Study Of Prion Protin Interacts With Calnexin By Biological Techniques

Prion diseases are fatal, neurodegenerative diseases that include scrapie in sheep; bovine spongiform encephalopathy (BSE) in cattle; Kuru, Gerstmann Straussler Scheinker disease (GSS), fatal familial insomnia (FFI), Creutzfeldt-Jakob disease (CJD), and variant Creutzfeldt-Jakob disease (vCJD)in human. Misfolding and aggregation of prion protein deposit on selected regions of the brain can result in prion diseases. A key event in all prion diseases appears to be the supreme structure changes of normal cellular form of prion protein (PrPc) into the infectious scrapie isoform (PrPSc).Calnexin(CNX) is a type I integral membrane protein in the endoplasmic reticulum (ER) that ensures the proper folding and quality control of newly synthesized N-linked glycoproteins. Previous studies showed us that the lectin chaperone CNX had two mechanisms of association with glycoproteins:lectin-binding only or lectin and polypeptide binding.In this work, we constructed prokaryotic and eukaryotic expression vectors of calnexin (CNX) and prion protein (PrP). Our findings for the first time confirmed that calnexin interacts with PrP. The immunoprecipitation and native polyacrylamide-gel electrophoresis results indicate that calnexin could bind PrP in vivo and in vitro. The E. coli expressed PrP was non-glycosylated, we guess the interaction of PrP with CNX maybe depend on polypeptide binding site.Some researchs demonstrated that CNX could suppresses thermal aggregation of non-glycosylated polypeptides in vitro. To assess whether CNX is capable of supressing aggregation of PrP in vitro, the turbidity assay was employed. Consistent with a molecular chaperone function, CNX effectively suppressed the aggregation of PrP in a concentration-dependent manner with maximal suppression occurring at a molar ratio of one CNX molecule to two PrP molecules. We can see at this molar ratio, the aggregation of PrP was almost suppressed. Then the atom force microscopy (AFM), light scattering and size exclusion chromatograph (SEC) assays proved our conclusion further.To investigate whether CNX could inhibit cytotoxicity induced by PrP, we employed MTT assay. The result showed that the viability of the cell expressed PrP dropped remarkably, while viability of the cells expressed both CNX and PrP kept unchanged compared with that blank control, when the concentration of CNX is the same as PrP, the viability of the cell did not changed significantly. In order to investigate whether CNX could inhibit the cell apoptosis induced by PrP in SK-N-SH cells, we employed flow cytometry analyses. The result showed CNX effectively suppressed the apoptosis induced by PrP in a concentration-dependent manner.Research on PrP interacts with CNX may have clinical benefits in prion-related diseases and other protein conformation diseases such as Alzheimer's, Parkinson's and Huntington's diseases, and other diseases especially resulted from protein misfolding and accumulation.