Mechanisms Of Glycosylation Modification Affected Prion Protein Misfolding

Prion diseases,also called transmissible spongiform encephalopathies(TSEs)are one of the most widespread and fatal neurodegenerative diseases,which can be found in most mammals especially in human being,including scrapie,bovine spongiform encephalopathy(BSE),the familial and the sporadic Creutzfeldt—Jakob disease(CJD),Gerstmann-Straussler-Scheinker(GSS),Fatal Insomina(FI)and Kuru of human.Now the concept of prion diseases has been extended to prion-like diseases,such as Parkinson's disease caused by a-Synuclein,Alzheimer disease caused by tau protein and A?,amyotrophic lateral sclerosis caused by TDP-43 and SOD1 and so on.The most widely accepted pathogenesis of prion diseases is that the normal cellular prion protein(PrPc)is misfolding and transformed into the pathological prion protein(PrPSc),then the monomer PrPSc aggregates and forms the dimer,the oligomer and finally the seeds which can induce the further transformation from PrPc to PrPSc.The process of seeds formation is the main rate-limiting step in vitro,but little is known about the factors that can influence the seeds formation in vivo.N-linked glycosylation modification is one of the most important modifications in prion protein,which palys a vital role in determining the prion strains,the the prion replication,the cytotoxicit and the timing of neuroinvasion.However,there is no direct evidence demonstrating that the abnormal glycosylation modification can lead to prion diseases,so is there any relationship between N-linked glycosylation and PrP location?What is the relationship between N-glycosylation and GPI-anchored modification?All these questions deserve to be investigated.First of all,we investigated whether the pathologic mutants around the glycosylation sites have abnormal glycosylation modification and its subcellular location.We identifed the glycosylation state of wild type PrP and pathologic mutants V1801 and F198S in insect sf9 cells,and found the N-glycosylation modification was normal in the WT PrP,but abnormal in the V1801 and F198S.Moreover,the location of WT PrP was mainly on the membrane,cytoplasm for F198S and V180I was partly located in the cytoplasm and some was located on the membrane.Therefor,we speculated the glycosylation can influence the PrP subcellular location in some degree.Then we constructed other glycosylation-related mutants(N181D,N197D,N181D/N197D,and T199N/N181D/N197D)and GPI-deletion mutants,and found all the monoglycosylated PrP(N181D,N197D,T199N/N181D/N197D)were attached on the membrane,the unglycosylated PrP(DM),the GPI-deletion PrP and the PrP treated with tunicamycin to remove the glycosylation modification were located in the cytoplasm.Our data demonstrated that GPI anchor played the crucial role in determining PrP location,glycosylation modification acted as a cofactor,but a necessary cofactor.We decided to clarify the question whether N-glycosylation modification can affect PrP aggregation by influencing PrP location,and finally lead to prion diseases in the following respects,PK-resistance ability,aggregation ability and cytotoxicity.Our PK gradient digestion assays and ultracentrifugation assays demonstrated that the lower level of glycosylation modification the PrP had,the stronger tendency for PrP to locate in the cytoplasm,the stronger PK-resistance ability and aggregation ability the PrP had.Besides,all the PrP almost had the same PK-resistance ability and aggregation ability after the N-glycosylation was inhibited by tunicamycin.Moreover,the MTT and flow cytometry data demonstrated that the oxidative stress and the cytotoxicity of glycosylation-related mutants were DM,V180I,N197D and WT PrP in order of strength,and all the prion proteins had the same cytotoxicity after tunicamycin treatment,demonstrating that the lower level of glycosylation modification in the PrP,the stronger tendency for PrP to aggregation,the stronger cytotoxicity the PrP has,and the easier to generate prion diseases.Finally,our ThT binding assays,sarcosyl-soluble SDS-PAGE and time-course TEM data shown that mannose,lactose and sucrose significantly inhibited the aggregation of WT PrP and F198S,while the maltose had no effects.ITC data demonstrated that lactose and maltose had a weak interaction with WT PrP,while the mannose didn't interact with PrP.We speculated that only the monosaccharides that composed of glycans in the N-glycosylation modification can suppress PrP aggregation,and some interactions were too weak to be detected.For other monosaccharides,though it can interact with PrP,it can't inhibite PrP aggregation.We take the PrP location as the pointcut,explain why there is so many pathologic mutants around the glycosylation sites and clarify the relationship between N-glycosylation modification and prion diseases in the following respects,PK-resistance ability,the aggregation ability and cytotoxicity.We conclude that the lower level of glycosylation modification,the easier to generate prion diseases.Moreover,our study suggest the glycosylation modification can act as measure of diagnosing the prion diseases when the patients are in incubation period.