Construction and Expression of Various Human Prion Protein Mutants with Modified N-glycosylation Sites in Mammalian Cells

Abstract To study the biological function of the N-glycosylation modification of prion proteins (PrP), various eukaryotic expression vectors for the mutants with N-glycosylation modification of human PrP were constructed and expressed. With the site-direct mutation technique, the human PRNP gene was mutated, and the obtained mutants were subcloned into eukaryotic expressing plasmid pcDNA3.1 and transiently expressed in human carcinoma cell line HeLa cells. The expression products of the mutated PrP were identified with the Western blotting assay and the PNGase digestion assay. Several mutants with specific glycosylation modification were identified from the expressed products by Western blot, including two mutants, one with a mutation at the glycosylation site and the other without. The expressed products were digested with PNGase F. The wild-type proteins and those with one of the glycosylation sites mutated were digested, resulting in their molecular weights being reduced, while the molecular weights of products with mutations at both glycosylation sites were not changed. The mutant of the wild-type human PRNP gene at the N-glycosylation modification sites and six modified mutants with mono- or non-N-glycosylation had been obtained successfully in the study. Moreover, the modified PrP with mono- and non-N-glycosylation could be expressed transiently in HeLa cells, which could be a useful means for studying prions.