Isolation and Structural Identification of a New T1-conotoxin with Unique Disulfide Connectivities Derived from

Overview

Journal J Venom Anim Toxins Incl Trop Dis

Date 2020 May 20

PMID 32425993

Citations 3

Authors

Nguyen Bao

Jean-Piere Lecaer

Ngo Dang Nghia

Phan Thi Khanh Vinh

Affiliations

Soon will be listed here.

Abstract

Background: Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of were determined by tandem mass spectrometry using collision-induced dissociation.

Methods: The venom glands of snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptide's cysteine connectivity was determined by rapid partial reduction-alkylation technique.

Results: The novel conotoxin, NGCC(I/L)VRECC, was firstly derived from sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C-C/C-C) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter.

Conclusion: We identified the first T1-conotoxin, denominated Bn5a, isolated from venom. However, Bn5a conotoxin exhibited unique C-C/C-C disulfide connectivity, unlike other T1-conotoxins (C-C/C-C). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.

Citing Articles

O1-conotoxin Tx6.7 cloned from the genomic DNA of that inhibits calcium currents.

Zhou M, Yang M, Wen H, Xu S, Han C, Wu Y J Venom Anim Toxins Incl Trop Dis. 2023; 29:e20220085.

PMID: 37283723 PMC: 10241523. DOI: 10.1590/1678-9199-JVATITD-2022-0085.

Challenges and Opportunities in Clinical Diagnostic Routine of Envenomation Using Blood Plasma Proteomics.

Cavalcante J, de Almeida D, Moraes M, Santos S, Moriel Pincinato P, Riciopo P Toxins (Basel). 2023; 15(3).

PMID: 36977071 PMC: 10056359. DOI: 10.3390/toxins15030180.

Prospecting for candidate molecules from toxins to develop new biopharmaceuticals.

Mohamed A, Nabil Z, El-Naggar M J Venom Anim Toxins Incl Trop Dis. 2022; 28:e20220028.

PMID: 36545288 PMC: 9761950. DOI: 10.1590/1678-9199-JVATITD-2022-0028.

References

Luo S, Zhangsun D, Wu Y, Zhu X, Xie L, Hu Y . Identification and molecular diversity of T-superfamily conotoxins from Conus lividus and Conus litteratus. Chem Biol Drug Des. 2006; 68(2):97-106. DOI: 10.1111/j.1747-0285.2006.00422.x. View

Lewis R, Dutertre S, Vetter I, Christie M . Conus venom peptide pharmacology. Pharmacol Rev. 2012; 64(2):259-98. DOI: 10.1124/pr.111.005322. View

Jakubowski J, Keays D, Kelley W, Sandall D, Bingham J, Livett B . Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry. J Mass Spectrom. 2004; 39(5):548-57. DOI: 10.1002/jms.624. View

Hu H, Bandyopadhyay P, Olivera B, 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. PMC: 3441800. DOI: 10.1186/1471-2164-13-284. View

Sharpe I, Gehrmann J, Loughnan M, Thomas L, Adams D, Atkins A . Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter. Nat Neurosci. 2001; 4(9):902-7. DOI: 10.1038/nn0901-902. View

Conticello S, 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):120-31. DOI: 10.1093/oxfordjournals.molbev.a003786. View

Luo S, Zhangsun D, Zhang B, Chen X, Feng J . Direct cDNA cloning of novel conotoxins of the T-superfamily from Conus textile. Peptides. 2006; 27(11):2640-6. DOI: 10.1016/j.peptides.2006.05.002. View

Kaas Q, Yu R, Jin A, Dutertre S, Craik D . ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Res. 2011; 40(Database issue):D325-30. PMC: 3245185. DOI: 10.1093/nar/gkr886. View

Balaji R, Ohtake A, Sato K, Gopalakrishnakone P, Kini R, Seow K . lambda-conotoxins, a new family of conotoxins with unique disulfide pattern and protein folding. Isolation and characterization from the venom of Conus marmoreus. J Biol Chem. 2000; 275(50):39516-22. DOI: 10.1074/jbc.M006354200. View

10.

Dutertre S, Jin A, Kaas Q, Jones A, Alewood P, Lewis R . Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom. Mol Cell Proteomics. 2012; 12(2):312-29. PMC: 3567856. DOI: 10.1074/mcp.M112.021469. View

11.

Peng C, Wu X, Han Y, Yuan D, Chi C, Wang C . Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning. Peptides. 2007; 28(11):2116-24. DOI: 10.1016/j.peptides.2007.08.026. View

12.

Livingstone C, Barton G . Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation. Comput Appl Biosci. 1993; 9(6):745-56. DOI: 10.1093/bioinformatics/9.6.745. View

13.

Nam H, Corneli P, Watkins M, Olivera B, Bandyopadhyay P . Multiple genes elucidate the evolution of venomous snail-hunting Conus species. Mol Phylogenet Evol. 2009; 53(3):645-52. DOI: 10.1016/j.ympev.2009.07.013. View

14.

Rigby A, Lucas-Meunier E, Kalume D, Czerwiec E, Hambe B, Dahlqvist I . A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx. Proc Natl Acad Sci U S A. 1999; 96(10):5758-63. PMC: 21933. DOI: 10.1073/pnas.96.10.5758. View

15.

Nguyen B, Le Caer J, Araoz R, Thai R, Lamthanh H, Benoit E . Isolation, purification and functional characterization of alpha-BnIA from Conus bandanus venom. Toxicon. 2014; 91:155-63. DOI: 10.1016/j.toxicon.2014.10.006. View

16.

Echterbille J, Quinton L, Gilles N, De Pauw E . Ion mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges. Anal Chem. 2013; 85(9):4405-13. DOI: 10.1021/ac303686w. View

17.

Kaas Q, Westermann J, Halai R, Wang C, Craik D . ConoServer, a database for conopeptide sequences and structures. Bioinformatics. 2007; 24(3):445-6. DOI: 10.1093/bioinformatics/btm596. View

18.

Galli A, Defelice L, Duke B, Moore K, Blakely R . Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants. J Exp Biol. 1995; 198(Pt 10):2197-212. DOI: 10.1242/jeb.198.10.2197. View

19.

Gray W . Disulfide structures of highly bridged peptides: a new strategy for analysis. Protein Sci. 1993; 2(10):1732-48. PMC: 2142270. DOI: 10.1002/pro.5560021017. View

20.

Liu J, Wu Q, Pi C, Zhao Y, Zhou M, Wang L . Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels. Peptides. 2007; 28(12):2313-9. DOI: 10.1016/j.peptides.2007.09.006. View