Study On Active Residues In BmK CT And Its Inhibition Effect On Migration Of Glioma

The first chlorotoxin (CTX)-like peptide, BmK CT, isolated from the venom gland of B. martensii Karsch, is a36mer peptide cross-linked by four disulfide bridges. Its sequence identity is68%similar to CTX isolated from the scorpion Leiurus quinquestriatus. The biological character of BmK CT has been well documented as a novel blocker of chloride channel and matrix metalloproteinase-2(MMP-2), rendering it an efficient inhibitor against the devastating invasiveness of glioma cancer cells.This research topic tried to find out the role of key active sites in BmK CT during the interaction process with its receptor-MMP-2. Site-directed mutagenesis of BmK CT, wound healing assay, gelatin zymography assay and computational simulation highlighted the importance of electrostatic contribution to BmK CT-MMP-2catalytic domain complex and a model of BmK CT-MMP-2catalytic domain complex was therefore proposed. BmK CT and its corresponding mutants (~5kDa) showed high expression level in in E. coli BL21(DE3), amounting to approximately20%of the total cellular proteins. According to the ellipticity values of208and222nm in the CD spectra analysis, we found that BmK CT and the mutants belonged to α+β type protein.0.15μM (for both BmK CT and its mutants) was selected as an appropriate concentration to compare the inhibition rates of C6cell migration. In the wound healing assay, the average inhibition rates of BmK CT, BmK CT*(denaturation and refolding), BmK CTK25A and BmK CTR35A were ranging from17to26%, which was two-fold higher than that of BmK CTR14k15AA and BmK CTR17A Gelatin Zymography assay suggested that BmK CT and its mutants could inhibit migration of glioma cells in a MMP-2dependent manner. The inhibitory capability of BmK CTR14K15AA and BmK CTR17A was weaker than that of BmK CT, BmK CTK25A, and BmK CTR35A. Based on the calculated electrostatic potential surface analysis, once the mutation occurred in α-helix as demonstrated in BmK CTR14K15AA and BmK CTR17A, the α-sheet2and C-terminal of a-helix were necessary in interacting with MMP-2catalytic domain. So, three important basic residues namely R14, R17and K15were the active residues of BmK CT. Collectively, this research topic may allow us to develop more potent MMP-2inhibitors with selective anti-glioma potential.We also tried to establish highly specific chlorine toxin molecular probes that target the receptor on glioma cell surface by developing novel molecular modeling of scorpion toxins and then using site-directed mutagenesis to optimize the mutual interactions. Based on modeling of the structure of BmK CT-MMP-2complex by using Swiss-Model, ZDOCK server and Insight II2000software, we designed a protein molecular model (named Op-BmK CT) with high selectivity and high specific activity for glioma cells. After expression and purification of Op-BmK CT, a electrophoretic homogeneity Op-BmK CT protein was obtained. Wound healing assay showed that the inhibition rate of the Op-BmK CT was twice that of the wild type BmK CT. The optimization of both the total energy and the protein surface electrostatic potential energy may be responsible for the observed results.Furthermore, the red fluorescent nanodiamonds (FND) have emerged as a new candidate material due to the obvious advantage of non-cytotoxicity, chemical stability and high affinity to biomolecules. The BmK CT coupled FND bioconjugates were produced via the formation of amide after FND surface functionalized with carboxyl groups. Assembled red upconversion fluorescence nanodiamonds could be detected in the cytoplasm of C6cells when treated with FND without FBS and FND-BmK CT, respectively, for5h at the particle concentration of5μg/ml, which demonstrated that MMP-2-mediated uptake of FND-BmK CT bioconjugated into rat C6glioma cells and direct tumor visualization were actualized by this delivery system. Further experiments indicated that glioma-specific multifunctional nanoparticles based on FND-BmK CT delivery vehicle could possibly be developed into more effective therapeutic agents in clinical treatment of glioma. To sum up, the study provided a theoretical foundation and experimental basis for anti-glioma drug research and development, and the experimental results implicated a scientific and economic significance.