Research About The Structure-function Relationship Of Two Kinds Of Jingzhaotoxins

Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. It is composed of 34 residues with three disulfide bonds. The primary structure of JZTX-XI is ECRKMFGGCSVDSDCCAHLGCKPTLKYCAWDGTF. The earlier stage’s studies showed that JZTX-XI could inhibit the currents from the Nav channel in the rat cardiac myocytes and the Kv2.1 channel expressed in Xenpus Laevis oocytes, but it had no effect on the Nav and Cav channels currents in adult rat dorsal root ganglion neurons. In order to investigate the structure-function relationship of JZTX-XI, the natural JZTX-XI and mutant R3 A-JZTX-XI were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorptionion ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding of synthetic linear peptides to find the optimal renaturation conditions of toxins. When samples (0.05 g/L) were dissolved in 0.1 mol/L Tris-HCl buffer containing 1.0 mmol/L reduced glutathion (GSH) and 0.1 mmol/L oxidized glutathion (GSSG) at pH 8.0 and 4℃, the best renaturation yields of synthetic peptides were obtained. The experiments also proved that the relative molecular masses of renatured peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak, the bioactivity of refolded JZTX-XI was almost the same as that of native toxin based on the results of electrophysiological experiments. JZTX-XI could only inhibit weakly rNav1.4 channel currents, but competely inhibit those of hNav 1.5 and rKv2.1 channels expressed in HEK293T cells with IC50 values of 438 nM and 95.8 nM respectively. The application of JZTX-XI affected the activation and inactivation characteristics of Navl.5 channel, and caused a depolarizing shift of the current-voltage relationship curve and a hyperpolarizing shift of the steady-state inactivation curve by approximately 10 mV and 8 mV respectively.Under the whole-cell patch-clamp mode, R3A-JZTX-XI could inhibit hNav 1.5 channel currents and rKv2.1 channel currents expressed in HEK293T cells with an IC50 value of 1.96μM and 1.22μM respectively. But the prohibitive level of R3A-JZTX-XI on Nav1.5 channels is only reduced by about 4.5 times and 12.4 times, indicating that Arg3 is not a key amino acid residue relative to the Nav1.5 channel bioactivity of JZTX-XI,but it is a key amino acid residue relative to the Kv2.1 channel bioactivity of JZTX-XI.Jingzhaotoxinl52 (JZTX152) is a novel small molecule neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. It can completely inhibit tetrodotoxin-resistant sodium channel currents in rat dorsal root ganglion neurons. Its molecular formula is C5H4N4O2 determined by combinating reverse-phase high performance liquid chromatography with high precision mass spectrometry. A firenew chemical structure of JZTX152 has been obtained by analyzing a series of 1-D NMR hydrogen spectra, carbon spectra, DEPT spectra and 2-D H-H COSY spectra, C-H HSQC spectra as well as C-H HMBC spectra. The pharmacoclogical experiments of JZTX152 are in progress.