Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom

Ying Fu Yu Zhang Shuang Ju Bokai Ma Wenwen Huang Sulan Luo About the authors

Abstract

Background:

Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea.

Methods:

C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique.

Results:

Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM.

Conclusion:

We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.

Keywords:
Conopeptides; Disulfide-poor conopeptides; Conus marmoreus ; nAChR; Cone snail; Conus venom

Background

Cone snails comprise a genus of carnivorous mollusks from the Conidae family that contains more than 700 Conus species in total [1Puillandre N, Duda TF, Meyer C, Olivera BM, Bouchet P. One, four or 100 genera? A new classification of the cone snails. J Molluscan Stud. 2015 Feb;81(1):1-23.-2Fallon PJ. Taxonomic review of tropical western Atlantic shallow water Drilliidae (Mollusca: Gastropoda: Conoidea) including descriptions of 100 new species. Zootaxa. 2016 Mar 14;4090(1):1-363.]. They live in the tropical and subtropical shallow seawater all over the world. These slow-moving mollusks rely on secreting and releasing venom for defense and predation [3Prashanth JR, Dutertre S, Lewis RJ. Pharmacology of predatory and defensive venom peptides in cone snails. Mol Biosyst. 2017 Nov 21;13(12):2453-65.-4Dutertre 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 Mar 24;5:3521-8.]. Typically, each cone snail venom contains at least 1000 neuropeptides, called conopeptides or conotoxins, and their composition differs from species to species [4Dutertre 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 Mar 24;5:3521-8.

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 Jul 21;112(29): E3782-91.
-6Davis J, Jones A, Lewis RJ. Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS. Peptides. 2009 Jul 4;30(7):1222-7.]. It is estimated that cone snails can produce up to 1 million different natural peptides. However, less than 0.1% of them has been structurally and functionally characterized to date [7Jin AH, Muttenthaler M, Dutertre S, Himaya SWA, Kaas Q, Craik DJ, Lewis RJ, Alewood PF. Conotoxins: Chemistry and biology. Chem Rev. 2019 Nov 13;119(21):11510-49.]. Thus, Conus venom has been considered an interesting source of peptide-based therapeutics because of their structural and functional diversity and their promising prospects for treating burdensome diseases including neuralgia, addiction, epilepsy, depression, cancer, etc. [8Lewis RJ, Dutertre S, Vetter I, Christie MJ. Conus venom peptide pharmacology. Pharmacol Rev. 2012 Apr 1;64(2):259-98.

Pennington MW, Czerwinski A, Norton RS. Peptide therapeutics from venom: Current status and potential. Bioorg Med Chem. 2018 Jun 1;26(10):2738-58.
-10King GF. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opin Biol Ther. 2011 Nov;11(11):1469-84.].

Conus marmoreus is a common species in the South China Sea. To date, 176 mature peptide sequences have been recorded in the online database “Conoserver” (http://www.conoserver.org/) [11Kaas Q, Westermann JC, Halai R, Wang CK, Craik DJ. ConoServer, a database for conopeptide sequences and structures. Bioinformatics. 2008 Feb 1;24(3):445-6.]. Dutertre et al. [12Dutertre S, Jin A-h, 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 Feb;12(2):312-29.] had identified 105 conopeptide precursor sequences from 13 gene superfamilies from the venom gland transcriptome of C. marmoreus, and discovered 2710 and 3172 peptides using MALDI-MS (matrix-assisted laser desorption ionization-mass spectrometry) and ESI-MS (electrospray ionization-mass spectrometry), respectively, from proteomic data of C. marmoreus venom. Lavergne et al. [13Lavergne V, Dutertre S, Jin AH, Lewis RJ, Taft RJ, Alewood PF. Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies. BMC Genomics. 2013 Oct 16;14:708.] had performed a reanalysis of C. marmoreus venom duct transcriptome using algorithm “ConoSorter” and revealed 158 novel conotoxins and 13 new gene superfamilies. These comprehensive transcriptomic and proteomic data showed the vast diversity of the conopeptides from C. marmoreus [14Dutt M, Dutertre S, Jin AH, Lavergne V, Alewood PF, Lewis RJ. Venomics Reveals Venom Complexity of the Piscivorous Cone Snail Conus tulipa. Mar Drugs. 2019 Jan 21;17(1):71.].

