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

• Published in: Journal of medicinal chemistry Vol. 47; no. 5; pp. 1234 - 1241
• Main Authors: LOUGHNAN, Marion L., NICKE, Annette, JONES, Alun, ADAMS, David J., ALEWOOD, Paul F., LEWIS, Richard J.
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 26.02.2004
• Subjects: Animal poisons toxicology. Antivenoms; Animals; Biological and medical sciences; Cholinergic Antagonists - chemistry; Cholinergic Antagonists - isolation & purification; Cholinergic Antagonists - pharmacology; Chromatography, Liquid; Conotoxins - chemistry; Conotoxins - isolation & purification; Conotoxins - pharmacology; In Vitro Techniques; Mass Spectrometry; Medical sciences; Mollusk Venoms - chemistry; Neurons - metabolism; Oligopeptides - chemical synthesis; Oligopeptides - chemistry; Oligopeptides - pharmacology; Oocytes - metabolism; Oocytes - physiology; Patch-Clamp Techniques; Protein Subunits; Rats; Receptors, Nicotinic - drug effects; Snails - chemistry; Structure-Activity Relationship; Toxicology; Xenopus; Peptides; Rat; Ligand; Central nervous system; Selectivity; Gastropoda; Cholinergic receptor; Organic sulfate; Nicotinic receptor; Rodentia; Animal origin; In vitro; Biological activity; Vertebrata; Biological fixation; Mammalia; Animal; Venom; Affinity; Isolation; Poison; Invertebrata; Mollusca; Subtype; Structural analysis
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm031010o

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-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 alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 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-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.