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

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 × 10 7 and 3 × 10 6 . 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 × 10 16 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.