AG, consistent with a GAG binding site in the N-terminal region of PrP. Mutations associated with familial prion disease have been shown to reveal a cryptic GAG binding site down stream of the residue 90, which 
may enable the C2 fragment of PrP to bind to GAGs. PNGaseF treatment revealed a truncated fragment of 101LmoPrP bound to heparin whereas the same fragment present in the 101P-moPrP expressing cells was not detected. This fragment was detected using antibodies 03R19 but not with the N-terminal antibody 03R17 indicating that it lacked Nterminal residues 230. Discussion Large negatively charged macromolecules have been implicated in the pathogenesis of prion diseases. Nucleic acids, and in particular RNA has been identified as a potential co-factor in the formation of the disease associated isoform of the prion protein, in hamster but not mouse models of prion disease. The current study using a August 2010 | Volume 5 | Issue 8 | e12351 Prion Protein Misfolding mouse adapted human prion strain, provides further insight into the prion strain and species specific requirements for prion propagation. We report that depletion of either nucleic acids or sGAG and in particular 18325633 heparan sulphate, prevent the cell free formation 25581517 of PrPres from murine PrPC seeded with mouse derived PrPSc. Changes to GAG sulphation through chlorate treatment increased the ability of PrPC encoding the P101L mutation linked with familial prion disease to form PrPres, which may be related to the ability of this molecule to associate with under sulphated GAG species. Recent reports have suggested that the cofactors required for efficient hamster PrPres formation may be species specific as the depletion of RNA from a murine derived substrate did not affect PrPres formation. In contrast prion infectivity can been generated from either hamster PrP or recombinant murine PrP, in the absence of a PrPSc seed, by the addition of RNA, albeit in the presence of lipids. Thus it would appear that RNA stimulation of de novo PrPres formation is species independent. Furthermore the data presented here showing that depletion of nucleic acids from the PrPC substrate of a mouse adapted model of human prion disease prevented PrPres formation, indicates that the requirement for endogenous RNA may be prion strain dependent. Moreover the use of a prion strain originally derived from a patient with GSS in the current study rather than scrapie or bovine spongiform encephalopathy strains raises the possibility that human prion strains have different cofactor requirements to animal prion strains. We are investigating this possibility further using a mouse adapted prion strain developed from a T2MM sporadic prion strain. A further species or strain specific effect is raised by the significant and specific effect of heparan sulphate depletion on conversion activity shown here. Unlike earlier reports, in which the heparinase III treatment did not affect the conversion activity of a hamster PrPC substrate seeded with 263K hamster adapted scrapie brain homogenate, we show that specific depletion of endogenous heparan sulphate inhibits the conversion activity of a mouse PrPC substrate when seeded with the M1000 mouse adapted human prion strain. The curing effect of heparinase III, but not heparinase I and chondroitinase ABC treatment of prion infected N2a cells has been previously MedChemExpress AZ-505 reported and proposed to relate to either the relative GAG content of N2a cells or relate to the cleavage specificity
