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

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 collision-induced 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, NGC1C2(I/L)VREC3C4, 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.(AU)

Isolation and characterization of five novel mini-M conotoxins from the venom of mollusk-hunter snail Conus bandanus

Objective: To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam. Methods: Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C18 column in order to screen small conotoxins. The primary structure of peptide was analyzed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman's degradation method. Results: Five new conotoxins were biochemically characterized from the crude venom of the mollusk-hunting cone snail Conus bandanus, which were collected at Ke Ga reef of the Nha Trang Bay (Vietnam). Each conotoxin had 15 or 16 amino acid residues and shared the same characteristic cysteine framework V as-CC-C-C-CC-. They were termed as Bn3b, Bn3c, Bn3d, Bn3e and Bn3f following the conotoxins nomenclature. Conclusions: The conotoxins Bn3b, Bn3e, and Bn3f are categorized in the mini-M conotoxins of the M1 branch, while conotoxins Bn3c and Bn3d are categorized in the mini-M conotoxins of the M2 branch. The homological analysis reveals that these conotoxins could serve as promising probe compounds for voltage-gated sodium channels.

Isolation and characterization of five novel mini-M conotoxins from the venom of mollusk-hunter snail Conus bandanus

Objective: To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam. Methods: Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C18 column in order to screen small conotoxins. The primary structure of peptide was analyzed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman's degradation method. Results: Five new conotoxins were biochemically characterized from the crude venom of the mollusk-hunting cone snail Conus bandanus, which were collected at Ke Ga reef of the Nha Trang Bay (Vietnam). Each conotoxin had 15 or 16 amino acid residues and shared the same characteristic cysteine framework V as -CC-C-C-CC-. They were termed as Bn3b, Bn3c, Bn3d, Bn3e and Bn3f following the conotoxins nomenclature. Conclusions: The conotoxins Bn3b, Bn3e, and Bn3f are categorized in the mini-M conotoxins of the M1 branch, while conotoxins Bn3c and Bn3d are categorized in the mini-M conotoxins of the M2 branch. The homological analysis reveals that these conotoxins could serve as promising probe compounds for voltage-gated sodium channels.