αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico

Abstract

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 μM, and it does not affect the hα3β2 subtype at 6.5 μM.

Conotoxin Diversity in the Venom Gland Transcriptome of the Magician’s Cone, Pionoconus magus

“…The defensive venom of R. vexillum is produced in the proximal region of the venom duct and it is almost exclusively composed of d conotoxins [54]. This superfamily is also found in other species of the genus including Rhizoconus capitaneus , Rhizoconus rattus , R. miles , and Rhizoconus mustelinus [54,57], but also in Strategoconus vitulinus , Strategoconus generalis [58], and Strategoconus planorbis [59], the Eastern Pacific Ductoconus princeps [60] and most strikingly in the Atlantic piscivorous C. ermineus [7] (Figure 3). The member of the D superfamily in P. magus showed no propeptide region and a larger and highly divergent mature region compared to those of R. vexillum .…”

A Novel Dimeric Conotoxin, FrXXA, from the Vermivorous Cone Snail Conus fergusoni, of the Eastern Pacific, Inhibits Nicotinic Acetylcholine Receptors

“…Particularly, most ions observed in modern mass spectrometry are adduct ions or pseudomolecular ions such as [M + H] + , [M + NH 4 ] + , [M + Na] + or [M + K] + [ 20 ]; Na + adducts are one of the most common in biological samples [ 22 ]. In addition, TFA adducts are reported as part of the repeating units commonly observed in background interferences [ 20 ]; these adducts have already been reported by Loughnan et al, and Hernández-Sámano et al, during the analysis of the αD-conotoxins from C. vexillum and C. princeps , respectively [ 15 , 18 ].…”

“…It is known most conotoxins present post-translational modifications. The three αD-conotoxins from C. vexillium : VxXXA, VxXXB, and VxXXC have proline hydroxylation and gamma glutamate carboxylation; on the other hand, PiXXA isolated from C. princeps shows the hydroxylation of proline [ 15 , 18 ]. This makes hydroxylation a common post-translational modification in αD-conotoxins and it could be that αD-FrXXA is not the exception; it might have oxygens according to the calculated values from the molecular masses mentioned above (fourth paragraph).…”

“…To the present, 25 αD-conotoxins have been reported from vermivorous species found in the Indo-Pacific [ 17 ]. However, in 2019 the first αD-conotoxin from a species that inhabits the Mexican Pacific Coast in the Eastern Pacific, C. princeps , was reported [ 18 ].…”

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

“…As an exception, we can mention the discovery of a new αS-conotoxin, GVIIIB, which was a highly selective inhibitor of α9α10 nAChR [ 101 ], αM-conotoxin MIIIJ, which, like ψ-conotoxins, belongs to the M-superfamily and also blocks muscle nAChR [ 102 ], αO-conotoxin GeXIVA [ 100 ], with good prospects for medical use (see below) and several αD-conotoxins. Of the two isolated dimeric αD-conotoxins, GeXXA [ 103 ] ( Figure 3 ) and PiXXA [ 104 ], the first was shown to be a non-selective blocker of both neuronal and muscle receptor subtypes, while for the second, inhibitory activity was revealed only towards human α7 nAChR (IC 50 6.2 µM). Another αD-conotoxin Lt28.1, with a cysteine pattern (-C-C-C-CC-C-C-C-C-C-) unusual for this family, was cloned from Conus litteratus , expressed in a yeast system, and showed low affinity (IC 50 3.0 µM) only towards α9α10 nAChR [ 105 ].…”