecific variation in the venom of the vermivorous cone snail Conus vexillum. Comp Biochem Physiol C Toxicol Pharmacol. 2011;154(4):318–325. doi: 10.1016/j.cbpc.2011.06.019. [DOI] [PubMed] [Google Scholar]
  32. Neves J, Campos A, Osório H, Antunes A, Vasconcelos V. Conopeptides from Cape Verde Conus crotchii. Mar Drugs. 2013;11(6):2203–2215. doi: 10.3390/md11062203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Biass D, Dutertre S, Gerbault A, Menou JL, Offord R, Favreau P, Stöcklin R. Comparative proteomic study of the venom of the piscivorous cone snail Conus consors. [2020 Nov 25];J Proteomics. 2009 72(2):210–218. doi: 10.1016/j.jprot.2009.01.019. [DOI] [PubMed] [Google Scholar]
  34. Dutertre S, Jin AH, Kaas Q, Jones A, Alewood PF, Lewis RJ. Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom. Mol Cell Proteomics. 2013;12(2):312–329. doi: 10.1074/mcp.M112.021469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Batista CVF, del Pozo L, Zamudio FZ, Contreras S, Becerril B, Wanke E, Possani LD. Proteomics of the venom from the amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;803(1):55–66. doi: 10.1016/j.jchromb.2003.09.002. [DOI] [PubMed] [Google Scholar]
  36. Batista CVF, Román-González SA, Salas-Castillo SP, Zamudio FZ, Gómez-Lagunas F, Possani LD. Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species. Comp Biochem Physiol C Toxicol Pharmacol. 2007;146(1-2):147–157. doi: 10.1016/j.cbpc.2006.12.004. [DOI] [PubMed] [Google Scholar]
  37. Abdel-Wahab M, Miyashita M, Kitanaka A, Juichi H, Sarhan M, Fouda M, Abdel-Rahman M, Saber S, Nakagawa Y. Characterization of the venom of the vermivorous cone snail Conus fulgetrum. Biosci Biotechnol Biochem. 2016;80(10):1879–1882. doi: 10.1080/09168451.2016.1176519. [DOI] [PubMed] [Google Scholar]
  38. Possani LD, Merino E, Corona M, Bolivar F, Becerril B. Peptides and genes coding for scorpion toxins that affect ion-channels. Biochimie. 2000;82(9-10):861–868. doi: 10.1016/s0300-9084(00)01167-6. [DOI] [PubMed] [Google Scholar]
  39. Rodríguez de la Vega RC, Possani LD. Overview of scorpion toxins specific for Na+ channels and related peptides: biodiversity, structure-function relationships and evolution. Toxicon. 2005;46(8):831–844. doi: 10.1016/j.toxicon.2005.09.006. [DOI] [PubMed] [Google Scholar]
  40. Fiske JL, Fomin VP, Brown ML, Duncan RL, Sikes RA. Voltage-sensitive ion channels and cancer. Cancer Metastasis Rev. 2006;25(3):493–500. doi: 10.1007/s10555-006-9017-z. [DOI] [PubMed] [Google Scholar]
  41. Gómez-Varela D, Zwick-Wallasch E, Knötgen H, Sánchez A, Hettmann T, Ossipov D, Weseloh R, Contreras-Jurado C, Rothe M, Stühmer W, Pardo LA. Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity. Cancer Res. 2007;67(15):7343–7349. doi: 10.1158/0008-5472.CAN-07-0107. [DOI] [PubMed] [Google Scholar]
  42. Wang W, Liu Z, Qian W, Fang Y, Liang S. Determination of disulfide bridges of spider peptide toxins: hainantoxin-III and hainantoxin-IV. J Venom Anim Toxins incl Trop Dis. 2009;15(2):268–277. [Google Scholar]
  43. Violette A, Biass D, Dutertre S, Koua D, Piquemal D, Pierrat F, Stöcklin R, Favreau P. Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach. J Proteomics. 2012;75(17):5215–5225. doi: 10.1016/j.jprot.2012.06.001. [DOI] [PubMed] [Google Scholar]
  44. Wedemeyer WJ, Welker E, Narayan M, Scheraga HA. Disulfide bonds and protein folding. Biochemistry. 2000;39(15):4207–4216. doi: 10.1021/bi992922o. [DOI] [PubMed] [Google Scholar]