In order to explore novel conopeptides and characterize their structure and function, we prepared and then fractionated the C. marmoreus venom. Meticulous purification process was conducted to obtain five novel disulfide-poor conopeptides. Their sequences were identified by integral analysis of MALDI-TOF tandem mass spectrometry data and Edman degradation result. They were named as Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPPNTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their inhibitory activities for several nAChR (nicotinic acetylcholine receptors) subtypes (α1β1δε, α3β2, α3β4, α4β2) were investigated.

Methods

Crude venom preparation and peptide isolation

Ten specimens of C. marmoreus were collected from shallow sea near Sansha City in South China and were frozen at −80 °C. The crude venom preparation and fractionation processes were conducted as previously described [15Zhang Y, Li C, Ju S, Fu Y, Luo SL. Blocking activity study of crude venom fractions of five cone snails from South China Sea against α3β2 and α4β2 nAChRs. Chin. J Mar Drugs. 2020 Oct;39(5):30-7.]. Briefly, the venom duct of the snail samples were dissected and then extracted by 60% acetonitrile aqueous solution to obtain crude venom powder. The venom powder was dissolved, fractionated by a preparative Waters HPLC e2535 separations module system equipped with a reverse-phase C18 column (Vydac Grace, 10 μm, 22 mm × 250 mm, 10 mL/min), and purified by preparative HPLC e2695 system with a reverse-phase C18 column (Vydac Grace, 5 μm, 4.6 mm × 250 mm, 0.8 mL/min). Solution A (0.1% TFA in ddH2O) and solution B (0.1% TFA in 90% acetonitrile aqueous solution) were used as the mobile phase. The monitoring wavelength was set at 214 nm throughout the fractionation and isolation process. The crude venom was fractionated to obtain 20 fractions named Mar-1−Mar-20 with isocratic elution of 5%−60% solution B in 60 min. Fraction Mar-4 was washed by 30% solution B and subjected to a 25 min isocratic elution of 10%−28% solution B to obtain Mr-2 at 9.86 min and Mr-3 at 10.92 min. Fraction Mar-9 eluted at 40% solution B was separated by a linear gradient of 18%−35% solution B in 20 min to gain Mr-4 at 12.3 min. Mr-5 was yielded at 14.82 min by gradient eluting program of 18%−35% solution B in 20 min from Mar-19, which was washed by 50% solution B. Mr-1 was obtained at 13.65 min by a 20 min gradient program of 23%−38% solution B from the 45% solution B eluted fraction Mar-15.

The purified conopeptides were subjected to LC-MS (Waters, Acquity I-Class/Xevo UPLC-ESI-TQD-MS, USA) analysis with a C18 column (Acquity UPLC Peptide BEH, 130 Å, 1.7 μm, 2.1 mm × 100 mm) with solution A (0.1% formic acid in ddH2O) and solution B (0.1% formic acid in acetonitrile) as mobile phase. The detection range of m/z ratio was set at 400−1500. The cone voltage and capillary voltage were 30 V and 3.5 kV, respectively. The desolvation temperature was 550 °C, and the ion source temperature was 150 °C. The gradient program of UPLC analysis was 5%~60% solution B for 6 min, and the flow rate was 0.5 mL/min.

Peptide sequencing by MALDI-TOF-MS/MS analysis and Edman degradation

The purified conotoxins were all subjected to total reduction by DTT (dithiothreitol) before sequencing. Each conopeptides (0.1 mM in 20% acetonitrile aqueous solution) was mixed with 200 mM DTT (in 20% acetonitrile aqueous solution). The mixtures were reacted at room temperature (25 °C) on a shaker for about 2 hours. Each reaction process was monitored by LC-MS every half an hour. Once the totally-reduced peptides were detected, the reaction solutions were separately purified by getting through a reverse-phase C18 column (Vydac Grace, 300 Å, 5 μm, 4.6 mm × 250 mm) to collect the reduced peptides for the follow-up sequence identification.

Mass spectrometry detection was carried out on a MALDI-TOF-MS/MS spectrometer (Bruker, Ultraflextreme, Germany). The mass axis was calibrated by a peptide mixture (Peptide Calibration Standards II, P/N 8222570, 700−4000 Da). For sample preparation, 1 μL of each reduced conotoxin solution (dissolved in 50% acetonitrile) and 1 μL of HCCA solution (1 mg in 250 μL standard solution) were successively dropped onto the target plate and then dried off. The laser frequency was 1000 Hz. The voltage was set at 19 kV. FlexControl was used to acquire the primary and secondary mass spectra, and flexAnalysis was applied to dispose the data and gain a series of b and y ions. For peptide sequence identification, Mascot search was performed with the online NCBI or Swiss-prot database.

