Determining sequences and post‐translational modifications of novel conotoxins in <i>Conus victoriae</i> using cDNA sequencing and mass spectrometry

Jakubowski, Jennifer A.; Keays, David A.; Kelley, Wayne P.; Sandall, David; Bingham, Jon Paul; Livett, Bruce G.; Gayler, Kenwyn R.; Sweedler, Jonathan V.

doi:10.1002/jms.624

Cited by 54 publications

(45 citation statements)

References 53 publications

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“…Vc1.1 contains an amidated C terminus, a post-translational modification common to most ␣-conotoxins, but it is not present in RgIA. Vc1.1 lacks the post-translationally modified hydroxyproline and ␥-carboxyglutamate residues present in the native peptide, vc1a, isolated from the venom duct of C. victoriae (18).…”

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confidence: 99%

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Halai

Clark

Nevin

et al. 2009

Journal of Biological Chemistry

113

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Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the ␣9␣10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp 5 -Arg 7 and Asp 11 -Ile 15 , because changes at these positions resulted in the loss of activity at the ␣9␣10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K؉N9A, all of which were more potent than Vc1.1 at both the rat ␣9␣10 and the human ␣9/rat ␣10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the ␣3␤2, ␣3␤4, and ␣7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the ␣9␣10 nAChR. These findings provide valuable insight into the interaction of Vc1

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confidence: 99%

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Halai

Clark

Nevin

et al. 2009

Journal of Biological Chemistry

113

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“…A polymerase chain reaction screen of cDNAs from the venom ducts of Conus victoriae was used to reveal ␣-conotoxin Vc1.1 (Sandall et al, 2003). The native peptide, designated vc1a, was subsequently identified in the venom of C. victoriae using MS analysis and has the two residues, Pro6 and Glu14, post-translationally modified to hydroxyproline and ␥-carboxyglutamate, respectively (Jakubowski et al, 2004). Synthetic Vc1.1 is a competitive antagonist of neuronal nAChRs in bovine adrenal chromaffin cells (Clark et al, 2006) and is most potent at recombinant ␣9␣10 nAChRs expressed in X. laevis oocytes (Vincler et al, 2006).…”

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confidence: 99%

Are α9α10 Nicotinic Acetylcholine Receptors a Pain Target for α-Conotoxins?

Nevin¹,

Clark²,

Klimis³

et al. 2007

Mol Pharmacol

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“…The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products.…”

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confidence: 99%

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

Thakur

Balaram

2008

J. Am. Soc. Mass Spectrom.

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The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of C ␣ H and C ␤ p H protons resulting in product ions derived by neutral loss of H 2 S 2 and H 2 S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. M ass spectrometric characterization of disulfide bonded peptides is generally achieved by the reduction of the S-S bond followed by alkylation [1][2][3][4] or by oxidation to sulfonic acid derivatives [5]. Subsequent gas-phase fragmentation results in readily identifiable backbone cleavage products. Several groups have focused on the mass spectrometric analysis of peptides containing intact disulfide bonds using both positive and negative ions for gas-phase fragmentation [6 -12]. In the case of positively charged peptide ions, disulfide bond fragmentation occurs with much lower efficiency than cleavage of backbone peptide bonds, under conditions of collision induced dissociation with non-mobile protons suggested to play an important role [11]. Restricted proton mobility along the polypeptide backbone has also been implicated in cases of disulfide cleavages in polypeptides, which have been cationized using gold (I) [7]. The use of metal ions as gas-phase disulfide cleavage agents has also been investigated [12]. In general, only limited information regarding structure can be derived from intact disulfide peptides in the positive ion mode. In contrast, several recent studies have emphasized the utility of negative ion mass spectrometry [7,10,[13][14][15][16][17][18]. Specifically, Bowie and coworkers have presented assignments of product ion spectra obtained by negative ion electrospray mass spectrometry of peptides containing both intra and inter molecular disulfide bond [10,18]. The present study complements the work of Bowie and coworkers and further provides mechanistic details by examining second generation product ions by using ion trap mass spectrometry.In the structural analysis of peptides containing intact disulfides, under negative ion conditions, proton abstraction from the C ␣ H and C ␤ H positions of the Cys residue results in diverse fragmentation reactions, leading to cleavage of disulfide linkages. In the case of peptides that contain labile peptide bonds, for example X-Pro sequences, preferential cleavage at these sites results in two linear chains linked by a disulfide bond, which then undergo further f...

show abstract

Determining sequences and post‐translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry

Cited by 54 publications

References 53 publications

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Are α9α10 Nicotinic Acetylcholine Receptors a Pain Target for α-Conotoxins?

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

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