[Construction and expression of various human prion proteins mutants with modified N-glycosylation sites in mammalian cells].
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 had been constructed and expressed. With site-direct mutation technique, human PRNP gene was mutated and the obtained mutants were subcloned into eukaryotic expressing plasmid pcDNA3.1 and transiently expressed in Hela cervical adenocarcinoma cell. The expression products of the mutated PrP were identified with 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 with one glycosylation site mutated and one without any mutation at glycosylation sites. The expressed products were digested with PNGase F. The wild type proteins and those with one of glycosylation sites mutated were digested, resulting in their molecular weights reduced, while the molecular weights of products with mutations at both glycosylation sites were not changed. The mutant of wild type human PRNP gene at 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 were able to be expressed transitantly in Hela cells, which could be a useful means for studying prions.
Yang
Yang
Y
National Laboratory of Medical Molecular Biology, Institute of Basic Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China.
Chen
Lan
L
Han
Bing-She
BS
Xu
Cai-Min
CM
Pan
Hua-Zhen
HZ
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000096:biosynthesis / Q000235:genetics; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics*; D011328:Prions / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection
D017127:Glycation End Products, Advanced; D050505:Mutant Proteins; D011328:Prions