Transmissible spongiform encephalopathys(TSEs),also known as prion diseases,involve the accumulation of abnormally aggregated form of the normal host prion protein(PrP).Prion protein is a glycoprotein anchoring in the outer membrane through glycosylphosphatidylinositol(GPI).However,many previous studies have found that amyloid deposits of most pathological mutants in TSEs exist in the cytoplasm,which prove that the subcellular localization of PrP is an important index to forecast whether PrP or PrP mutants can lead to prion diseases.Herein,by employing a cell model of pathological mutations beside the glycosylation sites of human PrP,V180I and F198S,which cause familiar Creutzfeldt-Jakob disease and Gerstmann-Str(a)ussler-Scheinker disease,respectively,we investigated the relationship between glycosylation modification and GPI anchor with the cellular location of human PrP.We also built other glycosylation site PrP mutants such as N181D,N197D,N181/197D,and GPI deletion mutants in SH-SY5Y neuroblastoma cells.As evidenced by confocal laser scanning microscopy,Sarkosyl-soluble SDS-PAGE,and Western blot,all GPI deletion mutants of PrP are located in the cytoplasm of SH-SY5Y cells,and two PrP pathological mutants V180I and F198S and the double glycosylation site mutant N181/197D are mainly or almost completely located in the cytoplasm.In contrast,the single glycosylation site mutants N181D and N197D as well as wild-type PrP are located on the membrane of SH-SY5Y cells.Moreover,we introduced a new glycosylation site into N181/197D and built a new mutant T199N/N181D/N197D,and found that such a mutant is located on the membrane.We thus suggest that both glycosylation modification and GPI anchor can affect the cellular location of human PrP,which could further affect abnormal aggregation of human PrP in neuronal cells.