Role of prion protein glycosylation in replication of human prions by protein misfolding cyclic amplification

Abstract

Prion diseases are transmissible neurological disorders associated with the presence of abnormal, disease-related prion protein (PrP^D). The detection of PrP^D in the brain is the only definitive diagnostic evidence of prion disease and its identification in body fluids and peripheral tissues are valuable for pre-mortem diagnosis. Protein misfolding cyclic amplification (PMCA) is a technique able to detect minute amount of PrP^D and is based on the conversion of normal or cellular PrP (PrP^C) to newly formed PrP^D, sustained by a self-templating mechanism. Several animal prions have been efficiently amplified by PMCA, but limited results have been obtained with human prions with the exception of variant-Creutzfeldt-Jakob-disease (vCJD). Since the total or partial absence of glycans on PrP^C has been shown to affect PMCA efficiency in animal prion studies, we attempted to enhance the amplification of four major sporadic-CJD (sCJD) subtypes (MM1, MM2, VV1, and VV2) and vCJD by single round PMCA using partially or totally unglycosylated PrP^C as substrates. The amplification efficiency of all tested sCJD subtypes underwent a strong increase, inversely correlated to the degree of PrP^C glycosylation and directly related to the matching of the PrP polymorphic 129 M/V genotype between seed and substrate. This effect was particularly significant in sCJDMM2 and sCJDVV2 allowing the detection of PK-resistant PrP^D (resPrP^D) in sCJDMM2 and sCJDVV2 brains at dilutions of 6 × 10⁷ and 3 × 10⁶. vCJD, at variance with the tested sCJD subtypes, showed the best amplification with partially deglycosylated PrP^C substrate reaching a resPrP^D detectability at up to 3 × 10¹⁶ brain dilution. The differential effect of substrate PrP^C glycosylations suggests subtype-dependent PrP^C-PrP^D interactions, strongly affected by the PrP^C glycans. The enhanced PMCA prion amplification efficiency achieved with unglycosylated PrP^C substrates may allow for the developing of a sensitive, non-invasive, diagnostic test for the different CJD subtypes based on body fluids or easily-accessible-peripheral tissues.