Isolation and structural identification of a new T1-conotoxin with unique disulfide connectivities derived from Conus bandanus

 

Nguyen Bao1, Jean-Pière Lecaer2, Ngo Dang Nghia3, Phan Thi Khanh Vinh1

 

1 Faculty of Food Technology, Nha Trang University, 02 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa, Vietnam.

2 Institut de Chimie des Substances Naturelles, Centre de Recherche de Gif, FRC3115, UPR 2301, F-91198 Gif-sur-Yvette, France.

3 Institute of Biotechnology and Environment, Nha Trang University, 02 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa, Vietnam.

 

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 Conus bandanus were determined by tandem mass spectrometry using collisioninduced dissociation.

 

Methods: The venom glands of C. bandanus 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 C18 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, NGC1 C2 (I/L)VREC3 C4 , was firstly derived from de novo 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 (C1 -C4 /C2 -C3 ) 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 C. bandanus 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 C. bandanus venom. However, Bn5a conotoxin exhibited unique C1 -C4 /C2 -C3 disulfide connectivity, unlike other T1-conotoxins (C1 -C3 /C2 -C4 ). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.

 

Keywords: T1-subfamily conotoxin Conus bandanus Bn5a Disulfide connectivity Cone snail venom

 

Correspondence: bao@ntu.edu.vn

 

Received: 03 December 2019; Accepted: 15 April 2020; Published online: 08 May 2020