DOI QR코드

DOI QR Code

Glycosylation modification of human prion protein provokes apoptosis in HeLa cells in vitro

  • Yang, Yang (National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Chen, Lan (National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Pan, Hua-Zhen (National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Kou, Yi (Health Science Center, Peking University) ;
  • Xu, Cai-Min (National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences and Peking Union Medical College)
  • Published : 2009.06.30

Abstract

We investigate the correlation between the glycosylation modified prion proteins and apoptosis. The wild-type PRNP gene and four PRNP gene glycosylated mutants were transiently expressed in HeLa cells. The effect of apoptosis induced by PrP mutants was confirmed by MTT assay, Hochest staining, Annexin-V staining and PI staining. ROS test detected ROS generation within the cells. The mitochondrial membrane potential was analyzed by the flow cytometry. The expression levels of Bcl-xL, Bax, cleaved Caspase-9 proteins were analyzed by Western Blot. The results indicated that the expressed non-glycosylated PrP in HeLa cells obviously induced apoptosis, inhibited the growth of cells and reduced the mitochondrial membrane potential, and more ROS generation and low levels of the apoptosis-related proteins Bcl-xL, the activated the cleaved Caspase-9 proteins were found. The apoptosis induced by non-glycosylated PrP demonstrates that its underlying mechanism correlates with the mitochondria-mediated signal transduction pathway.

