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 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.
Highlights
Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails
The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by tandem mass spectrometry (MS/mass spectrometry (MS))
The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides
Summary
Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Conopeptides (conotoxins) are peptides derived from the venomous salivary glands of cone snails consisting of 8–84 amino acids and zero to five disulfide bridges They are neuropharmacological probes and pharmacological development for G-protein-coupled receptors, ion channels (K+, Na+, Ca2+), and neurotransmitter receptors (such as N-methyl-d-aspartate receptor, 5-hydroxytryptamine, nicotinic acetylcholine receptor) with high degrees of specificity and potency [1,2]. Matrix-assisted laser desorption/ ionization time of flight (MALDI-TOF) mass spectrometry spectrometers combined with Edman degradation can provide the complete peptide sequence information from a small amount of sample The fragmentation capabilities, such as collisioninduced dissociation (CID) conferred by MALDI-TOF MS [9], along with the rapid partial reduction-alkylation procedure [10], are especially useful for determination of disulfide connectivity. We established the unusable disulfide pairing of a novel T1-subfamily conotoxin using the partial reduction-alkylation procedure
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