Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cyclosporin A Binding Protein Type-19 kDa Peptidyl-Prolyl Cis/Trans Isomerase from Euglena gracilis

  • SONG HYUK-HWAN (Department of Food Science and Technology, BET Research Institute, Chung-Ang University) ;
  • PARK SUNG-YONG (Department of Food Science and Technology, BET Research Institute, Chung-Ang University) ;
  • LEE CHAN (Department of Food Science and Technology, BET Research Institute, Chung-Ang University)
  • Published : 2005.10.01
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Abstract

Cyclosporin A binding protein type-19 kDa peptidyl-prolyl cis/trans isomerase (PPIases, EC 5.2.1.8) of Euglena gracilis was purified and some of its biochemical characters were elucidated. Purification of the PPIase was achieved by employing a series of steps involving ammonium sulfate precipitation, Superdex G-75 gel filtration chromatography, Mono­Q anion and Mono-S cation exchange chromatographies, and Superdex S-200 gel filtration chromatography on FPLC. Purified PPIase had a specific activity of 8,250 units/mg, showing a 27-fold increase compared with that of cell-free extract of Euglena gracilis. The enzyme consisted of a single polypeptide chain with a molecular mass of 19 kDa. It showed high substrate specificity to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and $k_{car}/K_{m}$, for this substrate was found to be $61.19{\times}10^5/sec$. The isomer distributions were investigated at an equilibrium of seven different peptide substrates, varying Xaa in Suc-Ala-Xaa-Pro-Phe-p-nitroanilide in dimethylsulfoxide. The cis/trans equilibrium constants were estimated to be from 0.14 (Ile) to 0.63 (Gly), which correspond to $12.00\%\;to\;38.52\%$ of the cis population, respectively, under experimental condition. The enzyme was highly sensitive to the immunosuppressive ligand cyclosporin A, but not to other immunosuppressants such as FK506 and rapamycin. Thus, it appears to belong to the class of cyclophilin.

