hrough a cryptic GAG binding site located between residues 10936. Consistent with this we report the increased binding affinity of 101L-moPrP for heparin and the preferential binding of a 22kDa fragment consistent with C2 from 101L but not 101P moPrP. The association of PrP with GAGs through this alternativebinding domain may play a role in the pathogenic process. It was surprising that modification of GAG sulphation with chlorate did not decrease the conversion activity of moPrP. Chlorate treatment does not change the PK-resistance or solubility of wild type 101P-moPrP, although it did increase the PrP levels, perhaps by altering the metabolism of PrPC. Chlorate competitively inhibits the formation of the sulphate donor 39-phosphoadenosine 59-phosphate Ariflo biological activity required for GAG sulphation. When cells are grown in medium containing normal sulphate supplementation, as performed here, sulphation of heparan sulphate is selectively inhibited, with 6-O-sulphation inhibited before 2-O-sulphation. Previous studies have highlighted the importance of 2-O but not 6-O sulphation for the interaction of wildtype PrP with heparin and the role of under sulphated GAGs in prion propagation. Therefore it is possible that under the conditions August 25581517 2010 | Volume 5 | Issue 8 | e12351 Prion Protein Misfolding used in this study sulphation required for the interaction of wild type 101P-moPrP with sGAG remained unaltered. Whereas due to the altered GAG binding pattern of 101L-moPrP, selective inhibition of sulphation may have increased the profile of GAGs that could bind and facilitate the conversion of mutant 101LmoPrP. Intriguingly, chlorate increased the solubility of mutant 101L-moPrP, which may have affected the ability of this species to be converted to PrPres. This study has revealed a further complexity to the role of cofactors in the propagation of prions. Although prion infectivity can be generated from PrP in the absence of cofactors it appears that the addition of cofactors may augment the conversion process. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms 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. Materials and Methods Ethics statement The use of tissue sourced from prion 
infected and uninfected mice was approved for this study by the University of Melbourne Animal Ethics Committee Preparation of prion infected brain homogenates Brains were collected from balb/c mice in the terminal stage of disease following intracerebral inoculation with M1000 prions. For use as a seed in the cell free assay of PrPres formation, 10% brain homogenates were prepared in calcium and magnesium free Dulbecco’s phosphate buffered saline or 20 mM Tris-HCl pH 7.4 supplemented with 1% Triton-X 100. Homogenates were prepared by passing tissue through a graded series of needles. The final sample was then cleared at 2006g for 2 minutes, the supernatant snap frozen in liquid nitrogen and stored at 280uC. Preparation of uninfected brain homogenates Brain tissues were collected from wild type, Prnp knock out and Prnp overexpressing mice. Brain homogenates were prepared in DPBS or 20mM Tris-HCl pH 7.4 supplemented with EDTA-free ecomplete mini protease inhibitors using a graded series of needles as described above. , snap frozen in liquid nit