Keywords

References

  1. Prusiner, S. B. (2004) Prion Biology and Diseases, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA
  2. Prusiner, S. B. (1982) Novel proteinaceous infectious particles cause scrapie. Science 216, 136-144 https://doi.org/10.1126/science.6801762
  3. Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383 https://doi.org/10.1073/pnas.95.23.13363
  4. Aguzzi, A. and Polymenidou, M. (2004) Mammalian prion biology: one century of evolving concepts. Cell 116, 313-327 https://doi.org/10.1016/S0092-8674(03)01031-6
  5. Sailer, A., Bueler, H., Fischer, M., Aguzzi, A. and Weissmann, C. (1994) No propagation of prions in mice devoid of PrPs. Cell 77, 967-968 https://doi.org/10.1016/0092-8674(94)90436-7
  6. Bueler, H., Aguzzi. A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M. and Weissmann, C. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73, 1339-1347 https://doi.org/10.1016/0092-8674(93)90360-3
  7. Harris, D. A. (1999) Cellular biology of prion diseases. Clin. Microbiol. Rev. 12, 429-444
  8. Winklhofer, K. F., Heller, U., Reintjes, A. and Tatzelt, J. (2003) Inhibition of complex glycosylation increases the formation of PrPsc. Traffic 4, 313-322 https://doi.org/10.1034/j.1600-0854.2003.00088.x
  9. Gao, J. M., Gao, C., Han, J., Zhou, X. B., Xiao, X. L., Zhang, J., Chen, L., Zhang, B. Y., Hong, T. and Dong, X. P. (2004) Dynamic analyses of PrP and PrP (Sc) in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms. Biomed Environ Sci. 17, 8-20
  10. Mange, A., Milhavet, O., Umlauf, D., Harris, D. and Lehmann, S. (2002) PrPs-dependent cell adhesion in N2a neuroblastoma cells. FEBS Lett. 514, 159-162 https://doi.org/10.1016/S0014-5793(02)02338-4
  11. Paitel, E., Alves da Costa, C., Vilette, D., Grassi, J. and Checler, F. (2002) Overexpression of PrPSc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti-PrPs antibodies. J. Neurochem. 83, 1208-1214 https://doi.org/10.1046/j.1471-4159.2002.01234.x
  12. Cronier, S., Laude, H. and Peyrin, J. M. (2004) Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. Proc. Natl. Acad. Sci. U.S.A. 101, 12271-12276 https://doi.org/10.1073/pnas.0402725101
  13. Dricu, A., Carlberg, M., Wang, M. and Larsson, O. (1997) Inhibition of N-linked glycosylation using tunicamycin causes cell death in malignant cells: role of down-regulation of the insulin-like growth factor 1 receptor in induction of apoptosis. Cancer Res. 57, 543-548
  14. Ning, Z. Y., Zhao, D. M., Yang, J. M., Cui, Y. L., Meng, L. P., Wu, C. D. and Liu, H. X. (2005) Quantification of prion gene expression in brain and peripheral organs of golden hamster by real-time RT-PCR. Anim. Biotechnol. 16, 55-65 https://doi.org/10.1081/ABIO-200053404
  15. Gauczynski, S., Krasemann, S., Bodemer, W. and Weiss, S. (2002) Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP + 90R (fCJD) reveal Proteinase K resistance. J. Cell Sci. 115, 4025-4036 https://doi.org/10.1242/jcs.00086
  16. Chasseigneaux, S., Haik, S., Laffont-Proust, I., De Marco, O., Lenne, M., Brandel, J. P., Hauw, J. J., Laplanche, J. L. and Peoc'h, K. (2006) V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation. Neurosci Lett. 408, 165-169 https://doi.org/10.1016/j.neulet.2006.08.008
  17. Lewis, V. and Collins, S. J. (2008) Analysis of endogenous PrPC processing in neuronal and non-neuronal cell lines. Methods Mol. Biol. 459, 229-239 https://doi.org/10.1007/978-1-59745-234-2_15
  18. Enrico, C., Frances, W., Nadia, L. T., Herbert, B., Paul, M., Lorraine, A., Jennifer, S. and Jean, C. M. (2005) Altered glycosylated PrP proteins can have different neuronal trafficking in brain but do not acquire scrapie-like properties. J. Biol. Chem. 280, 42909-42918 https://doi.org/10.1074/jbc.M509557200
  19. Sylvain, L. and David A. H. (1997) Blockade of glycosylation promotes acquistion of scrapie-like properties by the prion protein in cultured cells J. Biol. Chem. 272, 21479-21487 https://doi.org/10.1074/jbc.272.34.21479
  20. Winklhofer, K. F., Heske, J., Heller, U., Reintjes, A., Muranyi, W., Moarefi, I. and Tatzelt, J. (2003) Determinants of the in vivo folding of the prion protein. A bipartite function of helix 1 in folding and aggregation. J. Biol. Chem. 278, 14961-14970 https://doi.org/10.1074/jbc.M209942200
  21. Ma, J., Wollmann, R. and Lindquist, S. (2002) Neurotoxicity and neurodegeneration when PrP accumulates in the cytosol. Science 29, 1781-1785
  22. Jeong, M. S. and Kang, J. H. (2008) Acrolein, the toxic endogenous aldehyde, induces neurofilament-L aggregation. J. Biochem. Mol. Biol. 41, 635-639
  23. Watt, N. T. and Hooperl, N. M. (2005) Reactive oxygen species (ROS)-mediated $\beta$-cleavage of the prion protein in the mechanism of the cellular response to oxidative stress. Biochem. Soc. Trans. 33, 1123-1125 https://doi.org/10.1042/BST20051123
  24. Ma, J. and Lindquist, S. (2002) Conversion of PrP to a self-perpetuating PrPSclike conformation in the cytosol. Science 298, 1785-1788 https://doi.org/10.1126/science.1073619
  25. Liying, G., Fang, H., Zhushi, L., Bingshe, H., Qian, J., Yun, R., Yang, Y. and Caimin, X. (2008) P53 transcription-independent activity mediates selenite-induced acute promyelocytic leukemia NB4 cell apoptosis. J. Biochem. Mol. Biol. 41, 745-750
  26. Lobao-Soares, B., Bianchin, M. M., Linhares, M. N., Carqueja, C. L., Tasca, C. I., Souza, M., Marques, W. J., Brentani, R., Martins, V. R., Sakamoto, A. C., Carlotti, C. G. J. and Walz, R. (2005) Normal brain mitochondrial respiration in adult mice lacking cellular prion protein. Neurosci. Lett. 375, 203-206 https://doi.org/10.1016/j.neulet.2004.11.012
  27. Kim, B. H., Lee, H. G., Choi, J. K., Kim, J. I., Choi, E. K., Carp, R. I. and Kim Y. S. (2004) The cellular prion protein (PrPsC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation. Brain Res. Mol. Brain Res. 124, 40-50 https://doi.org/10.1016/j.molbrainres.2004.02.005
  28. Kurschner, C. and Morgan, J. I. (1996) Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein. Brain Res. Mol. Brain Res. 37, 249-258 https://doi.org/10.1016/0169-328X(95)00323-K
  29. Park, S. K., Choi, S. I., Jin, J. K., Choi, E. K., Kim, J. I., Carp, R. I. and Kim, Y. S. (2000) Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent. Neuroreport. 11, 1677-1682 https://doi.org/10.1097/00001756-200006050-00017
  30. Hachiya, N. S., Yamada, M., Watanabe, K., Jozuka, A., Ohkubo, T., Sano, K., Takeuchi, Y., Kozuka, Y., Sakasegawa, Y. and Kaneko, K. (2005) Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC. Neurosci. Lett. 374, 98-103 https://doi.org/10.1016/j.neulet.2004.10.044
  31. Lan Chen, Y. Y., Jun, H., Bao-Yun, Z., Lin, Z., Kai, N., Xiao-Fan, W., Feng, L., Chen, G., Xiao-Ping, D. and Cai-Min, X. (2007) Removal of the Glycosylation of Prion Protein Provokes Apoptosis in SF126. J. Biochem. Mol. Biol. 40, 662-669 https://doi.org/10.5483/BMBRep.2007.40.5.662

Cited by

  1. Resistance against apoptosis by the cellular prion protein is dependent on its glycosylation status in oral HSC-2 and colon LS 174T cancer cells vol.306, pp.1, 2011, https://doi.org/10.1016/j.canlet.2011.02.040
  2. Glycosylation Significantly Inhibits the Aggregation of Human Prion Protein and Decreases Its Cytotoxicity vol.8, pp.1, 2018, https://doi.org/10.1038/s41598-018-30770-6