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

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.

[1]  Hongxiang Liu,et al.  QUANTIFICATION OF PRION GENE EXPRESSION IN BRAIN AND PERIPHERAL ORGANS OF GOLDEN HAMSTER BY REAL-TIME RT-PCR , 2005, Animal biotechnology.

[2]  S. Prusiner.  Novel proteinaceous infectious particles cause scrapie. , 1982, Science.

[3]  V. Lewis,et al.  Analysis of endogenous PrPC processing in neuronal and non-neuronal cell lines. , 2008, Methods in molecular biology.

[4]  Xiao-bo Zhou,et al.  Dynamic analyses of PrP and PrP(Sc) in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms. , 2004, Biomedical and environmental sciences : BES.

[5]  M. Bianchin,et al.  Normal brain mitochondrial respiration in adult mice lacking cellular prion protein , 2005, Neuroscience Letters.

[6]  K. Kaneko,et al.  Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC , 2005, Neuroscience Letters.

[7]  A. Aguzzi,et al.  Mice devoid of PrP are resistant to scrapie , 1993, Cell.

[8]  Stanley B. Prusiner,et al.  Nobel Lecture: Prions , 1998 .

[9]  Yang Yang,et al.  P53 transcription-independent activity mediates selenite-induced acute promyelocytic leukemia NB4 cell apoptosis. , 2008, BMB reports.

[10]  S. Prusiner,et al.  Prion biology and diseases. , 1999, Harvey lectures.

[11]  H. Laude,et al.  Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Hauw,et al.  V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation , 2006, Neuroscience Letters.

[13]  Hyoung-Gon Lee,et al.  The cellular prion protein (PrPC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation. , 2004, Brain research. Molecular brain research.

[14]  Xiao-Fan Wang,et al.  Removal of the glycosylation of prion protein provokes apoptosis in SF126. , 2007, Journal of biochemistry and molecular biology.

[15]  O. Larsson,et al.  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. , 1997, Cancer research.

[16]  S. Lindquist,et al.  Neurotoxicity and Neurodegeneration When PrP Accumulates in the Cytosol , 2002, Science.

[17]  A. Aguzzi,et al.  Mammalian Prion Biology One Century of Evolving Concepts , 2004, Cell.

[18]  C. Kurschner,et al.  Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein. , 1996, Brain research. Molecular brain research.

[19]  I. Moarefi,et al.  Determinants of the in Vivo Folding of the Prion Protein , 2003, The Journal of Biological Chemistry.

[20]  A. Aguzzi,et al.  No propagation of prions in mice devoid of PrP , 1994, Cell.

[21]  Jung-Hoon Kang,et al.  Acrolein, the toxic endogenous aldehyde, induces neurofilament-L aggregation. , 2008, BMB reports.

[22]  N. Hooper,et al.  Reactive oxygen species (ROS)-mediated β-cleavage of the prion protein in the mechanism of the cellular response to oxidative stress , 2005 .

[23]  A. Mangé,et al.  PrP‐dependent cell adhesion in N2a neuroblastoma cells , 2002, FEBS letters.

[24]  R. Carp,et al.  Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent. , 2000 .

[25]  P. Monaghan,et al.  Altered Glycosylated PrP Proteins Can Have Different Neuronal Trafficking in Brain but Do Not Acquire Scrapie-like Properties* , 2005, Journal of Biological Chemistry.

[26]  D. Harris.  Cellular Biology of Prion Diseases , 1999, Clinical Microbiology Reviews.

[27]  F. Checler,et al.  Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti‐PrP antibodies , 2002, Journal of neurochemistry.

[28]  S. Krasemann,et al.  Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance , 2002, Journal of Cell Science.

[29]  D. Harris,et al.  Blockade of Glycosylation Promotes Acquistion of Scrapie-like Properties by the Prion Protein in Cultured Cells* , 1997, The Journal of Biological Chemistry.

[30]  K. Winklhofer,et al.  Inhibition of Complex Glycosylation Increases the Formation of PrPsc , 2003, Traffic.

[31]  S. Lindquist,et al.  Conversion of PrP to a Self-Perpetuating PrPSc-like Conformation in the Cytosol , 2002, Science.