A Cyclophilin from Griffithsia japonica Has Thermoprotective Activity and Is Affected by CsA

  • Cho, Eun Kyung (Institute of Molecular Biology and Genetics, Seoul National University) ;
  • Lee, Yoo Kyung (Polar BioCenter, Korea Polar Research Institute, Korea Ocean Research and Development Institute) ;
  • Hong, Choo Bong (Institute of Molecular Biology and Genetics, Seoul National University)
  • Received : 2005.04.13
  • Accepted : 2005.05.15
  • Published : 2005.08.31


Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, $H_6GjCyp-1$ maintained the stability of E. coli proteins up to $50^{\circ}C$. For a functional bioassay for recombinant $H_6GjCyp-1$, the viability of E. coli cells overexpressing $H_6GjCyp-1$ was compared with that of cells not expressing $H_6GjCyp-1$ at $50^{\circ}C$. After high temperature treatment for 1 h, E. coli overexpressing $H_6GjCyp-1$ survived about three times longer than E. coli lacking $H_6GjCyp-1$. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.


Supported by : Crop Functional Genomics Center


  1. Dolinski, K. J., Cardenas, M. E., and Heitman, J. (1998) CNS1 encodes an essential p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations and interacts with Hsp90. Mol. Cell. Biol. 18, 7344-7352
  2. Freskgard, P. O., Bergenhem, N., Jonsson, B. H., Svensson, M., and Carlson, U. (1992) Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase. Science 258, 466-468 https://doi.org/10.1126/science.1357751
  3. Kim, K. P., Joe, M. K., and Hong, C. B. (2004) Tobacco small heat-shock protein, NtHSP18.2, has broad substrate range as a molecular chaperone. Plant Sci. 167, 1017-1025 https://doi.org/10.1016/j.plantsci.2004.05.043
  4. Luan, S., Lane, W. S., and Schreiber, S. L. (1994) pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean. Plant Cell 6, 885-892 https://doi.org/10.1105/tpc.6.6.885
  5. Matheos, D. P., Kingsbury, T. J., Ahsan, U. S., and Cunningham, K. W. (1997) Tcn1p/Crzlp, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisiae. Genes Dev. 11, 3445-3458 https://doi.org/10.1101/gad.11.24.3445
  6. Ratajczak, T. and Carrello, A. (1996) Cyclophilin 40 (CyP-40), mapping of its hsp90 binding domain and evidence that FKBP52 competes with CyP-40 for hsp90 binding. J. Biol. Chem. 271, 2961-2965 https://doi.org/10.1074/jbc.271.6.2961
  7. Rutherford, S. L. and Zuker, C. S. (1994) Protein folding and the regulation of signaling pathways. Cell 79, 1129-1132 https://doi.org/10.1016/0092-8674(94)90003-5
  8. Stamnes, M. A., Shieh, B. H., Chuman, L., Harris, G. L., and Zuker, C. S. (1991) The cyclophilin homolog ninaA is a tissue- specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins. Cell 65, 219-227 https://doi.org/10.1016/0092-8674(91)90156-S
  9. Weisman, R., Creanor, J., and Fantes, P. (1996) A multicopy suppressor of a cell cycle defect in S. pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein. EMBO J. 15, 447-456
  10. Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., et al. (1991) Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes. Cell 66, 807- 815 https://doi.org/10.1016/0092-8674(91)90124-H
  11. Lee, Y. K., Hong, C. B., Suh, Y. B., and Lee, I. K. (2002) A cDNA clone for cyclophilin from Griffithsia japonica and phylogenetic analysis of cyclophilins. Mol. Cells 13, 12-20
  12. Fischer, G. and Schmid, F. X. (1990) The mechanism of protein folding. Implications of in vitro refolding models for de novo protein folding and translocation in the cell. Biochemistry 29, 2205-2212 https://doi.org/10.1021/bi00461a001
  13. Fejzo, J., Etzkorn, F. A., Clubb, R. T., Shi, Y., Walsh, C. T., et al. (1994) The mutant Escherichia coli F112W cyclophilin binds cyclosporin A in nearly identical conformation as human cyclophilin. Biochemistry 33, 5711-5720 https://doi.org/10.1021/bi00185a007
  14. Liu, X. D., Morano, K. A., and Thiele, D. J. (1999) The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone. J. Biol. Chem. 274, 26654-26660 https://doi.org/10.1074/jbc.274.38.26654
  15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  16. Sykes, K., Gething, M., and Sambrook, J. (1993) Proline isomerases function during heat shock. Proc. Natl. Acad. Sci. USA 90, 5853-5857
  17. Dolinski, K., Muir, R. S., Cardenas, M. E., and Heitman, J. (1997) All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94, 13093-13098
  18. Schneider, H., Charara, N., and Schmitz, R. (1994) Human cyclophilin C: primary structure, tissue distribution, and determination of binding specificity for cyclosporins. Biochemistry 33, 8218-8224 https://doi.org/10.1021/bi00193a007
  19. Duina, A. A., Marsh, J. A., Kurtz, R. B., Chang, H. J., Lindquist, S., et al. (1998) The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilin homolog Cpr7 is not required to support growth or glucocorticoid receptor activity in Saccharomyces cerevisiae. J. Biol. Chem. 273, 10819-10822 https://doi.org/10.1074/jbc.273.18.10819
