Study On The Chlorotoxin-like Neurotoxin From The Chinese Scorpion Buthus Martensii Karsch

Scorpion venoms are a particularly rich source of small, mainly neurotoxic proteins or peptides interacting specifically with various ionic channels in excitable membranes. Buthus martensii Karsch (BmK) is a representative species of scorpion in northwestern China, Mongolia and Korea. Its neurotoxins have been widely studied in recent years, including investigations into their physiological and biological functions, pharmaceutical properties and primary and three-dimensional structures. According to their molecular sizes, these neurotoxins can be divided into long- and short-chain neurotoxins. The accumulated data have shown that the most deadly components of them are long-chain peptides which are consisted of 60-70 amino acid residues cross-linked by four disulfide bridges and selectively block the voltage-gated sodium channels. Short-chain toxins are composed of 28-40 amino acid residues and are mainly cross-linked by three or four disulfide bridges and are mostly active on K~+ and/or Cl~- channels. The peptides, which have a potent block of small conductance Cl" channels inexcitable cells, are called chlorotoxin. The first chlorotoxin-like peptide gene has been cloned and sequenced from the venom of B. martensii Karsch. This 35 amino acid long peptide cross-linked by four disulfide bridges shares 66% of identity with the sequence of chlorotoxin, an inhibitor of small-conductance Cl" channels from the scorpion Leiurus quinquestriatus which was purified and characterized by DeBin et al.Primary brain tumors (gliomas) have the unusual ability to diffusely infiltrate the normal brain thereby evading surgical treatment. This type of chlorotoxin can specifically binds to the surface of glioma cells and impairs their ability to invade. The exact sub-type of Cl" channels that is affected by BmK CT remains to be identified.In this study, we synthesized the nucleotide sequence of this chlorotoxin-like peptide with a sequence optimized for codon usage in E. coll A simple and efficient purification protocol was designed to get a soluble and functional form of rBmK CTa, which could be used to study the pharmaceutical function of this neurotoxin. Cytotoxicity assays, overlay assays, whole cell patch-clamp recording and histological analysis were performed to determine the biochemical characterization and clinical significances of it.Part I Synthesis, expression and purification of this type of chlorotoxin-like peptide from the scorpion Buthus martensii KarschIn this section, the gene encoding a chlorotoxin-like peptide from thescorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in E. coli. In order to get high-level expression in E. coli, the BmK CT gene sequence was modified, in which 24 residues were replaced according to the codon usage of E. coli. This artificial synthesized chlorotoxin-like peptide gene, rBmK CTa, was over expressed in E. coli BL21 (DE3) using a secretive expression system - pExSecI. According to the characteristics of the pExSecI expression system, the IgG-binding domain-ZZ of protein A is fused to the iV-terminal of rBmK CTa. Because of the relatively protein-free property of the LB medium, it is easier for us to design a simple and efficient purification protocol as described blow. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form and was purified by IgG-Sepharose 6 Fast Flow and Superdex-75 gel filtration chromatography to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn-Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. The yield of affinity-purified protein was 1.4 mg/L of culture, estimated by the Bradford method. Western blot analysis was probed with rabbit anti-scorpion neurotoxin polyclonal antibody as the first antibody as prepared by the institute of Zoology in the Chinese Academy of Science, which demonstrated that this protein was rBmK CTa. This step shed light on the pharmaceutical function of this neurotoxin.xPart II Animal acute toxicity and cytotoxicity assays of rBmK CTa to cortical astrocytes and human glioma cell line SHG-44 and IC50 Determination Acute toxicity testing was performed using a modified LD50 assay. Nine 12-14 g male white mice were used for each test. Survival times after injection of increased doses (5-20 u g / g) of rBmK CTa in mice were recorded to the nearest 5 sec. According to the results of observed data including the calculated dose versus survival time (D/T) and the calculation scheme, the approximate LD50 value was 4.3 mg / kg. With this test it is possible to obtain an LD50 using only 8-10 experimental animals, instead of 30 or more. The main advantage of the method is that it permits the use of far fewer experimental animals. As classical LD50 experiments, owing to their high level of variability, are far from satisfactory, a change to the approximate method of LD50 assays is suggested for scientific, economic and ethical reasons.MTT assay, SCGE, morphological observation and internucleosomal DNA fragmentation electrophoresis were used in pre-experiment showed that recombinant rBmK CTa could have cytotoxicity to cortical astrocytes of rats. Then, human glioma cells (SHG-44) were used for examining the effects of rBmK CTa on cell growth by MTT assay. rBmK CTa at the increasing concentrations (30 nM -96 u M) was added to cell cultures (5X 104 cells / well) for 24 h without renewal of the medium. Inhibition index (11%) was calculated according to the following equation: II(%) = (1-T /C) X100%,where T and C represent the mean optical density of the treated group and vehicle control group, respectively. The results showed that rBmK CTa obviously inhibits the