Purification And Characterization Of Conopeptide Contryphan-bt And Search For Its Interacting Proteins

Conopeptides are a diverse group of bioactive peptides derived from cone snails venom.They can target a broad range of ion channels,membrane receptors and neurotransmitter transporters with high potency and specificity,which makes them valuable as pharmacological probes and drug leads.The sequencing of venom gland transcriptomes has facilitated larger,more rapid identification of novel venom peptides.However,only 0.1%conopeptides were pharmacologically characrized,which has become major obstacle to the drug development of conopeptides.In this research,a novel contryphan peptide was purified and identified from the venom of Conus betulinus,a vermivorous species distributed in the South China Sea.Its sequence,ZSGCOWKPWC-NH₂?Z,pyroglutamic acid,O:4-hydroxyproline,W:D-Trp?,was ascertained by a combination of de novo MS/MS sequencing,venom-duct transcriptome sequencing and chemical synthesis.This is the first occurrence of a pyroglutamic acid residue in the contryphans.According to the nomenclature of contryphans family,the peptide was named contryphan-Bt,with Bt from the word?betulinus‘.CD spectrum reveals that contryphan-Bt possess?-turn in solution.Like other contryphans,contryphan-Bt produces?stiff-tail‘syndrome in mice.Electrophysiological experiments,carried out on rat dorsal root ganglion neurons and hippocampal neurons,showed that contryphan-Bt weakly inhibits K⁺channels and has no effect on L-type Ca²⁺channels.Contryphan-Bt interacting protein was studied by traditional affinity chromatography.Contryphan-Bt and its derivatives contryphan-Bt-1 and Bt-2 were synthesized and covalently coupled to NH₂/COOH/NHS magnetic beads,CNBr activated Sepharose 4B and PEG resin via N/C terminal or Lys⁷ residue,respectively.The toxin-immobilized matrices were used to purify contryphan-Bt interacting protein from mouse brain membrane proteins,mouse brain homogenates and pig liver homogenates,but the results was undesirable and no protein was preyed.Contryphan-Bt interacting protein was then explored by PolyHis pull-down technique.The fusion peptide Bt-Acp-[His]₆,containing 6×His tag,6-aminocaproic acid and contryphan-Bt,was designed and synthesized.A 42 kDa protein was preyed by Bt-Acp-[His]₆ from rabbit brain membrane proteins,rabbit brain homogenates and pig liver homogenates.It was identified as glutamine synthetase by LC-MS/MS and confirmed by Western blot analysis.The binding between contryphan-Bt and glutamine synthetase was measured by Microscale Thermophoresis,and the dissociation constant K_d was about 79.23±9.21?M.The enzyme activity of glutamine synthetase,determined by?-glutamyl hydroxamate method,was not affected by the binding,suggesting that the binding site of contryphan-Bt was far from enzyme activity center.Finally,the membrane target of contryphan-Bt was studied using photoaffinity labeling technique.The photoaffinity probe biotin-Acp-Bpa-Bt was synthesized by introducing photoreactive Bpa?p-benzoyl-L-phenylalanine?,6-aminocaproic acid as space arm and biotin as purification tag into the peptide chain.HPLC analysis and immunofluorescence experiments showed that the probe could specifically bind to the membrane of mouse hippocampal neuronal cell line HT22 upon photoirradiation.After incubation with HT22 cells and subsequent irradiation with light of 365 nm,the probe labeled proteins were purified by streptavidin resin or directly digested on beads,and identified by LC-MS/MS.Combination with the MS/MS data and the general range of conopeptide targets,voltage-dependent anion channel protein 1 was scanned as the potential target of contryphan-Bt.