Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification

Abstract

The precise mechanism underlying the conversion of normal prion protein (PrP^C) into abnormal prion protein (PrP^Sc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrP^Sc, results in PrP^Sc replication that preserves the strain-specific characteristics of the input PrP^Sc; thus, PMCA mimics the process of in vivo PrP^Sc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrP^Sc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrP^Sc replication in vitro Here, we investigated whether GAGs play a role in the faithful replication of PrP^Sc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrP^Sc These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrP^Sc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrP^Sc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrP^C and/or PrP^Sc through their sulfate groups is critical for the faithful replication of prions.

Keywords: baculovirus-recombinant prion protein; heparan sulfate; heparin; prion; prion disease; protein folding; protein misfolding cyclic amplification.