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

摘要

pThe conformational conversion of the normal cellular prion protein (PrPsupC/sup) into the pathology-associated PrPsupSc/sup isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPsupC/sup molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPsupC/sup, PrPsupSc/sup is insoluble in non-ionic detergents and does not bind to Cusup2+/sup 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-β-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cusup2+/sup-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 PrPsupC/sup and denatured PrPsupSc/sup were retained by a Cusup2+/sup-loaded resin, native PrPsupSc/sup and PrPsupres/sup [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPsupC/sup 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 PrPsupC/sup from aggregated PrPsupSc/sup in infected brains. Our results indicate that in contrast with PrPsupSc/sup in uninfected as well as infected brains, PrPsupC/sup is predominantly present in the glycosylated forms./p