title: Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.
abstract: Shadoo (Sho) is an N-glycosylated glycophosphatidylinositol-anchored protein that is expressed in the brain and exhibits neuroprotective properties. Recently, research has shown that a reduction of Sho levels may reflect the presence of PrPSc in the brain. However, the possible mechanism by which prion infection triggers down-regulation of Sho remains unclear. In the present study, Western blot and immunohistochemical assays revealed that Sho, especially glycosylated Sho, declined markedly in the brains of five scrapie agent-infected hamsters and mice at the terminal stages. Analyses of the down-regulation of Sho levels with the emergence of PrPSc C2 proteolytic fragments did not identify close association in all tested scrapie-infected models. To further investigate the mechanism of depletion of Sho in prion disease, a Sho-expressing plasmid with HA tag was introduced into a scrapie-infected cell line, SMB-S15, and its normal cell line, SMB-PS. Western blot assay revealed dramatically decreased Sho in SMB-S15 cells, especially its glycosylated form. Proteasome inhibitor MG132 reversed the decrease of nonglycosylated Sho, but had little effect on glycosylated Sho. N-acetylglucosamine transferase inhibitor tunicamycin efficiently reduced the glycosylations of Sho and PrPC in SMB-PS cells, while two other endoplasmic reticulum stress inducers showed clear inhibition of diglycosylated PrPC, but did not change the expression level and profile of Sho. Furthermore, immunoprecipitation of HA-Sho illustrated ubiquitination of Sho in SMB-S15 cells, but not in SMB-PS cells. We propose that the depletions of Sho in scrapie-infected cell lines due to inhibition of glycosylation mediate protein destabilization and subsequently proteasome degradation after modification by ubiquitination.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (Zhejiang University), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Chang-Bai Road 155, Beijing, 102206, People's Republic of China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.forename: Wu-Ling
authors.initials: WL
authors.lastname: Xie
authors.forename: Yin
authors.initials: Y
authors.lastname: Xu
authors.forename: Ke
authors.initials: K
authors.lastname: Ren
authors.forename: Qi
authors.initials: Q
authors.lastname: Shi
authors.forename: Bao-Yun
authors.initials: BY
authors.lastname: Zhang
authors.forename: Cao
authors.initials: C
authors.lastname: Chen
authors.forename: Chan
authors.initials: C
authors.lastname: Tian
authors.forename: Chen
authors.initials: C
authors.lastname: Gao
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Dong
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D058851:GPI-Linked Proteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008647:Mesocricetus; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D012608:Scrapie / Q000378:metabolism* / Q000473:pathology; D025801:Ubiquitin / Q000502:physiology*
chemical_list: D058851:GPI-Linked Proteins; D009419:Nerve Tissue Proteins; C478971:SPRN protein, human; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex