Synthesis and disulfide structure determination of conotoxin GS, a ?-carboxyglutamic acid-containing neurotoxic peptide

Abstract

Conotoxin GS, a y-carboxyglutamic acid(Gla)-containing neurotoxic peptide composed of 34 amino acid residues with'one Gla residue and three intramolecular disulfide bonds, was synthesized in solution by the Boc strategy, using the cyclohexyl group to protect the y,y-dicarboxyl functional side chain of the Gla residue. All of the protecting groups were removed by the HF procedure. During the synthesis, the Gla residue was completely stable and no decarboxylated product was observed. The free peptide was subjected to the oxidative folding reaction. The reaction proceeded almost quantitatively in the presence of reduced and oxidized glutathione; however, no product was formed in the absence of redox reagents concomitant with the formation of disulfide isomers or intermediates. The final product was confirmed to be identical to natural conotoxin GS on reversed phase-and ion exchange-HPLC as well as capillary zone electrophoresis. The disulfide structure of synthetic conotoxin GS was determined by gas-phase sequencing and mass spectrometry of its proteolytic fragments and was found to be identical to those of other o~-conotoxins. The major disulfide isomer obtained during the oxidative folding reaction without redox reagents was determined in the same manner. To clarify the role of the Gla residue and the disulfide structure in the conotoxin GS molecule, decarboxylated conotoxin GS and its disulfide isomer were also synthesized, and the neurotoxic activities and circular dichroism spectra of these peptides were compared with those of conotoxin GS and its disulfide isomer. The results showed that the correct disulfide structure was necessary for expression of the toxicity; however, the presence of the Gla residue was not a prerequisite for both the activity and the calciumdependent conformational transition.

Solution structure of the sodium channel antagonist conotoxin GS: a new molecular caliper for probing sodium channel geometry

“…Disulfide bridges between residues Cys2-Cys14, Cys9-Cys19 and Cys13-Cys27 were indicated by the following NOEs: Cys2H␤-Cys14H␣ (strong intensity), Cys9H␤-Cys19H␤ (medium intensity) and Cys13H␤-Cys27H␤ (medium intensity). This pattern of disulfide bridges is consistent with that determined chemically [15],…”

“…Conotoxin GS and [Glu32]conotoxin GS were prepared by solid-phase peptide synthesis, and the disulfide bonds were introduced by air oxidation in the presence of reduced and oxidised glutathione, as described by Nakao et al [15]. Analytical reverse-phase HPLC and electrospray ionization mass spectrometry confirmed the purity and molecular weight of the synthetic peptides.…”

“…The conotoxins, a family of highly basic 22residue polypeptides (GIIIA, GIIIB and GIIIC), contain six cysteine residues that are paired in a 1-4, 2-5, 3-6 pattern to form three intramolecular disulfide bonds and a three-loop framework. Conotoxin GS has a strikingly different sequence and is 50% larger than the conotoxins; it contains six cysteine residues arranged in a similar 1-4, 2-5, 3-6 pattern [15], but differences in the spacings between cysteine residues results in a four-loop framework rather than a three-loop framework (Fig. 1).…”

Conotoxins and their potential pharmaceutical applications

“…Conotoxin GS has a strikingly different sequence and is 50% larger than the m-conotoxins. This polypeptide contains six cysteine residues arranged in a similar 1-4, 2-5, 3-6 pattern [Nakao et al, 1995]; however, differences in the spacings between cysteine residues results in a four-loop framework rather than a threeloop framework. Despite the low sequence identity, conotoxin GS binds competitively with m-conotoxin GIIIA, suggesting overlapping binding sites on the extracellular surface of skeletal muscle and eel electroplax sodium channels [Yanagawa et al, 1988].…”

Design and synthesis of cysteine-rich peptides

“…Reduced and oxidized glutathiones (GSH/GSSG) were shown to be crucial for the correct folding of many CRPs such as charybdotoxin, calciseptine and ω-conotoxin GS. [301][302][303] However,…”