  45. Hu H, Bandyopadhyay PK, Olivera BM, Yandell M. Characterization of the Conus bullatus genome and its venom-duct transcriptome. BMC Genomics. 2011;12:60. doi: 10.1186/1471-2164-12-60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Hu H, Bandyopadhyay PK, Olivera BM, Yandell M. Elucidation of the molecular envenomation strategy of the cone snail Conus geographus through transcriptome sequencing of its venom duct. BMC Genomics. 2012;13:284. doi: 10.1186/1471-2164-13-284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Dutertre S, Jin AH, Vetter I, Hamilton B, Sunagar K, Lavergne V, Dutertre V, Fry BG, Antunes A, Venter DJ, Alewood PF, Lewis RJ. Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails. Nat Commun. 2014;5:3521. doi: 10.1038/ncomms4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lavergne V, Harliwong I, Jones A, Miller D, Taft RJ, Alewood PF. Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks. Proc Natl Acad Sci U S A. 2015;112(29):E3782–E3791. doi: 10.1073/pnas.1501334112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Peng C, Yao G, Gao BM, Fan CX, Bian C, Wang J, Cao Y, Wen B, Zhu Y, Ruan Z, Zhao X, You X, Bai J, Li J, Lin Z, Zou S, Zhang X, Qiu Y, Chen J, Coon SL, Yang J, Chen JS, Shi Q. High-throughput identification of novel conotoxins from the chinese tubular cone snail (Conus betulinus) by multi-transcriptome sequencing. Gigascience. 2016;5(1):1–14. doi: 10.1186/s13742-016-0122-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Li X, Chen W, Zhangsun D, Luo S. Diversity of conopeptides and their precursor genes of Conus litteratus. Mar Drugs. 2020;18(9):464. doi: 10.3390/md18090464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Kumari A, Ameri S, Ravikrishna P, Dhayalan A, Kamala-Kannan S, Selvankumar T, Govarthanan M. Isolation and characterization of conotoxin protein from Conus inscriptus and its potential anticancer activity against cervical cancer (HeLa-HPV 16 Associated) cell lines. Int J Pept Res Ther. 2020;26:1051–1059. [Google Scholar]
  52. Barghi N, Concepcion GP, Olivera BM, Lluisma AO. High conopeptide diversity in Conus tribblei revealed through analysis of venom duct transcriptome using two high-throughput sequencing platforms. Mar Biotechnol (NY) 2015;17(1):81–98. doi: 10.1007/s10126-014-9595-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wickenden A, Priest B, Erdemli G. Ion channel drug discovery: challenges and future directions. Future Med Chem. 2012;4(5):661–679. doi: 10.4155/fmc.12.4. [DOI] [PubMed] [Google Scholar]
  54. Hurst R, Rollema H, Bertrand D. Nicotinic acetylcholine receptors: from basic science to therapeutics. Pharmacol Ther. 2013;137(1):22–54. doi: 10.1016/j.pharmthera.2012.08.012. [DOI] [PubMed] [Google Scholar]
  55. Jacob RB, McDougal OM. The M-superfamily of conotoxins: a review. Cell Mol Life Sci. 2010;67(1):17–27. doi: 10.1007/s00018-009-0125-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Heinemann SH, Leipold E. Conotoxins of the O-superfamily affecting voltage-gated sodium channels. Cell Mol Life Sci. 2007;64(11):1329–1340. doi: 10.1007/s00018-007-6565-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Robinson SD, Safavi-Hemami H, McIntosh LD, Purcell AW, Norton RS, Papenfuss AT. Diversity of conotoxin gene superfamilies in the venomous snail, Conus victoriae. PLoS One. 2014;9(2):e87648. doi: 10.1371/journal.pone.0087648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Schroeder CI, Lewis RJ. ω-conotoxins GVIA, MVIIA and CVID: SAR and clinical potential. Mar Drugs. 2006;4(3):193–214. [Google Scholar]
