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αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico.
Hernández-Sámano AC
,
Falcón A
,
Zamudio F
,
Batista CVF
,
Michel-Morfín JE
,
Landa-Jaime V
,
López-Vera E
,
Jeziorski MC
,
Aguilar MB
.
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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.
Figure 1. Fractionation by RP-HPLC of the C. princeps venom duct extract. An isocratic step at 0% Solution B for 10 min, followed by a linear gradient of 0% to 100% (v/v) of Solution B, over 200 min was used at 1 mL/min. The absorbance was measured at 220 nm. * Active fractions on human α7 nicotinic acetylcholine receptor (nAChR) inhibiting ≥25.0% the acetylcholine (ACh)-induced response.
Figure 2. Active peptidic fractions (at 0.07 µg/µL) from C. princeps venom on human α7 nAChRs. Experiments were performed at least 3 times and the values averaged. *Active fractions on hα7 nAChRs inhibiting ≥ 50.0% the ACh-induced response.
Figure 3. Representative current traces displaying the effect of active peptidic fractions (at 0.07 µg/µL) from C. princeps venom on human α7 nAChRs. (a) F7 fraction; (b) F16 fraction.
Figure 4. Purification of an active peptide of C. princeps venom by RP-HPLC. (a) F7 fraction: after an isocratic step at 10% Solution B for 10 min, followed by a linear gradient of 10% to 30% (v/v) of Solution B, over 200 min, at 1 mL/min was used. (b) F7-3 peptide: after an isocratic step at 0% Solution B for 10 min, followed by a linear gradient of 0% to 20% (v/v) of Solution B, over 200 min, at 0.8 mL/min was used. The absorbance was measured at 220 nm. Only the F7-3 peptide collected between the diagonal lines was used for further analyses.
Figure 5. Representative current traces displaying the effect of the purified F7-3 peptide (at 0.07 µg/µL) of C. princeps venom on two human nAChR subtypes. (a) hα7 (n = 4); and (b) hα3β2 (n = 3).
Figure 6. Concentration-response curve for the inhibition of human α7 nAChRs (circles) expressed in Xenopus laevis oocytes by the purified F7-3 peptide of C. princeps venom. The IC50 is 6.2 µM. The effect over human α3β2 (triangles) at 6.5 μM is also shown.
Figure 7. (a) Original mass spectrum of the native purified F7-3 peptide from C. princeps venom, the m/z signal (assuming z = 10) at 1074.56 corresponds to an average mass of 10,735.50 Da, which agrees very well with the m/z signal (z = 9) at 1193.95 which corresponds to an average mass of 10,735.54 Da. “?” denotes that the charge of the ion was not automatically determined by the software. (b) Deconvoluted mass spectrum of the native purified F7-3 peptide from C. princeps venom.
Figure 8. Top line, sequence of the purified F7-3 (αD-PiXXA) peptide from C. princeps obtained by automated Edman degradation; O is hydroxy-Pro. Bottom line, sequence TR34549_4_2 from the transcriptome of C. princeps venom duct; * indicates identical residues at the same position.
Figure 9. (a) Sequences of the precursors of PiXXA and of the alphaD-conotoxins whose biological activity has been demonstrated experimentally. (b) Comparison of the αD-CTX that have been characterized electrophysiologically. %I, percent identity with respect to the first αD-CTX studied at this level, αD-VxXXB [13]. The prefixes “r” and “h” stand for rat and human, respectively. nAChRs: mf, α1β1γδ; ma, α1β1δε. Cp and Ms, αD-Cp (major sequence) and αD-Ms, respectively [17]. GeXXA, αD-GeXXA [2], PiXXA (bold face), αD-PiXXA [this work]. Post-translational modifications: O, hydroxy-Pro; γ, gamma-carboxyglutamic acid. In both panels, positions with identical residues (*) and conserved (:) or semiconserved (.) substitutions are indicated at the bottom of the alignments. In panel B, with the exception of Cys, identical residues present in two or more sequences are highlighted by black background when at least one conserved substitution (highlighted by gray background) is present at the same position.
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