The Edman degradation sequencing was performed in a PPSQ-53A Protein sequencer (Shimadzu, Japan). Each reduced peptide was dissolved by 20 μL ddH2O. Ten microliter of the testing solution was added to the PVDF (polyvinylidene fluoride) film to dry off, and then the film was transferred to the reactor. A certain PTH (phenylthiohydantoin)-amino acid was yielded after the N-terminal amino acid degradation in each cycle. HPLC analysis of the PTH-amino acid derived from each cycle was detected using a Wakopak Wakosil-PTH-II column (wako, S-PSQ, 4.6 mm× 250 mm) with 40% acetonitrile (< 2.5% acetic acid) as mobile phase. The retention time (t R ) of the certain PTH-amino acid yielded in each cycle was compared with the t R of the standards to identify the amino acid until the intact sequence was obtained.

Peptide synthesis

According to the identified sequences, the five conopeptides were synthesized on a polypeptide synthesis reactor (Shanghai Aladdin Biochemical Technology Co., LTD) by a stepwise solid-phase method using Fmoc (N-9-flurenylmethoxycarbonyl) chemistry. The 2-Cl(Trt)-Cl Resin (Tianjin Nankai University Resin Co., LTD) and the Fmoc-L-amino acids [Cishi Biotechnology (Shanghai) Co., LTD] were used. Ninhydrin chromogenic method was applied to detect the Fmoc group on the resin. The synthesized peptide was released from the resin by a mixture of TFA/phenol/thioanisole/water 90/7.5/2.5/5 on a shaker for 0.5−2 hours in the dark. The resin was then removed by filtration and rinsed with TFA three times. The obtained filtrate was added drop by drop into cold diethyl ether (4 °C) to precipitate the peptide. After centrifugation, the crude peptide was subjected to preparative reverse-phase HPLC for purification with gradient elution of 10−35% solution B. The homogeneity of retention time, molecular weight and sequence between the synthesized and the isolated native peptides was individually confirmed by analytical HPLC and MALDI-TOF-MS/MS.

Electrophysiological measurements for nAChR blockage

Rat α1, α3, α4, β1, β2, β4, δ and ε nAChR subunit clones were kindly provided by Utah University (Salt Lake City, Utah, USA). In vitro cRNA synthesis was conducted as previously reported [16Ren J, Zhu X, Xu P, Li R, Fu Y, Shuai D, Zhangsun, D, Wu Y, Luo S. D-Amino Acid Substitution of α-Conotoxin RgIA Identifies its Critical Residues and Improves the Enzymatic Stability. Mar Drugs. 2019 Feb 28;17(3):142.]. Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). Mature female X. Laevis frogs were anesthetized on ice and dissected for the oocytes, which were subjected to enzymolysis (25 °C, 40 min) by 20 mg trypsin in 40 mL OR-2 buffer (82.5 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl2·6H2O, 5 mM HEPES, pH 7.5) to obtain individual oocyte. Each subunit cRNA (10−20 ng in 46−59 nL of water) were individually injected into the oocytes to obtain several nAChR subtypes (α4β2, α3β2, α1β1δε, α3β4). α1β1δε cRNA was formed by mixing α1, β1, δ, ε subunits at 2:1:1:1 ratio. α1 and β1 subunit cRNAs were separately mixed at 4:2, 3:2 and 3:4 ratios to obtain α4β2, α3β2 and α3β4 cRNAs. All oocytes were then incubated at 17 °C in ND96 buffer (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, at pH 7.1−7.5) supplemented with 50 mg/L gentamicin for 2−5 days. For electrophysiological measurements, ACh was used to obtain a control response before incubating with conopeptides Mr-1−Mr-5. The control response by ACh was measured under a two electrode voltage clamp amplifier (Axon 900A, Molecular Devices MD, Sunnyvale, CA, USA), at a holding potential of −70 mV. An amount of 10 µM ACh was applied for α1β1δε subtype, and 100 µM ACh was for rat α4β2, α3β2, α3β4 subtypes. Micropipettes were filled with 3 M KCl and had resistances of 0.5−2 MΩ. The elicited current responses were recorded and analyzed using pClamp10 software (MD, Sunnyvale, CA), filtered at 10 Hz, and digitized at 200 Hz. All tested conopeptides (5 µL of 1 mmol/L in ND96) were separately added into the 50 µL cell chamber and incubated for 5 min. The activities of Mr-1−Mr-5 at the heterologously expressed rat nAChRs were determined by comparing the ACh-induced current response after a 5 min incubation with Mr-1−Mr-5 to the average ACh-induced responses before the incubation. α-conotoxin GID (IRDECCSNPACRVNNOHVC) was used as a positive control for blocking α4β2 nAChR. All data were presented as mean ± SEM of 4−9 oocytes.

