Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

Abstract

BackgroundThe accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.Methodology/Principal FindingIn this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.Conclusions/SignificanceCofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

Modulation of Glycosaminoglycans Affects PrPScMetabolism but Does Not Block PrPScUptake

“…Alternatively, changes in the intracellular distribution or trafficking kinetics of PrP Sc and endogenous GAGs could contribute to the antiprion activity of GAG modulators. In vitro studies demonstrate a direct effect of GAGs and GAG analogues on PrP Sc amplification, potentially by providing a scaffold for PrP C -PrP Sc interactions and conversion (13,45,66). The observed reduction in endogenous GAGs could thus directly affect the conversion process and thereby abrogate prion propagation.…”

“…Sodium chlorate, a competitive inhibitor of the cellular 3=-phosphoadenosine 5=-phosphosulfate, prevents both HS and CS sulfation (41)(42)(43) and also decreases PrP Sc accumulation in persistently infected cells (31,44). GAG sulfation also affects PrP Sc formation in in vitro assays and thus directly acts on PrP Sc amplification (45). So far, a comparative analysis of the effects of GAG modulators on host cell PrP C , on endogenous sulfated GAGs, and on the individual stages of infection by different strains has not been performed.…”

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

“…PrP Sc accumulation also influences the metabolism of GAGs and results in their accumulation and secretion in urine (34). Furthermore, sulfated GAGs containing HS stimulate the conformational change of PrP C into a PrP Sc -like proteinase K (PK)-resistant form even in a cell-free conversion assay (9,12). Thus, GAG involvement in prion disease pathogenesis and PrP C conversion into PrP Sc has been widely reported (35)(36)(37), but whether GAGs promote PrP Sc replication in PMCA, which mimics in vivo prion replication, is unknown.…”

“…Sulfate Groups on HP Are Critical for Bac-PrP res Amplification-GAG length and degree of sulfation were previously reported to affect the conversion of PrP C or recPrP into PrP Sc (9,10,(35)(36)(37)(38). Therefore, to examine the relative effects of GAG length and sulfation degree on Bac-PrP conversion into Bac-PrP res , we added chemically modified HPs to iPMCA reaction mixtures under nucleic acid-depleted conditions (Fig.…”

Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

“…Cells were lysed via passage through small-gauge needles and the resulting homogenate inoculated into the left parietal regions of Tga20 mice (37). Tga20 mice, which overexpress PrP approximately 4-fold relative to wild-type mice, act as a sensitive and physiologically relevant detector of prion infectivity (56). As expected, mice inoculated with M1000 MoPrP-RK13 lysate developed symptoms of prion disease, which progressed to terminal illness after a mean of 126 days Ϯ 6 days (standard error of the mean [SEM]) ( Table 1).…”