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

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.


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][6][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][9][10][11][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][15][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, highperformance liquid chromatography (HPLC) fractionation combined with LC/mass spectrometry (LC-MS) and offline matrix-assisted laser desorption/ionization (MALDI)-time-offlight (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.

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, freezedried, and then saved at −80°C until use.

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 H 2 O (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-hydroxycinnamic 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 TripleTOF TM 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.

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    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.

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 disulfidepoor ( Figure 4B and Additional file 3).
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.

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][25][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 + , Ca 2+ , K + , and Clion 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 disulfiderich, with the highest possibility of 3 disulfide bridges. Disulfiderich 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][46][47][48][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] and Psytallia species [64], and in the crude venom extract of Pteromalus puparum [65], Diversinervus elegans [66] and Cotesia 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 of C. 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.