3. Results

Peptide isolation and sequence identification

For the venom extraction, 9 mg venom powder of C. marmoreus was obtained and subjected to the systematic separation, which led to the isolation of five novel disulfide-poor conopeptides named Mr-1−Mr-5. Their HPLC peaks were illustrated in Figure 1. The five purified conopeptides were individually reduced by DTT. The molecular weights of both the intact peptides (shown in Figure 2) and their reduced forms were respectively determined by UPLC-ESI-TQD-MS (Table 1), which indicated that none of them contains disulfide bonds. Thus, the isolated native peptides were subjected to MALDI-TOF-MS/MS detection and Edman degradation experiment. Their sequences were assigned by comprehensive analysis of mass spectrum and Edman degradation data. In the MALDI-TOF-MS/MS spectrum, b/y ions were generated by CID (collision-induced dissociation) fragmentation at peptide bond, while a/x ions were produced by breaking the Cα−C=O bond. The sequencing results were listed in Table 1.

Figure 1.
UPLC profiles of C. marmoreus venom and the purified conopeptides. The black curve indicates the UPLC profile of crude venom. The red, blue, green, pink and yellow curves represent the UPLC profiles of the purified Mr-1 to Mr-5, respectively.

Figure 2.
MS spectra of conopeptides Mr-1 to Mr-5. (A) Peak at m/z 958.74 [M+2H]2+ showed that the molecular weight of Mr-1 was 1915.48 Da. (B) Peaks at m/z 701.75 [M+2H]2+ and 1401.95 [M+H]+ indicated that the molecular weight of Mr-2 was 1400.95 Da. (C) 1695.62 Da for Mr-3 was deduced from the peak at 848.81 [M+2H]2+. (D) Peaks at m/z 471.68 [M+3H]3+ and 707.93 [M+2H]2+ revealed a molecular weight of 1413.86 Da for Mr-4. (E) 1108.95 Da for Mr-5 was confirmed by signal at m/z 555.21 [M+2H]2+.

Table 1.
Sequences, molecular weights and numbers of disulfide bonds of the purified conopeptides.

Peak at m/z 958.74 [M+2H]2+ observed in the ESI-TQD-MS spectrum (Figure 2A) coincided with the parent ion peak at m/z 1915.001 in the MALDI-TOF-MS/MS spectrum (Figure 3). The Edman degradation sequencing (Additional file 1) of Mr-1 illustrated the sequence to be DWEYHAHPKPNSFWT. Two fragments of DWEYHAH and PNSFWT were deduced from the consecutive b ions (b 5b 7, b 9b 14) and y ions (y 6 , y 8y 14) in the MS/MS spectrum, which was compatible with the Edman degradation result. Hence, the primary sequence of Mr-1 was undoubtedly identified to be DWEYHAHPKPNSFWT, which was a novel disulfide-poor conomarphin peptide. Through sequence alignment, Mr-1 was found to be similar to peptide conomarphin-Mr1 (DWEYHAHPKONSfWT, O: hydroxyproline, f: D-phenylalanine), which was previously purified from C. marmoreus venom [17Han Y, Huang F, Jiang H, Liu L, Wang Q, Wang Y, Shao X, Chi C, Du W, Wang C. Purification and structural characterization of a D-amino acid-containing conopeptide, conomarphin, from Conus marmoreus. FEBS J. 2008 May;275(9):1976-87.]. Thus, Mr-1 can be named as conomarphin-Mr3.

Figure 3.
MALDI-TOF-MS/MS spectrum of Mr-1. The primary sequence of Mr-1 was determined by the consecutive b/y ions generated from CID fragmentation.

