Chemical and Functional Identification and Characterization of Novel Sulfated α-Conotoxins from the Cone Snail Conus anemone

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Abstract
An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the α-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated α-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, α-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat α3β2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat α7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at α3β2 (IC50 0.6 nM) and α7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the α7 (IC50 367 nM) than at the α3β2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-α-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for α3β2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor α7 affinity.