  20. Lee, G. J. and Vierling, E. (1998) Expression, purification and molecular chaperone activity of plant recombinant small heat shock proteins: Protein Folding: catalysts, accessory proteins, and chaperones; in Methods in Enzymology, Lorimer, G. and Baldwin, T. O. (eds.), Vol. 29, pp. 350-365
  21. Wang, P., Cardenas, M. E., Cox, G. M., Perfect, J. R., and Heitman, J. (2001) Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans. EMBO Rep. 2, 511-518 https://doi.org/10.1093/embo-reports/kve017
  22. Breuder, T., Hemenway, C. S., Movva, N. R., Cardenas, M. E., and Heitman, J. (1994) Calcineurin is essential in cyclosporin A- and FK506-sensitive yeast strains. Proc. Natl. Acad. Sci. USA 91, 5372-5376
  23. Stathopoulos, A. M. and Cyert, M. S. (1997) Calcineurin acts through the CRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast. Genes Dev. 11, 3432-3444 https://doi.org/10.1101/gad.11.24.3432
  24. Hartl, F. U. and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-1858 https://doi.org/10.1126/science.1068408
  25. Stathopoulos-Gerontides, A., Guo, J. J., and Cyert, M. S. (1999) Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes Dev. 13, 798-803 https://doi.org/10.1101/gad.13.7.798
  26. Ondek, B., Hardy, R. W., Baker, E. K., Stamnes, M. A., Shieh, B. H., et al. (1992) Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA. J. Biol. Chem. 267, 16460-16466
  27. Stamnes, M. A., Rutherford, S. L., and Zuker, C. S. (1992) Cyclophilins: a new family of proteins involved in intracellular folding. Trends Cell Biol. 2, 272-276 https://doi.org/10.1016/0962-8924(92)90200-7
  28. Clubb, R. T., Ferguson, S. B., Walsh, C. T., and Wagner, G. (1994) Three-dimensional solution structure of Escherichia coli periplasmic cyclophilin. Biochem. 33, 2761-2772 https://doi.org/10.1021/bi00176a004
  29. Foor, F., Parent, S. A., Morin, N., Dahl, A. M., Ramadan, N., et al. (1992) Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from $\alpha$-factor arrest in yeast. Nature 360, 682-684 https://doi.org/10.1038/360682a0
  30. Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E., and Schreiber, S. L. (1992) Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex. Science 256, 1315-1318 https://doi.org/10.1126/science.1376003
  31. Handschumacher, R. E., Harding, M. W., Rice, J., Drugge, R. J., and Speicher, D. W. (1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226, 544-547 https://doi.org/10.1126/science.6238408
  32. Crabtree, G. R. (2001) Calcium, calcineurin and the control of transcription. J. Biol. Chem. 276, 2313-2316 https://doi.org/10.1074/jbc.R000024200
  33. Galat, A. (1993) Peptidylproline cis-trans-isomerases: immunophilins. Eur. J. Biochem. 216, 689-707 https://doi.org/10.1111/j.1432-1033.1993.tb18189.x
  34. Chang, H. C. and Lindquist, S. (1994) Conservation of Hsp90 macromolecular complexes in Saccharomyces cerevisiae. J. Biol. Chem. 269, 24983-24988
  35. Edvardsson, A., Eshaghi, S., Vener, A. V., and Andersson, B. (2003) The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20. FEBS Lett. 542, 137-141 https://doi.org/10.1016/S0014-5793(03)00366-1
  36. Mayer, M. P., Brehmer, D., Gassler, C. S., and Bukau, B. (2001) Protein folding in the cell; in Advances in Protein Chemistry, Horwich, A. (ed.), pp. 1-44, Academic Press, San Diego
  37. Groenendyk, J., Lynch, J., and Michalak, M. (2004) Calreticulin, $Ca^{2+}$, and calcineurin-signaling from the endoplasmic reticulum. Mol. Cells 17, 383-389
  38. Kimura, Y., Yahara, I., and Lindquist, S. (1995) Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways. Science 268, 1362-1365 https://doi.org/10.1126/science.7761857
  39. Tesic, M., Marsh, J. A., Cullinan, S. B., and Gaber, R. F. (2003) Functional interactions between Hsp90 and co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae. J. Biol. Chem. 278, 32692-32701 https://doi.org/10.1074/jbc.M304315200
  40. Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., et al. (2003) Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J. Biol. Chem. 278, 43202-43213 https://doi.org/10.1074/jbc.M305414200
  41. Kern, G., Kern, D., Schimid, F. X., and Fischer, G. (1994) Reassessment of the putative chaperone function of prolylcis/ trans-isomerase. FEBS Lett. 348, 145-148 https://doi.org/10.1016/0014-5793(94)00591-5
  42. Steinmann, B., Bruckner, P., and Superti-Furga, A. (1991) Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cistrans isomerase. J. Biol. Chem. 266, 1299-1303
  43. Yu, H. S., Kong, H. H., and Chung, D. I. (2002) Cloning and characterization of Giardia intestinalis cyclophilin. Korean J. Parasit. 40, 131-138 https://doi.org/10.3347/kjp.2002.40.3.131