The successive b ions (b 2b 3, b 5b 7), a ions (a 1a 2, a 4a 6, a 8, a 9, a 11) and y ions (y 6y 8) observed in the MALDI-TOF-MS/MS spectrum (Figure 4) revealed a YPTRAYPSNKF fragment in the sequence of Mr-2. The detection of b 10 ion (m/z 1178.5953) and a 11 ion (m/z 1297.6688) indicated a phenylalanine (Phe, F, 147.1739) residue at position 11. For the assignment of residue at the C-terminal of Mr-2, a glycine residue (57.0513) could be easily speculated from the mass difference (57.5097) between the parent ion (m/z 1400.969) and fragment YPTRAYPSNKF (m/z 1343.4863). Thus, the primary sequence of Mr-2 was determined to be YPTRAYPSNKFG, which was perfectly consistent with the Edman degradation sequencing result (YPTRAYPSNKFG, Additional file 2).

Figure 4.
MALDI-TOF-MS/MS spectrum of Mr-2. Serial b/y ions and a ions generated from CID fragmentation confirmed the primary sequence of Mr-2.

The molecular weight of Mr-3 was determined to be 1695.62 Da based on the observation of peak at m/z 848.81 [M+2H]2+ in the ESI-MS spectrum (Figure 2C), which was in accordance with the parent ion (m/z 1695.766) presented in MALDI-TOF-MS/MS spectrum (Figure 5). A doubtless fragment sequence of NVIQAPAQSVAP*N*** in Mr-3 was confirmed by the Edman degradation sequencing (Additional file 1). The series of a ions (a 10a 16 ) observed in MALDI-TOF-MS/MS spectrum illustrated that the residues at positions 10−16 were VAPPNTS. Based on the mass difference (101.1) between NVIQAPAQSVAPPNTS (m/z 1593.737) and the parent ion mass (m/z 1695.766), a threonine (Thr, m/z 101.10392) residue was deduced at the C-terminus (position 17). Thus, the intact sequence of Mr-3 was assigned as NVIQAPAQSVAPPNTST.

Figure 5.
MALDI-TOF-MS/MS spectrum of Mr-3. The observation of y and a ions help confirming the partial sequence of Mr-3.

Peak at m/z 707.93 [M+2H]2+ in the MS spectrum (Figure 2D) of Mr-4 indicated the peptide mass to be 1413.86 Da, which was consistent with the parent ion (m/z 1413.872) in MALDI-TOF-MS/MS spectrum (Figure 6). The Edman degradation sequencing of Mr-4 showed an unambiguous sequence fragment of KENVLNKLKS** (Additional file 4). The observation of successive b ions (b 6b 8) and few y ions (y 5, y6, y 9) were also detected in MS/MS spectrum to verify the Edman degradation data. KENVLNKLKS** confirmed the residues at positions 5 and 8 to be Leu (L), which could not be identified by MS/MS sequencing. The y 1 (m/z 132.108) and b 11 (m/z 1282.758) ions revealed the residues at positions 11 and 12 should be lysine (K) and L/I, respectively. MS/MS data could not distinguish between Leu and Ile residues. Thus, Mr-4 was a novel 12-residue peptide whose complete sequence was KENVLNKLKSK(L/I).

Figure 6.
MALDI-TOF-MS/MS spectrum of Mr-4. Partial sequence of Mr-4 was assigned by the observed b/y ions, corresponding with the Edman degradation sequencing. L/I at position 12 could not be distinguished by MS/MS analysis.

Similarly, the sequential b ions (b 5b 11) and y ions (y 4y 6) recorded in the MALDI-TOF-MS/MS spectrum (Figure 7) revealed the fragment AN(L/I)PG(L/I)V at positions 6−12 of Mr-5. The fragment NAVA**N***** was speculated from Edman degradation sequencing (Additional file 5). The result combined with the observation of the series of b ions (b 1b 2 and b 5) showed that the residues at positions 1−5 were NAVAA. Therefore, Mr-5 was ascertained to be NAVAAAN(L/I)PG(L/I)V.

Figure 7.
MALDI-TOF-MS/MS spectrum of Mr-5. Partial sequence of Mr-5 was ascertained by the b/y ions generated from CID fragmentation. L/I at positions 8 and 11 could not be distinguished by MS/MS analysis.

