In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrPSc. Prion-infected neurons generate PrPSc from a host glycoprotein designated PrPC through a process of induced conformational change, but the molecular mechanism by which PrPC undergoes conformational change into PrPSc remains unknown. We employed an in vitro PrPSc amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrPSc from diluted scrapie-infected brain homogenate can be amplified > 10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrPSc amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrPSc displays an optimum pH of similar to7, whereas amplification of CD-1 mouse RML PrPSc is optimized at pH similar to6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrPSc amplification in vitro, indicating that the conformational change from PrPC to PrPSc requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrPC to PrPSc.