Chinese Abstract
在現代醫學上，從動物毒液中所提煉的毒素具有相當高的潛力作為藥物設計的模板。蠍氯毒素（CTX），一種由36個氨基酸所組成且含有四對雙硫鍵的短胜肽，已在癌症的治療和在顯像劑開發上有許多的應用。由於其對於脊椎動物幾乎無毒和能夠專一性結合到腫瘤細胞的特性(尤其是腦瘤細胞)；CTX被普遍認為在未來的醫療上具有很好的價值。對於癌細胞的專一性可能肇因於能選擇與氯離子通道、基質金屬蛋白酶-2（MMP-2）和膜聯蛋白A2 (Annexin A2)進行結合。據報導，CTX可透過抑制氯離子通道來降低人類臍靜脈內皮細胞（HUVEC）以及膠質瘤細胞的遷移力，如：U-87 MG和U-373 MG。在過去的研究中，透過使用膜片鉗垂體測量GH3細胞的全細胞電容，我們發現CTX及其突變株可以抑制單元電容。為了確定CTX是如何參與神經膠質瘤細胞遷移，我們將CTX與其同源蛋白進行序列比對；結果顯示在N端區域（位點1-9）、第二迴圈（位點22-25）和C端（位點的36-39）除了氨基酸序列組成不同之外，連長度都有所變異。另外，根據結構分析，這三個區域在空間中面對相近的方向。因此，我們推測它們可能彼此協同於CTX與其目標受體的相互作用中。迄今為止，我們使用酵母菌株(P. pastoris)作為表現蛋白的宿主且已成功生產出高純度的CTX與十三個相關的突變株，產率約為1-11 mg/L。這些重組蛋白可藉由不同的劑量對腦瘤細胞遷移進行抑制，以達到研究其功能與結構之間的關係。從突變株R36APY, M1R, Y29K於抑制腦瘤細胞遷移實驗與結晶學的結果，說明CTX之N, C端及loop-2的鄰近區域對其功能很有可能存在重要的影響；而且這些結果與之前對於細胞電容值的研究十分相似。在我們成功解出正常的CTX及其突變株M1R, Q11N及Y29K等四個三維結構中，解析度均小於2 Å；RMSD均小於0.1 Å，且具有相似的三級結構。有趣的是，11Gln, 14Arg, 15Lys, 23Lys等位向有差異的支鏈通常位於結構上較穩定的區域；而對腦瘤遷移有影響的位點卻通常在較有彈性的區域。這些結果協助我們了解CTX的結構對於其功能的影響，也期望在未來對抗癌藥物的設計上提供參考。
關鍵字：蠍氯毒素、神經膠質瘤細胞、細胞遷移、結構分析

English Abstract
Toxins from venom can serve as potential drugs or scaffolds for drug design. Chlorotoxin (CTX), a 36 amino acids small peptide with 4 disulfide bonds, has potential therapeutic applications in the treatment of cancer and in the development as an an imaging agent due to its low toxicity to vertebrates and its specific binding to cancer cells, especially glioma. This targeting specificity is due to its selective binding to chloride channel, matrix metalloproteinase-2 (MMP-2), and annexin A2. It was reported that CTX could inhibit the migration of malignant glioma cells, U-87 MG, and U-373 MG, as well as human umbilical vein endothelial cell (HUVEC) that may be due to the cellular volume change mediated by ion channels. We found that CTX and its mutants also inhibited cell capacitance, which was measured by the whole-cell capacitance of pituitary GH3 cells using patch clamp. To identify the residues of CTX involved in the inhibition of glioma cell migration, we have analyzed primary and tertiary structures of CTX homologous proteins. The sequence alignment of CTX and its homologous peptides showed that N-terminal region (the residues 1-9), the loop-2 region (the residues 22-25) and C-terminus (the residue 36-39) exhibited diverse amino acid sequence. We also found that three regions faced the same direction according to structural analysis. Therefore, we hypothesized that they may cooperatively interact with CTX-targeting proteins. To date, I have successfully expressed wild-type and thirteen CTX mutants in Pichia pastoris and purified them to homogeneity with the yields of 1-11 mg/L. Functional analysis showed that recombinant CTX inhibited FBS- and PDGF-induced glioma cells (U-87 and U-373) migration in a dose-dependent manner. The relative inhibitory activity of glioma cell migration by CTX mutants were R36APY<M1R<Y29K, showing that C- and N-terminal regions, as well as loop-2 of CTX, may play an important role in inhibiting glioma migration. This was very similar to the results of cell capacitance inhibition by CTX. We have successfully determined 3D structures of wild-type, M1R, Q11N, and Y29K of CTX using X-ray crystallography. Superimposition of the backbone atoms of the four solved structures resulted in the root mean square values (RMSD) of 0.072, 0.048, 0.082 Å, indicating that they have similar tertiary fold. In contrast, the sidechain orientations of 11Gln, 14Arg, 15Lys, 23Lys and 36Arg were different. According the results of B-factor, N- and C-terminal regions were the most flexible region in CTX; loop-1 and loop-3 regions have minor flexibility. Interestingly, we found that the residues with different sidechain orientations were not located at flexible regions, while the residues involved in glioma cell migration were located in flexible regions. Results of this study will serve as the basis for the design of CTX mutant in the treatment of cancer.

