Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC)

Abstract

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-beta-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.

Pronounced cytosolic aggregation of cellular prion protein in pancreatic β-cells in response to hyperglycemia

“…The specificity of these antibodies has previously been reported. 21,22 We confirmed further the specificity of the PrP antibodies in our laboratory using Western blots of purified PrP C . All other primary antibodies are shown in Supplementary QImaging, Burnaby, BC, Canada) was used for fluorescence imaging.…”

Prion adsorption to stainless steel is promoted by nickel and molybdenum

“…In addition, steel 410L incubated with the RML strain To get an idea of whether the binding of PrP Sc to the metals was ionic in character or not, we used a metal ion-chelating pulldown assay (metal ions immobilized to Sepharose gel). Undigested PrP derived from RML-infected cells was bound to chelating Sepharose loaded to Cu 2+ and Ni 2+ in accordance with previous studies (Brown et al, 1997;Hornshaw et al, 1995;Muller et al, 2005;Shaked et al, 2001;Stockel et al, 1998;Taraboulos et al, 1992) (data not shown). Copper ions are known to bind to PrP C at the Nterminal octarepeat region, which can bind up to four Cu 2+ (Burns et al, 2003; Wells et al, 2006).…”

Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent

“…Another unexpected finding in this study was the retention of nondenatured PrP Sc by the copper ion, because failure to bind to immobilized copper ion has been reported by others to be a distinctive trait of nondenatured PrP Sc (56,57). In our experimental conditions, the binding of both FL PrP Sc and PrP res was reproducible, and plainly ion-dependent (data not shown).…”