Mass Spectral Identification of Vc1.1 and Differential Distribution of Conopeptides in the Venom Duct of Conus victoriae. Effect of Post-Translational Modifications and Disulfide Isomerisation on Bioactivity
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Molluscs of the genus Conus (cone shells) are carnivorous, feeding on marine worms, small fish and other marine molluscs. They capture their prey by injecting venom containing hundreds of neurally active peptide components. These peptides are classed as conotoxins and consist of small disulfide-bonded peptides exhibiting a high degree of post-translational modifications (PTMs). The functional roles of these modifications remain largely unknown. Two of the most frequently observed modifications are γ-carboxylation of glutamate and hydroxylation of proline (Buczek et al. Cell Mol Life Sci 62:3067, 2005). Vc1.1 is an α-conotoxin from Conus victoriae (Sandall et al. Biochemistry 42(22):6904–6911, 2003) and the only form of this peptide which has been detected in the venom is the γ-glutamate and hydroxyproline (Vc1.1.P6O:E14Gla) version of the molecule (Jakubowski et al. Toxicon 47(6):688–699, 2006). In order to investigate the role of PTMs, we did mass spectral profiling of the venom duct of C. victoriae looking at changes in mass and the number of peptides detected. We synthesised a number of predicted Vc1.1-PTM peptides together with the three possible disulfide isoforms of Vc1.1 and assessed the possible functional role of the PTM conopeptides by measuring the in vitro activity at the cognate neuronal nicotinic acetylcholine receptors (nAChRs). In addition we looked for their presence Vc1.1 venom by mass spectrometry and by this approach we were able to detect unmodified Vc1.1 in C. victoriae venom for the first time.
KeywordsConotoxins Post-translational modification PTM Disulfide isoforms Vc1.1 Neuronal nicotinic acetylcholine receptor
We thank Dr. Jennifer Jakubowski for their advice on optimisation of mass spectral conditions for conopeptides. Thanks also to Dr. Robyn Bradbury for provision of the C. victoriae specimens, and to Mr John Ahern of the Department of Zoology, University of Melbourne, for maintaining the snails in captivity. We thank Dr. Tom Hennessy from Agilent for software analysis.
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