Identification of glutamine synthetase as a contryphan-Bt binding protein by His-tag pull-down

Background: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. Methods: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Results: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Conclusions: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

Identification of glutamine synthetase as a novel contryphan-Bt binding protein by his-tag pull down

Abstract(1)BackgroundContryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance.(2)MethodsHis-tag pull down assays were performed to investigate binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings.(3)ResultsGlutamine synthetase was identified as a potential contryphan-Bt binding protein by pull down assays and subsequent LC-MS/MS. The binding of contryphan-B to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center.(4)ConclusionsGlutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins.Key ContributionThis is the first report that the conopeptide binds to an intracellular protein, therefore offering a new concept and methodology for developing peptide toxins.