Keywords

References

  1. Benneta, P. C., L. G. Singaretnamb, W.-Q. Zhao, A. Lawenb, and K. T. Ng. 1998. Peptidyl-prolyl-cisltrans-isomerase activity may be necessary for memory formation. FEBS Lett. 431: 386-390 https://doi.org/10.1016/S0014-5793(98)00795-9
  2. Colly, N. J., E. K. Baker, M. A. Stamnes, and C. S. Zuker. 1991. The cyclophilin homolog nina A is required in the secretory pathway. Cell 67: 255-263 https://doi.org/10.1016/0092-8674(91)90177-Z
  3. Dennis, E. B. 1968. The Biology of Euglena. Academic Press. Burlington. U.S.A
  4. Fisher, G. 1994. Peptidyl-prolyl cisltrans isomerases and their effectors. Angew. Chem. Int. Ed. Engl. 33: 1415-1436 https://doi.org/10.1002/anie.199414151
  5. Fischer, G., B. Wittmann-Lieborld, K. Lang, T. Kiefgaber, and F. K. Schmid. 1989. Cyclophilins and peptidyl-prolyl cisltrans isomerase are probably identical proteins. Nature 34: 953-955
  6. Fischer, G., H. Bang, E. Berger, and A. Schelenberger. 1984. Conformational specificity of chymotrypsin toward proline-containing substrates. Biochim. Biophys. Acta 791: 87-97 https://doi.org/10.1016/0167-4838(84)90285-1
  7. Fischer, G. and F. X. Schmid. 1990. The mechanism of protein folding: Implications of in vitro refolding models for de novo protein folding and translocation in the cell. Biochemistry 29: 2205-2216 https://doi.org/10.1021/bi00461a001
  8. Fischer, G., T. Tradler, and T. Zarnt. 1998. The mode of action of peptidyl proly cis/trans isomerases in vivo: Binding vs. catalysis. FEEs Lett. 429: 17-20 https://doi.org/10.1016/S0014-5793(98)00505-5
  9. Franziska, P. 2000. Three-step purification of a fragment of the large immunophilin. J. Chromatogr. 737: 71- 76 https://doi.org/10.1016/S0378-4347(99)00413-2
  10. Friedman, J. and I. Weissman. 1991. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin; One in the presence and one the absence of CsA. Cell 66: 799-806 https://doi.org/10.1016/0092-8674(91)90123-G
  11. Furutani, M., T. Iida, S. Yamano, K. Kamino, and T. Maruyama. 1998. Biochemical and genetic characterization of an FK506-sensitive peptydyl prolyl cis-trans isomerase from a Thermophilic archaeom, Methanococcus thermolithotrophicus. J. Bacteriol. 80: 388-394
  12. Galat, A. and S. M. Metcalfe. 1995. Peptidyl proline cis/trans isomerase. Prog. Biophys. Molec. Biol. 63: 67 -118 https://doi.org/10.1016/0079-6107(94)00009-X
  13. Harrison, R. K. and R. L. Stein. 1990. Substrate specificites of the peptidyl-prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: Evidence for the existence of a family of distinct enzymes. Biochemistry 29: 3813-3816 https://doi.org/10.1021/bi00468a001
  14. Iida, T., T. Iwabuchi, A. Ideno, S. Suzuki, and T. Maruyama. 2000. FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum k. Gene 256: 319-326 https://doi.org/10.1016/S0378-1119(00)00378-4
  15. Jorg, H. and G. Fischer. 1993. Immunophi\ins: Structure-function relationship and possible role in microbial pathogencity. Molec. Microbiol. 10: 445-456 https://doi.org/10.1111/j.1365-2958.1993.tb00917.x
  16. Kim, S. J. and C. Lee. 1996. Kinetic studies of peptidyl-prolyl cis-trans isomerase from Porcine spleen. J. Biochem. Mol. 29: 519-524
  17. Kim, W. S., S. Davis, G. Wong, and A. L. Demain. 2003. Nutritional studies on the growth ofthe rapamycin-producing Streptomyces hygroscopicus. J. Microbiol. Biotechnol. 13: 560-563
  18. Kofron, J. L., P. Kuzmic, V. Kischore, B. E. Colon, and D. H. Rich. 1991. Determination of kinetic constants for peptidyl-prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry 30: 6127-6134 https://doi.org/10.1021/bi00239a007
  19. Lang, K., F. X. Schmid, and G. Fischer. 1987. Catalysis of protein folding by prolyl isomerase. Nature 329: 268-270 https://doi.org/10.1038/329268a0
  20. Lodish, H. F. and N. Kong. 1991. Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum. J. Biol. Chem. 266: 14835-14838
  21. Masse, K., S. Bhamra, C. E. Haldin, and E. A. Jones. 2004. Cloning and characterisation of the immunophilin X-CypA. Gene Express. Patterns 5: 51-60 https://doi.org/10.1016/j.modgep.2004.06.007
  22. Moss, M. L., R. E. Palmer, B. E. Dunlap, W. Henzel, J. I. Kofron, W. S. Mellon, C. A. Rowyer, and D. H. Rich. 1992. Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays. J. Biol. Chem 267: 22054-22059
  23. Peterson, M. R., D. H. Hall, M. Berriman, J. A. Nunes, G. A. Leonard, A. H. Fairlamb, and W. N. Hunter. 2000. The three-dimensional structure of a Plasmodium falciparum cyclophilin in complex with the potent anti-malarial cyclosporin A. J. Mol. Biol. 298: 123-133 https://doi.org/10.1006/jmbi.2000.3633
  24. Philip, S. S. and A. V. Michael. 1996. Purification and characterization of cytosolic and microsomal cyclophilins from maize. Biochem. J. 315: 965-969 https://doi.org/10.1042/bj3150965
  25. Price, E. R., L. D. Zydowsky, M. Jin, C. M. Boker, F. D. Mckeon, and C. T. Walsh, 1991. Human cyclophilin B; A second cyclophilin gene encodes a peptidyl-prolyl isomerase with signal sequence. Proc. Natl. Acad. Sci. 88: 1903-1907
  26. Rahfeld, J. U., K. P. Rucknagel, G. Schelbert, B. Ludwig, J. Hacker, K. Mann, and G. Fisher. 1994. Confirmation of the existence of a third family among peptidyl-prolyl cis/trans isomerases: Amino acid sequence and recombinant production of parvulin. FEBS Lett. 352: 180-184 https://doi.org/10.1016/0014-5793(94)00932-5
  27. Rainer, Z., U. Keller, C. Lee, and K. Hoffman. 1992. A seventeen kilodaltons peptidyl-prolyl cis/trans isomerase of the cyclosporin-producer Tolpocladium inflatum is sensitive to cycloporin A. J. Antibiot. 45: 265-268 https://doi.org/10.7164/antibiotics.45.265
  28. Saul, F. A., J.-P. Arie, B. Vulliezle-Ie Normand, R. Kahn, J.-M. Betton, and G. A. Bentley. 2004. Structural and functional studies of Fkbp A from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity. J. Mol. Biol. 335: 595-608 https://doi.org/10.1016/j.jmb.2003.10.056
  29. Schreiber, S. L. 1991. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251: 283-287 https://doi.org/10.1126/science.1702904
  30. Schreiber, S. L. and G. R. Crabtree. 1992. The machanism of action of cyclosporin A and FK506. lmmunol. Today 13: 136-142 https://doi.org/10.1016/0167-5699(92)90111-J
  31. Steinmann, B., P. Burckner, and A. Superti-Furga. 1991. Cyclosporin A slows collagen triple-helix formation in vivo: Indirect evidence for a physiologic role of peptidyl-prolyl cis-trans isomerase. J. Biol. Chem. 266: 1299-1303
  32. Such ira, B., M. Mattias, and B. F. Robert. 1994. The charactrization of a cyclophilin-type peptidyl-prolyl cis-trans isomerase from the endoplasmic-reticulum lumen. Biochem. J. 300: 871-875 https://doi.org/10.1042/bj3000871
  33. Tropschug, M., I. B. Barthelmess, and W. Neupert. 1989. Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae. Nature 342: 953-955 https://doi.org/10.1038/342953a0
  34. Walsh, C. T, L. D. Zydowsky, and F. D. Mckeon. 1992. Cyclosporin A, the cyclophilin class of peptidyl-prolyl isomerases and blockade of T cell signal transduction. J. Biol. Chem. 267: 13115-13118