Study On The Biological Activity Of Chloride Channel Toxin From The Chinese Scorpion Buthus Martensii Karsch

Scorpion is an animal which preys on mollusk insect.Its venom is rich sources of toxic poly peptides that affect ion-channel function of excitable cells,containing 20-90 amino acids linked by three or four disulfide bridges. Four different families of scorpion toxins have been described,which interact with ion channels:Na⁺,K⁺,Cl^- and Ca²⁺.They are proved to be important tools for clarifying the physical effects and functional mapping of ion-channels.Scorpion venoms are contain rich source of small,mainly neurotoxic proteins which interacting with ionic channels in excitable cell membranes.In our previous work,a choride channel peptide BmK CT(Accession Number:AF 135821) was synthesized with a sequence optimized for codon usage in E.coli encoding.This 35 amino acids peptide linked by four disulfide bridges,and shares 68%of identity with the sequence of chlorotoxin,an inhibitor of Cl^- channels from the Leiurus quinquestriatus which was purified and characterized by DeBin et al.The results determined by MTT assay showed that rBmK CTa could inhibited the survival of glioma cellsand had less toxicity on cortical astrocytes.In this study,we expressed rBmK CTa in E.coli BL21(DE3).The protein was purified by Ni-NTA resin to analyze the influence of rBmK CTa on the migration and invasion of glioma cells.Gelatin zymography,western blot, and pull down assay were used to determine the interaction between rBmK CTa and its putative receptors of glima cells—MMP-2(matrix metalloproteinase-2).The ¹³¹I labeled ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma,to investigate the biodistribution and pharmacokinetics of BmK CT.The results were obtained as followed:1.The recombinant expression plasmid pRSETc-rBmK CTa was constructed and the fusion protein rBmK CTa(Recombinant BmK CT artifact) was expressed in E.coli BL21(DE3).rBmK CTa was eluted after purificated on the Ni-NTA resin.However,it was found that most of rBmK CTa was existed as monomer Western blot.Finally,purified protein was obtained by centrifugation.The final product by this procedure was about 1.5-2.1 mg/L cultured cells.This step suggested the pharmaceutical function of rBmK CTa.2.The wound healing assay and matrigel invasion assay observation were showed that rBmK CTa decreased human glioma cell SHG-44 migration and cell invasion(IC₅₀ is 0.28μM).Immunochemistry,zymography and western blot results demonstrated that rBmK CTa could inhibit expression of MMP-2. Otherwise,the results of flow cytometry showed that the cell cycle of glioma was arrested in G0/G1 phase after treated.And the rate of S phase was decreased.The apoptosis rate of glioma cell was approximately 50%when treated with 3.2μM rBmK CTa.These result suggested that neurotoxin BmK CT may have a therapeutic role on the medical application of glioma.3.The gene sequence MMP-2C was encoding from the total RNA of human glioma cell SHG-44 by RT-PCR and was cloned into prokaryotic expression vector pRSETc for expression in E.coli.The product was found in inclusion bodies.After denaturation and renaureation procedure,soluble MMP-2C was obtained.The results indicated MMP-2C and rBmK CTa could interact in vitro by using in vitro pull down assay.4.The fusion protein,ZZ-rBmK CTa-Tyr,was expressed in E.coli BL21 (DE3) using a pExSecI expression system and was purified using IgG sepharose column.The ZZ-rBmK CTa-Tyr was then radiolabeled with ¹³¹I using standard Iodogen Bead method.After purified with Sepharose G-25,the radio-chemical purity of the sample was reach to 90%.¹³¹I-ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma SHG-44 at the dose of 2μCi and 17.9μCi.SPECT analysis were performed on the mice received ¹³¹I-ZZ-rBmk CTa at 10 period of timeinjection.The results showed that ZZ-rBmk CTa can cross blood and tissue barriers immediately.Mice were sacrificed 24 h later,the blood,heart, liver,spleen,lung,kidney,stomach,small intestine,skeleton,muscle and tumors were weighed and counted to determine ¹³¹I-ZZ-rBmk CTa concentration.The binding amount of ¹³¹I- ZZ-rBmk CTa in the right brain was much higher than the left brain,and T/NT is 1.25 and 4.13 respectively. Suggesting that ZZ-rBmk CTa can binds to brain glioma cells by the high affinity action power with glioma cells,reflecting non-specific uptake of rBmk CTa in normal tissues in the brain.