  59. McGivern JG. Ziconotide: a review of its pharmacology and use in the treatment of pain. Neuropsychiatr Dis Treat. 2007;3(1):69–85. doi: 10.2147/nedt.2007.3.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Peng C, Ye M, Wang Y, Shao X, Yuan D, Liu J, Hawrot E, Wang C, Chi C. A new subfamily of conotoxins belonging to the A-superfamily. Peptides. 2010;31(11):2009–2016. doi: 10.1016/j.peptides.2010.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Alegre-Cebollada J, Oñaderra M, Gavilanes JG, del Pozo AM. Sea anemone actinoporins: the transition from a folded soluble state to a functionally active membrane-bound oligomeric pore. Curr Protein Pept Sci. 2007;8(6):558–572. doi: 10.2174/138920307783018686. [DOI] [PubMed] [Google Scholar]
  62. Noiva R. Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum. Semin Cell Dev Biol. 1999;10(5):481–493. doi: 10.1006/scdb.1999.0319. [DOI] [PubMed] [Google Scholar]
  63. Colinet D, Anselme C, Deleury E, Mancini D, Poulain J, Azéma-Dossat C, Belghazi M, Tares S, Pennacchio F, Poirié M, Gatti JL. Identification of the main venom protein components of Aphidius ervi, a parasitoid wasp of the aphid model Acyrthosiphon pisum. BMC Genomics. 2014;15(1):342. doi: 10.1186/1471-2164-15-342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Mathé-Hubert H, Colinet D, Deleury E, Belghazi M, Ravallec M, Poulain J, Dossat C, Poirié M, Gatti JL. Comparative venomics of Psyttalia lounsburyi and P. concolor, two olive fruit fly parasitoids: a hypothetical role for a GH1 β-glucosidase. Sci Rep. 2016;6:35873. doi: 10.1038/srep35873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yan Z, Fang Q, Wang L, Liu J, Zhu Y, Wang F, Li F, Werren JH, Ye G. Insights into the venom composition and evolution of an endoparasitoid wasp by combining proteomic and transcriptomic analyses. Sci Rep. 2016;6:1–12. doi: 10.1038/srep19604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Liu NY, Wang JQ, Zhang ZB, Huang JM, Zhu JY. Unraveling the venom components of an encyrtid endoparasitoid wasp Diversinervus elegans. Toxicon. 2017;136:15–26. doi: 10.1016/j.toxicon.2017.06.011. [DOI] [PubMed] [Google Scholar]
  67. Teng ZW, Xiong SJ, Xu G, Gan SY, Chen X, Stanley D, Yan ZC, Ye GY, Fang Q. Protein discovery: combined transcriptomic and proteomic analyses of venom from the endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae) Toxins (Basel) 2017;9(4):135. doi: 10.3390/toxins9040135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Safavi-Hemami H, Li Q, Jackson RL, Song AS, Boomsma W, Bandyopadhyay PK, Gruber CW, Purcell AW, Yandell M, Olivera BM, Ellgaard L. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides. Proc Natl Acad Sci U S A. 2016;113(12):3227–3232. doi: 10.1073/pnas.1525790113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Violette A, Leonardi A, Piquemal D, Terrat Y, Biass D, Dutertre S, Noguier F, Ducancel F, Stöcklin R, Križaj I, Favreau P. Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms. Mar Drugs. 2012;10(2):258–280. doi: 10.3390/md10020258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Turano C, Coppari S, Altieri F, Ferraro A. Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell Physiol. 2002;193(2):154–163. doi: 10.1002/jcp.10172. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1. Monoisotopic molecular masses (MM) of native components in Conus taeniatus venom detected by LC/MS analysis.
Click here for additional data file. (131KB, pdf)
Additional file 2. Monoisotopic molecular masses (MM) of reduced and alkylated components in Conus taeniatus venom detected by LC/MS analysis.
Click here for additional data file. (137.5KB, pdf)
Additional file 3. Estimation of the number of disulfide bridges included in each component in Conus taeniatus venom.
Click here for additional data file. (142.8KB, pdf)
Additional file 4. List of peptide sequences detected in C. taeniatus venom by using MALDI/TOF/MS.
Click here for additional data file. (214.5KB, pdf)

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