The Stoichiometry of Host PrP^C Glycoforms Modulates the Efficiency of PrP^Sc Formation in Vitro

Abstract

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.

Formation of native prions from minimal components in vitro

“…Building on this advance, we investigated whether purified PrP C molecules could function as a substrate for the propagation of PrP Sc molecules and infectious prions in vitro. To perform these studies, it was necessary to develop a protocol to purify PrP C from native brain tissue because recombinant PrP produced in Escherichia coli is an inefficient substrate for PMCA reactions, even when reconstituted with crude brain homogenates (25). During the course of these studies, we identified conditions under which purified substrates could propagate infectious prions in vitro and also unexpectedly discovered that infectious prions could be generated spontaneously from purified, noninfectious components.…”

Prion Protein Glycosylation Is Not Required for Strain-Specific Neurotropism

“…Unglycosylated PrP C was purified as previously described (26). Briefly, 12 mouse brains were Potter homogenized in 40 ml of cold buffer A (20 mM MOPS [morpholinepropanesulfonic acid] [pH 7.0] and 150 mM NaCl) with Complete EDTA-free protease inhibitor (Roche, Indianapolis, IN).…”

“…A series of recent advances has provided a new opportunity to produce infectious unglycosylated PrP Sc molecules in vitro by using serial protein misfolding cyclic amplification (sPMCA) reactions (7,26), a cell-free prion amplification technique that faithfully preserves the strain properties of the initial seed material (6). Here, we use reconstituted sPMCA reactions to produce unglycosylated PrP Sc molecules derived from two mouse prion strains associated with easily distinguishable patterns of neurotropism (6).…”

Les prions, agents d’encéphalopathies transmissibles chez les mammifères

“…Les domaines structuraux et/ou les modifications post-traductionnelles de la PrP Sc impliqués dans le déterminisme de souche, ainsi que les mécanismes de transfert à la PrP C restent cependant à déterminer. Il est possible que des ces mécanismes soient spécifique de souche (Nishina et al, 2006, Moudjou et al, 2016. Des données du laboratoire suggèrent que le déterminant structural de souche serait déjà présent dans l'unité de base composant les agrégats de PrP Sc (Igel-Egalon et al, en révision).…”