survival of glioma cells in a dose-dependent manner, and the 50% inhibitory concentration (IC50) value was approximately 0.28 u M while as high as 8 u M on cortical astrocytes, which demonstrated that the cytotoxicity of this toxin to normal astrocytes must have been extremely low. This result showed that the rBmK CTa could induce the apoptosis of glioma cells and therefore is functional at least in this respect, which suggest that it may have a potentially therapeutic role for human glioma.Part III Whole cell patch-clamp recording and confirmation of electrophysiological characterization of rBmK CTaThe chloride current (ICi), kalium current (IK) and sodium current (Iua) of single gliomas cell (SHG-44) was recorded respectively using the whole cell voltage technique. Firstly, the effect of rBmK CTa (0.07 uM and 0.14uM, which is 1/4 IC50 and 1/2 IC50 respectively) on chloride current-voltage (I-V) relationship was examined between -105 and +170 mV in 25 mV steps. The results showed that the chloride current was observably inhibited under control conditions in the presence of rBmK CTa. The average inhibition was 17.64% + 3.06% and 55.86%±2.83% (n=5), respectively. Secondly, the effect of rBmK CTa (0.07 u M and 0.14 u M) on kalium current-voltage (I-V) relationship was examined between -80 and +100 mV in 20 mV steps. The results showed thatthe kalium current was not inhibited under control conditions in the presence of rBmK CTa. Thirdly, the effect of rBmK CTa (0.07 uM and 0.14uM) on sodium current-voltage (I-V) relationship was examined between -100 and +0 mV in 20 mV steps. The results showed that the sodium current was not inhibited under control conditions in the presence of rBmK CTa. In conclusion, the whole cell patch-clamp recording analysis showed that chloride current of gliomas cells (SHG-44) was observably inhibited under control conditions in the presence of rBmK CTa, but this inhibition was not presented in kalium current and sodium current, which was identical with the predicted function based on its sequence homology with chlorotoxin.Part IV Preparation of polyclonal antibody and detection of putative receptors of SHG-44 by overlay assay (Far western blot)Polyclonal antibody against expressed rBmK CTa was produced by one basal subcuticular injection to rats. The specificity for the polyclonal antibody against scorpion's natural secretions collected by electrical stimulation was detected by western blot analysis according to the manufacturer's instructions (Bio-Rad). The titer of the polyclonal rBmK CTa antibody, estimated by ELISA assay, was about 1:7000. Immunospecificity was observed for the scorpion's natural secretions.According to MTT assays, we infer that toxin rBmK CTa bind to and act with some putative receptors in SHG-44 cells, which would result in thedestruction of these cells. To detect these putative rBmK CTa receptors, total cell lysates of human SHG-44 glioma cells were probed with rBmK CTa and showed binding to two prominent proteins with a corresponding molecular weight of about 80 kDa and 35 kDa. But these proteins were not present in cultured rat cortical astrocytes. Gel purified 35 kDa receptor was subjected to automated protein sequence analysis. The first six cycles of an Edman degradation gave the sequence: Ser-His-Lys-Gln-Glu-Glu.Part V Effects of rBmK CTa on seven normal organs of adult miceThis study was conducted in three adult mice with body weight about 20g. The toxin, rBmK CTa, was resuspended in PBS and injected celiac at a dose of 1.4 pmol /kg, 2.8 (Jmol /kg body weight respectively and a control mouse with PBS. After 24 h, seven organs, including of kidneys, brain, spleen, liver, leg muscle, lung and cardiac muscle, were dissected and were used for histological analysis. The reagents employed and the sequence of transfers from fixation to paraffin-ribbon mounts are as normal process. The ribbons were stained with Ehrlich's hematoxylin and eosin (H&E). Histological analysis was performed.Histological analysis showed that the brain, cardiac muscle and leg muscle were the target organs of this toxin. The uptakes of rBmK CTa in cardiac muscle and leg muscle was most likely due to the up-regulation ofvotage-gated chloride channels (CLC family) in the muscle tissues, especially C1C-1, which had higher affinity for rBmK CTa. Although penetration of extrinsic proteins through intact blood-brain barrier is very difficult, observed responses using this toxin were promising. Two logic important features were presumed: (1) rBmK CTa is a small, 36 amino acid, peptide that can cross blood and tissue barriers, the effects of rBmK CTa were observed within one day post injection. (2) It can binds to brain glial cells with an unknown action power. rBmK CTa is such a novel targeting agent that had both above features. According to these hypotheses and the results of cytotoxicity and overlay assays, rBmK CTa can bind to human gliomas tumor tissues intensively, but faintly or tinily to non-tumor brain tissues, or to other non-brain tissues in human body.Part VI Crystallization of rBmK CTaCrystallization of rBmK CTa was performed using the hanging-drop method. 51 conditions (Crystal Screening Kit, Hampton Research) were used in order to get good quality, well repeat crystals. The results showed that it could be obtained from a condition containing 1.4 M Sodium Acetate, 0.1 M Na Cacodylate, pH 6.51, at 4°C and the protein was dissolved in ddH2O.In conclusion, the findings presented in this study are essential for the further exploration of this peptide. These results suggested that rBmK CTa hadclinical significances in human gliomas cancer and it may be a useful therapeutic drug to prevent gliomas cancer.