Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates

Glycobiology Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates Laura J Ellett 1 Bradley M Coleman 0 Mitch C Shambrook 0 Vanessa A Johanssen 1 Steven J Collins 1 Colin L Masters 2 Andrew F Hill 0 Victoria A Lawson 1 0 Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute 1 Department of Pathology 2 The Florey Institute of Neuroscience and Mental Health, The University Of Melbourne , Parkville, VIC 3010 , Australia Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrPC, to a misfolded isoform called PrPSc. Although PrPSc is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrPSc. Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrPSc deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrPC and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a β-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding. glycosaminoglycan; heparan sulfate; heparin; prions; sulfation - Prion diseases are a family of transmissible, neurodegenerative disorders associated with the misfolding of the normal cellular prion protein (PrPC) into a disease-associated form (PrPSc) (Prusiner 1982). Relative to PrPC, PrPSc is resistant to protease digestion, has an increased β-sheet content and propensity to form detergent insoluble aggregates (Bolton et al. 1982; Caughey, Dong, et al. 1991; Caughey, Raymond, et al. 1991; Pan et al. 1993). The most widely accepted mechanism for the transmissibility of prion disease is that PrPSc can act as a template for the misfolding of the mainly α-helical PrPC (Prusiner et al. 1990; Kocisko et al. 1994). How this misfolding and aggregation occurs and how it results in neurotoxicity, neurodegeneration and the classic spongiform change observed in disease-affected brain remains unknown. PrPSc is a necessary component of the infectious agent ( prion) of prion disease; however, additional factors, or cofactors, have been shown to contribute to the efficient formation of the transmissible PrPSc (reviewed Silva et al. 2010). Recombinant PrP can be misfolded into a form that induces disease when inoculated into susceptible animals (Legname et al. 2004; Kim et al. 2010); however, high titres of infectivity have not been reported for unseeded misfolding of recombinant PrP in the absence of cofactors. Polyanions, negatively charged polymers in particular glycosaminoglycans (GAGs), lipids and nucleic acids (RNA and DNA) have been shown to contribute to PrP misfolding and are viewed as potential cofactors in prion disease (Brimacombe et al. 1999; Deleault et al. 2005; Boshuizen et al. 2007; Deleault et al. 2007; Geoghegan et al. 2007; Lawson et al. 2010). Wang et al. (2010) demonstrated that RNA and lipids induce the misfolding of PrP that could be subsequently amplified over a period of time to generate PrPSc that could induce disease when inoculated into mice. It has since been reported that different animal species may require different cofactors in order for prions to propagate, including the finding that some species do not require a nucleic acid cofactor (Deleault et al. 2010). GAGs, linear, unbranched polysaccharides, have been implicated in prion disease and make plausible candidates for the prion cofactor (Diaz-Nido et al. 2002). GAGs exist in all tissues of the body and have many roles in ligand binding, cell–cell communication, extracellular matrix adhesion and blood clotting (Rabenstein 2002). Sulfation of GAGs can occur within the Golgi apparatus via highly regulated enzymatic processes with the resulting sulfation patterns being tissuespecific and an important determinant of GAG function (Esko and Selleck 2002). Heparan sulfate proteoglycans (HSPGs) are produced by all mammalian cells whereas heparin, a hypersulfated analog of heparan sulfate (HS), is stored in secretory granules in mast cells (Rabenstein 2002). Sulfation of HSPGs occurs via both N-linked sulfation (at the number 2 carbon of the glucosamine residue sugar (...truncated)