Key words: Chlorotoxin, glioma cell migration, structural analysis.

Adams, D.J., Callaghan, B., and Berecki, G. (2012). Analgesic conotoxins: block and G protein-coupled receptor modulation of N-type (Ca(V) 2.2) calcium channels. British journal of pharmacology 166, 486-500.
Alam, M., Ali, S.A., Abbasi, A., Kalbacher, H., and Voelter, W. (2012). Design and Synthesis of a Peptidyl-FRET Substrate for Tumor Marker Enzyme human Matrix Metalloprotease-2 (hMMP-2). International Journal of Peptide Research and Therapeutics 18, 207-215.
Ali, S.A., Alam, M., Abbasi, A., Undheim, E.A., Fry, B.G., Kalbacher, H., and Voelter, W. (2016). Structure-Activity Relationship of Chlorotoxin-Like Peptides. Toxins 8, 36.
Catacuzzeno, L., Aiello, F., Fioretti, B., Sforna, L., Castigli, E., Ruggieri, P., Tata, A.M., Calogero, A., and Franciolini, F. (2011). Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration. Journal of cellular physiology 226, 1926-1933.
Cheng, C.H., Chen, Y.C., Shiu, J.H., Chang, Y.T., Chang, Y.S., Huang, C.H., Chen, C.Y., and Chuang, W.J. (2012). Dynamics and functional differences between dendroaspin and rhodostomin: insights into protein scaffolds in integrin recognition. Protein science : a publication of the Protein Society 21, 1872-1884.
Cheng, Y., Zhao, J., Qiao, W., and Chen, K. (2014). Recent advances in diagnosis and treatment of gliomas using chlorotoxin-based bioconjugates. Am J Nucl Med Mol 4, 385-405.
Craik, D.J., Fairlie, D.P., Liras, S., and Price, D. (2013). The future of peptide-based drugs. Chemical biology & drug design 81, 136-147.
Cuddapah, V.A., and Sontheimer, H. (2010). Molecular interaction and functional regulation of ClC-3 by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in human malignant glioma. The Journal of biological chemistry 285, 11188-11196.
Cuddapah, V.A., Turner, K.L., Seifert, S., and Sontheimer, H. (2013). Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3. The Journal of neuroscience : the official journal of the Society for Neuroscience 33, 1427-1440.
Dardevet, L., Rani, D., Aziz, T.A., Bazin, I., Sabatier, J.M., Fadl, M., Brambilla, E., and De Waard, M. (2015). Chlorotoxin: a helpful natural scorpion peptide to diagnose glioma and fight tumor invasion. Toxins 7, 1079-1101.
Debin, J.A., Maggio, J.E., and Strichartz, G.R. (1993). PURIFICATION AND CHARACTERIZATION OF CHLOROTOXIN, A CHLORIDE CHANNEL LIGAND FROM THE VENOM OF THE SCORPION. American Journal of Physiology 264, C361-C369.
Deshane, J., Garner, C.C., and Sontheimer, H. (2003). Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2. The Journal of biological chemistry 278, 4135-4144.
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta crystallographica Section D, Biological crystallography 60, 2126-2132.
Ernest, N.J., Weaver, A.K., Van Duyn, L.B., and Sontheimer, H.W. (2005). Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells. American journal of physiology Cell physiology 288, C1451-1460.
Gerke, V., Creutz, C.E., and Moss, S.E. (2005). Annexins: linking Ca2+ signalling to membrane dynamics. Nature reviews Molecular cell biology 6, 449-461.
Heldin, C.H. (2013). Targeting the PDGF signaling pathway in tumor treatment. Cell communication and signaling : CCS 11, 97.
Hockaday, D.C., Shen, S., Fiveash, J., Raubitschek, A., Colcher, D., Liu, A., Alvarez, V., and Mamelak, A.N. (2005). Imaging glioma extent with 131I-TM-601. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 46, 580-586.
Huys, I., Waelkens, E., and Tytgat, J. (2004). Structure-function study of a chlorotoxin-chimer and its activity on Kv1.3 channels. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 803, 67-73.
Jacoby, D.B., Dyskin, E., Yalcin, M., Kesavan, K., Dahlberg, W., Ratliff, J., Johnson, E.W., and Mousa, S.A. (2010). Potent pleiotropic anti-angiogenic effects of TM601, a synthetic chlorotoxin peptide. Anticancer research 30, 39-46.
Jentsch, T.J., Gunther, W., Pusch, M., and Schwappach, B. (1995). Properties of voltage-gated chloride channels of the ClC gene family. The Journal of physiology 482, 19s-25s.
Kesavan, K., Ratliff, J., Johnson, E.W., Dahlberg, W., Asara, J.M., Misra, P., Frangioni, J.V., and Jacoby, D.B. (2010). Annexin A2 is a molecular target for TM601, a peptide with tumor-targeting and anti-angiogenic effects. The Journal of biological chemistry 285, 4366-4374.
Kordis, D., and Gubensek, F. (2000). Adaptive evolution of animal toxin multigene families. Gene 261, 43-52.
Leader, B., Baca, Q.J., and Golan, D.E. (2008). Protein therapeutics: a summary and pharmacological classification. Nature reviews Drug discovery 7, 21-39.
Li, C., Wen, A., Shen, B., Lu, J., Huang, Y., and Chang, Y. (2011). FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnology 11, 92.
Lima e Silva, R., Shen, J., Gong, Y.Y., Seidel, C.P., Hackett, S.F., Kesavan, K., Jacoby, D.B., and Campochiaro, P.A. (2010). Agents that bind annexin A2 suppress ocular neovascularization. Journal of cellular physiology 225, 855-864.
Lippens, G., Najib, J., Wodak, S.J., and Tartar, A. (1995). NMR sequential assignments and solution structure of chlorotoxin, a small scorpion toxin that blocks chloride channels. Biochemistry 34, 13-21.
Lovell, S.C., Davis, I.W., Arendall, W.B., 3rd, de Bakker, P.I., Word, J.M., Prisant, M.G., Richardson, J.S., and Richardson, D.C. (2003). Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50, 437-450.
Lyons, S.A., O'Neal, J., and Sontheimer, H. (2002). Chlorotoxin, a scorpion-derived peptide, specifically binds to gliomas and tumors of neuroectodermal origin. Glia 39, 162-173.
Mamelak, A.N., and Jacoby, D.B. (2007). Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin (TM-601). Expert opinion on drug delivery 4, 175-186.
Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A.A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta crystallographica Section D, Biological crystallography 67, 355-367.
Olsen, M.L., Schade, S., Lyons, S.A., Amaral, M.D., and Sontheimer, H. (2003). Expression of voltage-gated chloride channels in human glioma cells. The Journal of neuroscience : the official journal of the Society for Neuroscience 23, 5572-5582.
Onishi, M., Ichikawa, T., Kurozumi, K., Inoue, S., Maruo, T., Otani, Y., Fujii, K., Ishida, J., Shimazu, Y., Yoshida, K., et al. (2015). Annexin A2 regulates angiogenesis and invasion phenotypes of malignant glioma. Brain tumor pathology 32, 184-194.
Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Macromolecular Crystallography, Pt A 276, 307-326.
Qin, C., He, B., Dai, W., Lin, Z., Zhang, H., Wang, X., Wang, J., Zhang, X., Wang, G., Yin, L., et al. (2014). The impact of a chlorotoxin-modified liposome system on receptor MMP-2 and the receptor-associated protein ClC-3. Biomaterials 35, 5908-5920.
Schägger, H., von Jagow, G. (1987). Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa ANALYTICAL BIOCHEMISTRY 166, 368-379
Soroceanu, L., Gillespie, Y., Khazaeli, M.B., and Sontheimer, H. (1998). Use of chlorotoxin for targeting of primary brain tumors. Cancer research 58, 4871-4879.
Soroceanu, L., Manning, T.J., Jr., and Sontheimer, H. (1999). Modulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers. The Journal of neuroscience : the official journal of the Society for Neuroscience 19, 5942-5954.
Suzuki, M., Morita, T., and Iwamoto, T. (2006). Diversity of Cl(-) channels. Cellular and molecular life sciences : CMLS 63, 12-24.
Tamborini, M., Locatelli, E., Rasile, M., Monaco, I., Rodighiero, S., Corradini, I., Comes Franchini, M., Passoni, L., and Matteoli, M. (2016). A Combined Approach Employing Chlorotoxin-Nanovectors and Low Dose Radiation To Reach Infiltrating Tumor Niches in Glioblastoma. ACS nano 10, 2509-2520.
Tatenhorst, L., Rescher, U., Gerke, V., and Paulus, W. (2006). Knockdown of annexin 2 decreases migration of human glioma cells in vitro. Neuropathology and applied neurobiology 32, 271-277.
Terlau, H., and Olivera, B.M. (2004). Conus venoms: a rich source of novel ion channel-targeted peptides. Physiological reviews 84, 41-68.
Turner, K.L., and Sontheimer, H. (2014). Cl- and K+ channels and their role in primary brain tumour biology. Philosophical transactions of the Royal Society of London Series B, Biological sciences 369, 20130095.
Turpin, E., Russo-Marie, F., Dubois, T., de Paillerets, C., Alfsen, A., and Bomsel, M. (1998). In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner. Biochimica et biophysica acta 1402, 115-130.
Vaguine, A.A., Richelle, J., and Wodak, S.J. (1999). SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta crystallographica Section D, Biological crystallography 55, 191-205.
Veiseh, M., Gabikian, P., Bahrami, S.B., Veiseh, O., Zhang, M., Hackman, R.C., Ravanpay, A.C., Stroud, M.R., Kusuma, Y., Hansen, S.J., et al. (2007). Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci. Cancer research 67, 6882-6888.
Watkins, S., and Sontheimer, H. (2011). Hydrodynamic cellular volume changes enable glioma cell invasion. The Journal of neuroscience : the official journal of the Society for Neuroscience 31, 17250-17259.
Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G., McCoy, A., et al. (2011). Overview of the CCP4 suite and current developments. Acta crystallographica Section D, Biological crystallography 67, 235-242.
Yin, L.T., Fu, Y.J., Xu, Q.L., Yang, J., Liu, Z.L., Liang, A.H., Fan, X.J., and Xu, C.G. (2007). Potential biochemical therapy of glioma cancer. Biochemical and biophysical research communications 362, 225-229.
Zhai, H., Acharya, S., Gravanis, I., Mehmood, S., Seidman, R.J., Shroyer, K.R., Hajjar, K.A., and Tsirka, S.E. (2011). Annexin A2 promotes glioma cell invasion and tumor progression. The Journal of neuroscience : the official journal of the Society for Neuroscience 31, 14346-14360.
Zhang, Y., Huang, Z., Wu, Z., Yin, G., Wang, L., and Gao, F. (2016). Functionalized magnetic nanochains with enhanced MR imaging: A novel nanosystem for targeting and inhibition of early glioma. Colloids and surfaces B, Biointerfaces 140, 437-445.
Zhao, L., Shi, X., and Zhao, J. (2015). Chlorotoxin-conjugated nanoparticles for targeted imaging and therapy of glioma. Current topics in medicinal chemistry 15, 1196-1208.