So far, 176 mature peptides have been reported from C. marmoreus, 21 of them are disulfide-poor conopeptides (Table 2). Except for conomarphins and contryphan-M, the rest of them were deduced from transcriptomic and proteomic data. Their conopeptide class and pharmacological activity have not been identified yet.

Table 2.
Disulfide-poor conopeptides discovered from C. marmoreus species.

Biological effect on nAChRs

In order to investigate the nAChR-associated activity of the five disulfide-poor conopeptides (Mr-1−Mr-5), they were synthesized according to the identified sequences. Since Ile and Leu are isomers whose hydrophobicity, dispersion and ionic properties are quite similar, we chose Leu to substitute I/L in the sequences of Mr-4 and Mr-5. Mr-4 and Mr-5 were synthesized as KENVLNKLKSKL and NAVAAANLPGLV, respectively. If the sequences show certain activity, the sequences contained Ile would be synthesized for further investigation. Several rat nAChR subtypes (α4β2, α3β2, α1β1δε, α3β4) were expressed in the X. Laevis oocytes, and subjected to electrophysiological experiments (Figure 8). Both Mr-2 and Mr-5 separately showed about 30 ± 6.5 % of ACh-evoked currents mediated by α3β2 nAChR (Figure 8A and 8B), while Mr-1 inhibited 20 ± 8.5 % of α4β2 nAChR ACh-evoked currents (Figure 8C), at the concentration of 100 µM. On the other hand, 1 µM GID inhibited 55 ± 4.3 % of α4β2 nAChR ACh currents (Figure 8D). No activity was observed against α3β4 and α1β1δε nAChRs for all the five conopeptides testing at 100 µM. Thus, all the five novel conopeptides presented no significant activity against the above nAChRs.

Figure 8.
Biological effect on nAChRs of Mr-1, Mr-2 and Mr-5. Current trace of inhibition of α3β2 nAChR by (A) Mr-2 and (B) Mr-5. Current trace of inhibition of α4β2 nAChR by (C) Mr-1 and (D) GID. GID was used as a positive antagonist control for α4β2 nAChR.

Discussion

nAChRs are a class of trans-membrane ligand-gated ion channel receptors and have been identified as targets for various diseases such as pain, addiction, depression and cancer, etc [8Lewis RJ, Dutertre S, Vetter I, Christie MJ. Conus venom peptide pharmacology. Pharmacol Rev. 2012 Apr 1;64(2):259-98., 20Hurst R, Rollema H, Bertrand D. Nicotinic acetylcholine receptors: from basic science to therapeutics. Pharmacol Ther. 2013 Jan;137(1):22-54.-21Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol. 2007 Feb 10;47:699-729.]. The α4β2 subtype, a neurotype nAChR, has been proven to be a crucial target associated with addiction [22Tapper AR, McKinney SL, Nashmi R, Schwarz J, Deshpande P, Labarca C, Whiteaker P, Marks MJ, Collins AC, Lester HA. Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science. 2004 Nov 5;306(5698):1029-32.]. For the weaker withdrawal reaction and less relapsing rate, α4β2 nAChR antagonist have become an research hot spot on searching potential candidates for smoking cessation drug [22Tapper AR, McKinney SL, Nashmi R, Schwarz J, Deshpande P, Labarca C, Whiteaker P, Marks MJ, Collins AC, Lester HA. Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science. 2004 Nov 5;306(5698):1029-32.

Wu J, Cippitelli A, Zhang Y, Debevec G, Schoch J, Ozawa A, Yu Y, Liu H, Chen W, Houghten RA, Welmaker GS, Giulianotti MA, Toll L. Highly selective and potent alpha4beta2 nAChR antagonist inhibits nicotine self-administration and reinstatement in rats. J Med Chem. 2017 Dec 28;60(24):10092-104.
-24Rollema H. and Hurst RS. The contribution of agonist and antagonist activities of alpha4beta2* nAChR ligands to smoking cessation efficacy: a quantitative analysis of literature data. Psychopharmacology. 2018 Jul 7;235(9):2479-505.]. Several smoking cessation drugs such as bupropion and varenicline, have launched into the market for years [25Tobey KM, Walentiny DM, Wiley JL, Carroll FI, Damaj MI, Azar MR, Koob GF, George O, Harris LS, Vann RE. Effects of the specific alpha4beta2 nAChR antagonist, 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine, on nicotine reward-related behaviors in rats and mice. Psychopharmacology. 2012 Apr 22;223(2):159-68.-26Aubin HJ, Luquiens A, Berlin I. Pharmacotherapy for smoking cessation: pharmacological principles and clinical practice. Br J Clin Pharmacol. 2014 Feb 11;77(2):324-36.]. However, most of them have ended up with unsatisfactory therapeutic effects due to the low selectivity and potency for α4β2 nAChR [27Mills EJ, Wu P, Lockhart I, Thorlund K, Puhan M, Ebbert JO. Comparisons of high-dose and combination nicotine replacement therapy, varenicline, and bupropion for smoking cessation: a systematic review and multiple treatment meta-analysis. Ann Med. 2012 Sep 10;44(6):588-97.

