Abstract
Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH2-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R.
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Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V. V., Furth, M. E., Squinto, S. P. and Yancopoulos, G. D., 1991. The receptor for ciliary neurotrophic factor.Science,253(5015): 59–63.
Davis, S., Aldrich, T. H., Ip, N. Y., Stahl, N., Scherer, S., Farruggella, T., DiStefan, P. S., Curtis, R., Panayotatos, N., Gascan, H., Chevalier, S. and Yancopoulos, G. D., 1993a. Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses.Science,259 (5102): 1736–1739.
Davis, S., Aldrich, T. H., Stahl, N., Pan, L., Taga, T., Kishimoto, T., Ip, N. Y. and Yancopoulos, G. D., 1993b. LIFR beta and gp130 as heterodimerizing signal transducers of the tripartile CNTF receptor.Science,260(5115): 1805–1808.
Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J. P., Kallen, K. J., Wollmer, A., Grötzinger, J. and Rose-John, S., 1997. A bioactive designer cytokine for human hematopoietic progenitor cell expansion.Nature Biotech.,15(2): 142–145.
Hirano, T., 1999. Molecular basis underlying functional pleiotropy of cytokines and growth factors.Biochem. Biophys. Res. Commun.,260(2): 303–308.
Ip, N. Y., Li, Y. P., van de Stadt, I., Alderson, R. F. and Lindsay, R. M., 1991. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures.J. Neurosci.,11 (10): 3124–3134.
Larkfors, L., Lindsay, R. M., Alderson, R. F., 1994. Ciliary neurotrophic factor enhances the survival of Purkinje cells in vitro.Eur. J. Neurosci.,6 (6): 1015–1025.
Kallen, K. J., Grötzinger, J., Lelièvre, E., Vollmer, P., Aasland, D., Renne, C., Müllberg, J., Myer zum Buschenfelde, K. H., Gascan, H. and Rose-John, S., 1999. Receptor recognition sites of cytokines are organized as exchangeable modules: transfer of the LI-FR binding site from CNTF to IL-6.J. Biol. Chem.,274 (17): 11859–11867.
Kruttgen, A., Grötzinger, J., Kurapkat, G., Weis, J., Simon, R., Thier, M., Schröder, M., Heinrich, P., Wollmer, A., Comeau, M., Müllberg, J. and Rose-John, S., 1995. Human ciliary neurotrophic factor: a structure-function analysis.Biochem. J.,309(Pt 1): 215–220.
McDonald, N. Q., Panayotatos, N., and Hendrickson, W. A., 1995. Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing.EMBO J.,14(12): 2689–2699.
März, P., Otten, U. and Rose-John, S., 1999. Neuronal activities of IL-6 type cytokines often depend on soluble cytokine receptors.Eur. J. Neurosci.,11(9): 2995–3004.
Pflanz, S., Tacken, I., Grötzinger, J., Jacques, Y., Dahmen, H., Heinrich, P. C. and Müller-Newen, G., 1999. A fusion protein of interleukin-11 and soluble interleukin-11 receptor acts as a superagonist on cells expressing gp130.FEBS Lett.,450(1–2): 117–122.
Sambrook, J., Fritsch, E. F. and Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Senaldi, G., Varnum, B. C., Sarmiento, U., Starnes, C. and Lile, J., 1999. Novel neurotrophin-1/B cell-stimulating factor-3: a cytokine of the IL-6 family.Proc. Natl. Acad. Sci.,96(20): 11458–11463.
Sendtner, M., Schmalbruch, H., Stockli, K. A., Kreutzberg, G. W. and Thoenen, H., 1992. Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy.Nature,358(6386): 502–504.
Sleeman, M. W., Anderson, K. D., Lambert, P. D., Yancopoulos, G. D. and Wiegand, S. J., 2000. The ciliary neurotrophic factor and its receptor, CNTFR alpha.Pharm. Acta Helv.,74(2–3): 265–272.
Taga, T. and Kishimoto, T., 1997. Gp130 and the interleukin-6 family of cytokines.Annu. Rev. Immunol.,15: 797–819.
Vollmer, P., Oppmann, B., Voltz, N., Fischer, M. and Rose-John, S., 1999. A role for the immunoglobulin-like domain of the human IL-6 receptor: intracellular protein transport and shedding.Eur. J. Biochem.,263(2): 438–46.
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Project supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholar, Zhejiang Province, China and SFB415, TP B5, Germany.
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Sun, Y., Pia, M., Uwe, O. et al. Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity. J. Zhejiang Univ. Sci. A 4, 340–345 (2003). https://doi.org/10.1631/jzus.2003.0340
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DOI: https://doi.org/10.1631/jzus.2003.0340