Human Prion Protein N-glycosylation On In Vitro Induced Apoptosis Of A Preliminary Study

Prion diseases are a group of transmissible neurodegenerative encephalopathies involved in human and animals. The pathogen of this kind of diseases is believed to be a unique infectious protein particle devoid of nucleic acid.Our previous studies show the correlation between the ratio of the prion protein and its biological functions. The present study investigates the effects of the glycosylation of PrP on biological functions in cells. Firstly, human PRNP gene was amplified from peripheral white blood cells. Two mutants PRNP N181Q, PRNP N197Q in which only one glycosylation site (the first or the second glycosylation site) within the PRNP gene was mutated and a PRNP N181Q/N197Q mutant in which both N-linked glycosylation sites were removed (two glycosylation sites were mutated) were obtained by site-directed mutagenesis. The plasmids were introduced into the mammalian cells expression systems, such as SF126 and HeLa cells. By Western blot assay, it was found that the expressed wild-type PrP proteins exhibit a variety of glycosylation forms with a molecular weights between 30 and 35 kDa. After treated with PNGase F, the molecular weights of the proteins were reduced. Two mutants with only one N-glycosylation site mutated were expressed to obtain the forms which are not fully glycosylated with a molecular weights between 29 and 30 kDa, while the mutant with N-linked glycosylation sites removed was expressed to generate a protein with a molecular weight of 27 kDa without any glycosylation. The glycosylated PrPs could be digested by glycosidase and their molecular weights would be reduced, which indicated the successful expression of wild-type PrP and the constructed mutants with one glycosylation site mutated or N-linked glycosylation sites removed.The conventional methods for detecting apoptosis, such as Hoechst 33342/PI staining method, MTT method, Annexin V/PI and PI assay were applied to demonstrate in vitro in the SF126 and HeLa cells, the expression of PrP with N-linked glycosylation sites removed was much easier to induce apoptosis than the wild-type PrP, while the expressions of PrPs with only one N-glycosylation site mutated have a apoptosis-inducing effect similar to that of the wild-type. The further study was carried out to analyze Reactive Oxygen Species(ROS) in cells in order to investigate the mechanism of the apoptosis. It was found that the PrP with N-linked glycosylation sites removed was easier...