The glycopeptide CcTx, isolated from the venom of the Indo-Pacific fish-hunting cone snail Conus consors, belongs to the κA-family of conopeptides. These toxins elicit excitotoxic responses in the prey by acting on voltage-gated sodium channels. The structure of CcTx, a first in the κA-family, has been determined by a combination of analytical technologies, including mass spectrometry (MALDI-TOF-MS and ESI-MS), sugar composition analysis (GLC-EI-MS), monosaccharide absolute configuration determination (GLC-EI-MS), sugar methylation analysis (GLC-EI-MS), and NMR spectroscopy.
Peptide analysis revealed a glycopeptide of 30 amino acids with three disulfide bridges, a C-terminal cysteine residue, three 4-hydroxyproline residues, and an O-glycan with the composition Hex3HexNAc2 at Ser7.
AOWLVP(Hex3HexNAc2)SQITTCCGYNOGTMCOSCMCTNTC
Glycan analysis revealed a novel type of glycopeptide O-glycan core, here registered as core type 9, containing two terminal α-L-galactose units.
α-L-Galp-(1→4)-α-D-GlcpNAc-(1→6)
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α-D-GalpNAc-(1→O)-Ser
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α-L-Galp-(1→2)-β-D-Galp-(1→3)
Analysis of the NMR solution structure of CcTx showed that the backbone of the C-terminal region was well defined with three disulfide bridges, a series of turns, including a Type I' β-turn for Cys12-Tyr15 and a partially distorted Type I β-turn for Asn16-Thr19, preceding the short α-helix Ser23-Thr27.
A sequence comparison to other putative members of the κA-family suggests that O-linked glycosylation might be more common than previously thought.
Acknowledgements
This work has been supported by a grant of the European Commission: CONCO, the cone snail genome project for health. Integrated Project ref. LSHB-CT-2007-037592.
