Liberation of GPI-Anchored Prion from Phospholipids Accelerates Amyloidogenic Conversion

Abstract

Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed.

Quercetin Disaggregates Prion Fibrils and Decreases Fibril-Induced Cytotoxicity and Oxidative Stress

“…The pET101/D-TOPO plasmid encoding mouse prion 23-230 (moPrP) was transformed into competent Escherichia coli BL21(DE3) cells and over-expressed in inclusion bodies by induction with isopropyl β- d -thiogalactopyranoside. The expressed moPrP proteins were purified on a Ni-Sepharose column based on a previously described method [ 35 ] with some modifications [ 36 ]. MoPrP was further purified with reversed-phase C4-high performance liquid chromatography (HPLC) column [ 20 ].…”

Simulations of membrane‐bound diglycosylated human prion protein reveal potential protective mechanisms against misfolding

“…) nor denaturants (Lin et al . ) have been shown to induce misfolding and oligomerization of membrane‐bound PrP C . Furthermore, unlike bare recombinant PrP, membrane‐bound PrP C appears to be resistant to PrP Sc ‐induced misfolding unless it is released from membranes by phospholipase C digestion (Baron et al .…”

The Effect of Octapeptide Repeats on Prion Folding and Misfolding

“…Subsequent addition of 1 mM isopropyl β-D-1-thiogalactopyranoside induced expression of proteins. The PrP-5oct and PrP-8oct proteins were purified by immobilized metal affinity chromatography and reversed-phase C4-HPLC as modified from previously reported procedures [ 32 , 33 , 34 ]. The PrP-Δoct and PrP-1oct proteins were purified by cation exchange chromatography with SP Sepharose and reversed-phase C4-HPLC as previously described [ 35 ].…”