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Mutation of Angiogenesis Inhibitor TK1-2 to Avoid Antigenicity In Vivo  

Lee Sang-Bae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
Kim Hyun-Kyung (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
Oh Ho-Kyun (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
Hong Yong-Kil (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
Joe Young-Ae (Department of Biomedical Sciences, Cancer Research Institute, The Catholic University of Korea)
Publication Information
Biomolecules & Therapeutics / v.14, no.1, 2006 , pp. 30-35 More about this Journal
Abstract
Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.
Keywords
tissue-type plasminogen activator; kringle doamin; angiogenesis; N-glycosylation; site-directed mutagenesis; Pichia pastoris;
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1 Aoki, S., Shimizu, N., Shimonishi, M., Kitagawa, M., Okumura, K., and Tanigawara, Y. (2001). Influence of sugar chain on fibrin affinity of recombinant t-PA. Biol Pharm Bull 24, 295-298   DOI   ScienceOn
2 Asselbergs, F. A., Burgi, R., and van Oostrum, J. (1993). Functional effects of kringle 2 glycosylation in a hybrid plasminogen activator. Blood Coagul Fibrinolysis 4, 27-33   DOI
3 Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1, 27-31   DOI
4 Hamilton, S. R., Bobrowicz, P., Bobrowicz, B., Davidson, R. C., Li, H., Mitchell, T., Nett, J. H., Rausch, S., Stadheim, T. A., Wischnewski, H., Wildt, S., and Gemgross, T. U. (2003). Production of complex human glycoproteins in yeast. Science 301, 1244-1246
5 Hanahan, D. (1997). CELL BIOLOGY: Signaling Vascular Morphogenesis and Maintenance. Science 277, 48-50   DOI   ScienceOn
6 Hanahan, D., and Folkman, J. (1996). Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis. Cell 86, 353-364   DOI   ScienceOn
7 Larsen, G. R., Henson, K., and Blue, Y. (1988). Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin finger-like and/or the epidermal growth factor domains. J Biol Chem 263, 1023-1029
8 Narhi, L. O., Arakawa, T., Aoki, K. H., Elmore, R., Rohde, M. F., Boone, T., and Strickland, T. W. (1991). The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem 266, 23022-23026
9 Risau W. (1997). Mechanisms of angiogenesis. Nature. 386, 671-674   DOI   ScienceOn
10 Ballou, C. E. (1990). Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol 185, 440-470   DOI
11 Bause, E. (1983). Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331-336   DOI
12 Bennett, W. F., Paoni, N. F., Keyt, B. A., Botstein, D., Jones, A. J., Presta, L., Wurm, F. M., and Zoller, M. J. (1991). High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem 266, 5191-5201
13 Berg, D. T., Burck, P. J., Berg, D. H., and Grinnell, B. W. (1993). Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties. Blood 81, 1312-1322
14 Browder, T., Folkman, J., and Pirie-Shepherd, S. (2000). The Hemostatic System as a Regulator of Angiogenesis. J. Biol. Chem. 275, 1521-1524   DOI   ScienceOn
15 Elliott, S., Chang, D., Delorme, E., Eris, T., and Lorenzini, T. (2004). Structural Requirements for Additional N-Linked Carbohydrate on Recombinant Human Erythropoietin. J. Biol. Chem. 279, 16854-16862   DOI   ScienceOn
16 Folkman, J. (1971). Tumor angiogenesis: therapeutic implications. N Engl J Med. 285, 1182-1186   DOI   ScienceOn
17 Berg, D. T., and Grinnell, B. W. (1993). Pro to Gly (P219G) in a silent glycosylation site results in complete glycosylation in tissue plasminogen activator. Protein Sci 2, 126-127
18 Folkman, J., and D'Amore, P. A. (1996). Blood Vessel Formation: What Is Its Molecular Basis? Cell 87, 1153-1155   DOI   ScienceOn
19 Sage, EH. (1997). Pieces of eight: bioactive fragments of extracellular proteins as regulators of angiogenesis. Trends Cell Biol 7, 182-186   DOI   ScienceOn
20 Helenius, A., and Aebi, M. (2001). Intracellular functions of Nlinked glycans. Science 291, 2364-2369   DOI   ScienceOn
21 Imperiali, B., and O'Connor, S. E. (1999). Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 3, 643-649   DOI   ScienceOn
22 Ingber, D. E., and Folkman, J. (1989). How does extracellular matrix control capillary morphogenesis? Cell 58, 803-805   DOI   ScienceOn
23 Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973). Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52, 2745-2756   DOI   ScienceOn
24 Khanna, R., Myers, M. P., Laine, M., and Papazian, D. M. (2001). Glycosylation Increases Potassium Channel Stability and Surface Expression in Mammalian Cells. J. Biol. Chem. 276, 34028-34034   DOI   ScienceOn
25 Kim, HK, Lee, SY, Oh, HK, Kang, BH, Ku, HJ, Lee, Y, Shin, JY, Hong YK, and Joe YA, (2003). Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissuetype plasminogen activator. Biochem Biophys Res Commun. 304, 740-746   DOI   ScienceOn
26 Roitsch, T., and Lehle, L. (1989). Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence. Eur J Biochem 181, 525-529   DOI   ScienceOn
27 Shim, BS, Kang, BH, Hong, YK, Kim, HK, Lee, IH, Lee, SY, Lee, YJ, Lee, SK, and Joe, YA. (2005). The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth. Biochem Biophys Res Commun. 327, 1155-1162   DOI   ScienceOn
28 Stack, M. S., Gately, S., Bafetti, L. M., Enghild, J. J., and Soff, G. A. (1999). Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation. Biochem J 340, 77-84   DOI
29 Wilhelm, J., Kalyan, N. K., Lee, S. G., Hum, W. T., Rappaport, R., and Hung, P. P. (1990). Deglycosylation increases the fibrinolytic activity of a deletion mutant of tissue-type plasminogen activator. Thromb Haemost 63, 464-471   DOI
30 Wujek, P., Kida, E., Walus, M., Wisniewski, K. E., and Golabek, A. A. (2004). N-glycosylation is crucial for folding, trafficking, and stability of human tripeptidyl-peptidase I. J Biol Chem 279, 12827-12839   DOI   ScienceOn
31 Imperiali, B., and Shannon, K. L. (1991). Differences between Asn-Xaa-Thr-containing peptides: a comparison of solution conformation and substrate behavior with oligosaccharyltransferase. Biochemistry 30, 4374-4380   DOI   ScienceOn