Monarrez-Espino J, Galanti MR, Hansson J, Janszky I, Soderberg-Lofdal K, Moller J. Treatment with bupropion and varenicline for smoking cessation and the risk of acute cardiovascular events and injuries: a swedish case-crossover study. Nicotine Tob Res. 2018 Apr 2;20(5):606-13.
-29Cinciripini PM, Robinson JD, Karam-Hage M, Minnix JA, Lam C, Versace F, Brown VL, Engelmann JM, Wetter DW. Effects of varenicline and bupropion sustained-release use plus intensive smoking cessation counseling on prolonged abstinence from smoking and on depression, negative affect, and other symptoms of nicotine withdrawal. JAMA Psychiatry. 2013 May;70(5):522-33.]. The main reason for the low selectivity is that α4β2 nAChR antagonists (such as GIC and GID) also show potent inhibitory activity on α3β2 nAChR [30McIntosh JM, Dowell C, Watkins M, Garrett JE, Yoshikami D, Olivera BM. Alpha-conotoxin GIC from Conus geographus, a novel peptide antagonist of nicotinic acetylcholine receptors. J Biol Chem. 2002 Sep 13;277(37):33610-5.-31Nicke A, Loughnan ML, Millard EL, Alewood PF, Adams DJ, Daly NL, Craik DJ, Lewis RJ. Isolation, structure, and activity of GID, a novel alpha 4/7-conotoxin with an extended N-terminal sequence. J Biol Chem. 2003 Jan 31;278(5):3137-44.]. Thus, we had investigated the α4β2 and α3β2 nAChRs inhibitory effects of the C. marmoreus venom fractions. Several fractions had been shown with certain α4β2/α3β2 nAChRs selectivity [15Zhang Y, Li C, Ju S, Fu Y, Luo SL. Blocking activity study of crude venom fractions of five cone snails from South China Sea against α3β2 and α4β2 nAChRs. Chin. J Mar Drugs. 2020 Oct;39(5):30-7.], which indicated C. marmoreus venom as a good natural source for discovering novel α4β2 nAChR antagonist with low selectivity against α3β2 nAChR. In this work, we purified the venom fractions of C. marmoreus collected from the South China Sea and structurely characterized and functionally identified five novel disulfide-poor conopeptides. Their sequences were assigned by comprehensive analysis of MALDI-TOF tandem mass data and Edman degradation sequencing. They were named Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPPNTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. Mr-1 is structurely similar to conomarphin-Mr1, which have been discovered from C. marmoreus venom since 2008. No activity study have been recorded for conomarphin-Mr1. Mr-1−Mr-5 were first reported and synthesized by Fmoc-SPPS chemistry, and their activity at several nAChR subtypes (α1β1δε, α3β2, α3β4, α4β2) were investigated. All the five conopeptides showed no significant activity against the above nAChR subtypes. α1β1δε nAChR, a muscular type nAChR, is associated with the muscle contraction and is considered as a target contributing to the venom toxicity for cone snail predation. Mr-1−Mr-5 showed no activity against α1β1δε nAChR, which meant that they do not affect the muscle contraction.

