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

Abstract

Conotoxin GS, a γ-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 γ,γ-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 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 ω-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 calcium-dependent conformational transition.