| Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann-Stra'ussler-Scheinker disease (GSS), and fatal familial insomnia (FFI) in humans.and scrapie, bovine spongiform encephalopathy (BSE, mad cow disease)), transmissible mink encephalopathy(TME), and chronic wasting disease (CWD) in animals. In 1982, according to abundant experimental evidence, Prusiner raised a new hypothesis on TSE, Prion Hypothesis, which presumed that Prion Protein witin humans and animals suffering conformational change caused prion disease. The infectious agent (prion) that causes theses diseases is composed primarily of the abnormal prion protein-PrPSc. PrPSc is a conformationally altered isoform of a normal, cell-surface glycoprotein called prion protein (PrPC). A wealth of evidence indicates that PrPC is essential for the development of prion disease, since it serves as substrate for conversion to PrPSc. Thus, mice lacking the endogenous gene (prn-p) encodimg PrP are reisitant to experimental scrapie inoculation. In addition, all known familial prion diseases are due to mutations in the Prn-p gene that are thought to favor spontaneous conversion of PrPC to PrPSc.Although a great deal of information now available about the role PrPSc in the disease process, relatively little known about the normal, physiological function of PrPC and about how the prion agent enter into the cells and propagate in vitro and in vivo. Several papers suggested that the endocytosis of prion protein is crucial step in both infectious cell lines and normal function. Blocking endocytosis of the infectious cells will reduce prion titer. The previous report showed that the N-terminal domain of prion protein was an essential for its endocytosis via clathrin-coated pits. Deletion 23-30 residuals of prion protein dramatically reduce the activity of prion protein for rescuing yeast from Bax mediated cell death. Deletion 23-28 of prion protein nullified the neuroprotective activity of PrPC. Thus, N-terminal charged 23-30 residuals may be an essential domain for the endocytosis of prion protein.The aim of this study is to discuss the role of 23-30 residuals of PrP on endocytosis of prion protein, anti-oxidant and prion propagation.PCR primers were designed according to the published sequence of mouse PrP gene. Four gene fragments were amplified via PCR and SOE-PCR including PrP gene(PrP), deletion of 23-30 residuals in N-terminus PrP gene (PrP△23-30), mutation on 108 and 111 residuals PrP gene (PrP3F4) and the PrP gene which was deleted of 23-30 residuals and mutated on 108 and 111 residuals(PrP△23-30-3F4). Green fluorescence protein(GFP) gene was also amplified via PCR according to the published sequence.The amplified DNA fragments were inserted into the PMD18-T vector separately to generate 5 recombinant plasmids, named PMD18-T-wtPrP,PMD18-T-PrP△23-30,PMD18-T-PrP3F4,PMD18-T-PrP△23-30-3F4和PMD18-T-GFP. After sequence analysis, the eukaryotic expression plasmid PCDNA3.1(-)and 5 plasmids and were digested with corresponding restriction enzyme. The recovered bands were ligated with T4 ligase to produce a fusion expression plasmid, named PCDNA3.1(-)-wtPrP PrP-GFP,PCDNA3.1(-)-PrP△23-30-GFP,PCDNA3.1(-)-PrP3F4和PCDNA3.1(-)-PrP△23-30-3F4.PCDNA3.1(-)-wtPrP-GFP and PCDNA3.1(-)-PrP△23-30-GFP were transfected into N2a cells with liposome under the selection pressure of G418 and limiting dilution.N2a cells expressing PrP-GFP and PrP△23-30-GFP were identified by laser confocal microscopy and western-blotting. The results of laser confocal microscopy analysis demonstrate wtPrP-GFP and PrP△23-30-GFP were both cell membrance protein, and distribute in cell membrance and perinuclear compartment. wt PrP-GFP fluorescence in the plasmamembrane decreases by 71.8% during 30 min perfusion of 500 500μM Cu2-.but PrP△23-30-GFP fluorescence in the plasmamembrane decreases by 41.3%, which suggest deletion of 23-30 residuals disturb the encytosis of prion protein not deprive of all. Colocalization of this two protein with ER, golgi body and lysosome using cell tissue staining suggest wtPrP-GFP and PrP△23-30-GFP share the same secretory pathway demonstrating the two protein were modified similarly.PCDNA3.1(-)-PrP3F4 and PCDNA3.1(-)-PrP△23-30-3F4 were transfected into CHO cells with liposome under the selection pressure of G418 and limiting dilution. CHO cells steadyly expressing PrP3F4 and PrP△23-30-3F4 were identified by western-blotting. inhibition ratio of H2O2 to untransfected CHO cells, CHO cells expressing PrP3F4 and CHO cells expressing PrP△23-30-3F4 which was detected with MTT, were different, suppression to untransfected CHO cells is the most obvious, and the suppression to CHO cells expressing PrP3F4 is the least.ROS of the three kinds of cell was detected with DCFH-DA kit, under the 100μM H2O2, ROS of CHO cells expressing PrP3F4 does not increase (P(?)0.05), ROS of CHO cells and CHO cells expressing PrP△23-30-3F4 increase(P<0.05). The SOD activity of untransfected CHO cells. CHO cells expressing PrP3F4 and CHO cells expressing PrP△23-30-3F4 is respectively 42.3±1.9U/mg, 68.5±2.8 U/mg和55.75±4.4U/mg, detected with SOD kit, and the GSH-Px activity of untransfected CHO cells, CHO cells expressing PrP3F4 and CHO cells expressing PrP△23-30-3F4 is respectively 222.5±12.4 U/mg,354.7±22.3 U/mg和278.9±15.9 U/mg, detected with GSH-Px kitPCDNA3.1(-)-PrP3F4 and PCDNA3.1(-)-PrP△23-30-3F4 were transfected into N2a cells with liposome under the selection pressure of G418 and limiting dilution. N2a cells steadyly expressing PrP3F4 and PrPA23-30-3F4 were identified by western-blotting. N2a cells steadyly expressing PrP3F4 and PrP△23-30-3F4 was infected with RML PrPSc, which is adapt to mouse, after 15 passages, cells was identified by western-blotting taking 3F4 as the first antibody. Prion protein which is resistant to PK could be detected in both two kinds of cell, suggesting PrP without 23-30 residuals could be converted to PrPSc.With the method described above, we found infected N2a cells expressing PrP3F4 and PrPA23-30-3F4 exhibit following change, the viability of cells decrease, ROS of cells increase, activity od SOD and GSH-Px decrease.We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac2O), which specifically target accessible tyrosine and lysine residues, respectively, to modify Syrian hamster recombinant PrP (90-231) (rPrP) and PrP 27-30, aiming at finding locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin, and subsequent analysis by mass spectrometry (MALDI-TOF). These results suggest that:1) PrPSc exhibits important conformational differences in the C-terminal region with respect to rPrP, resulting in loss of solvent accessibility of Y225 and Y226, very solvent-exposed in the latter conformation; since other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in PrPSc; 2) contrarily, tyrosines contained in the stretch spanning approximately from Y149 to R164 are more accessible in PrPSc, suggesting rearrangements inα-helix HI and the shortβ-sheet of PrP; 3) the amino terminal region of PrPSc is very accessible. These data should help validate and construct structural models of PrPSc. |
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