Generally, disulfide-rich conopeptides, conotoxins, are considered to be the dominant component of the Conus venom and preferentially present the neuroactive pharmacology [8Lewis RJ, Dutertre S, Vetter I, Christie MJ. Conus venom peptide pharmacology. Pharmacol Rev. 2012 Apr 1;64(2):259-98., 32Halai R, Craik DJ. Conotoxins: natural product drug leads. Nat Prod Rep. 2009 Apr;26(4):526-36.]. Although disulfide-poor conopeptides occupy a minor portion of Conus venom, they still have caught interest for intensive investigation [33Giribaldi J, Ragnarsson L, Pujante T, Enjalbal C, Wilson D, Daly NL, Lewis RJ, Dutertre S. Synthesis, pharmacological and structural characterization of novel conopressins from Conus miliaris. Mar Drugs. 2020 Mar 6;18(3):150.]. Disulfide-poor conopeptides can be divided into many subgroups, including contulakins, conantokins, conorfamides, conolysins, conopressins, contryphans, conophans, conomarphins, conomaps, conoCAPs, conoNPYs, conoGAYs and hormone-like conopeptides, which have been found to interact with diverse targets (such as ion channels, vasopressin receptor, NMDA receptor, neurotensin receptor) and have shown potential prospects as drug candidates for cardiovascular disease, epilepsy, mood control and pain release [34Lebbe EK and Tytgat J. In the picture: disulfide-poor conopeptides, a class of pharmacologically interesting compounds. J Venom Anim Toxins Incl Trop Dis. 2016 Nov 7;22:30. doi: 10.1186/s40409-016-0083-6.eCollection2016.
https://doi.org/10.1186/s40409-016-0083-...
]. To date, two of them (contulakin-G and conantokin-G) have already reached clinical trials for alleviating pain, which indicates disulfide-poor conopeptides as promising leads for drug discovery [35Malmberg AB, Gilbert H, McCabe RT, Basbaum AI. Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T. Pain. 2003 Jan;101(1-2):109-16.-36Lee HK, Zhang L, Smith MD, Walewska A, Vellore NA, Baron R, McIntosh JM, White HS, Olivera BM, Bulaj G. A marine analgesic peptide, contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties. Front Pharmacol. 2015 Feb 10;6:11.].

Mr-1, which was identified as a conomarphin, does not present the common post-translational modification (PTM) as previous conomarphins. The hydroxylation of 10-Pro residue and the epimerization of Phe-13 residue in Mr-1 were absent, suggesting the maturation of conomarphins could be accomplished without the above PTMs. As for Mr-2−Mr-5, they differ from each other and do not belong to any category of the known disulfide-poor conopeptides. Their conopeptide class could not be ascertained until their pharmacological targets are identified.

Conclusion

We purified and structurally characterized five novel disulfide-poor conopeptides (Mr-1 to Mr-5) from C. marmoreus crude venom and investigated their activity at the rat nAChRs. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from C. marmoreus venom, which provided new information for their further exploring. Their activities against other types of receptors (such as ion channels, vasopressin receptor, NMDA receptor, neurotensin receptor) and effects on animal models remain to be further studied.

Abbreviations

CID: collision-induced dissociation; ddH2O: double distilled water; ESI: electrospray ionization; HCCA: cyano-4-hydroxycinnamic acid; HEPES: 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid; HPLC: high-performance liquid chromatography; LC-MS: liquid chromatography-mass spectrometry; MALDI-TOF: matrix-assisted laser desorption ionization-time of flight; MS: mass spectrometry; N-methyl-D-aspartic acid receptor; Na2-EDTA: ethylenediaminetetraacetic acid disodium salt dihydrate; nAChRs: nicotinic acetylcholine receptors; NET: norepinephrine transporter; NMDA: PTH: phenylthiohydantoin; PTM: post-translational modification; TCEP: tris-(2-carboxyethyl)-phosphine; TFA: trifluoroacetic acid; TQD: triple quadrupole; UPLC: ultra-performance liquid chromatography; VGCC: voltage-gated calcium channel; VGPC: voltage-gated potassium channel.

Acknowledgments

We thank Prof. Mei Zhang (Beijing Key Laboratory of Organic Materials Testing Technology & Quality Evaluation, Institute of Analysis and Testing, Beijing Academy of Science and Technology) for invaluable advice in the peptide sequence interpretation

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  • 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 National Natural Science Foundation of China (no. 81903492) and Young Elite Scientist Sponsorship Program of Beijing Association for Science and Technology (2021-2023)
  • Ethics approval

    We declared that the cone snail specimens in this research do not involve endangered or protected species. All the experiments for X. laevis were performed strictly according to the approved protocol (IACUC-HaiDaBan-2020-17) of the Care and Use Guideline of Laboratory Animal at Ethics Committee of Hainan University

Publication Dates

  • Publication in this collection
    18 May 2022
  • Date of issue
    2022

History

  • Received
    10 Nov 2021
  • Accepted
    29 Mar 2022
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