Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type Tao Pan,* Ruliang Li,* Shin-Chung Kang,* Manuela Pastore,* Boon-Seng Wong,  James Ironside,à Pierluigi Gambetti* and Man-Sun Sy§ , ¶ *Division of Neuropathology, Case Western Reserve University, Cleveland, Ohio, USA  Division of Neuropathology, University of Edinburgh, Edinburgh, UK àNational CJD Surveillance Unit, School of Clinical and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK §Institute of Pathology and ¶Department of Neuroscience, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA Abstract The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrP Sc ) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two- dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also sepa- rately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrP Sc , but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP frac- tion suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrP Sc of vCJD in one- dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases. Keywords: 2-D electrophoresis, Prion CJD, sCJD, vCJD. J. Neurochem. (2005) 92, 132–142. Prion diseases are neurodegenerative diseases that affect humans and animals. The human diseases are unique as they include not only sporadic and familial forms, like other neurodegenerative diseases, but also forms acquired by infection from either affected humans or animals. Regardless of form, prion diseases are believed to share a common pathogenic mechanism, which is based on the conversion of a normal cellular prion protein (PrP C ) into a pathogenic, infectious isoform, commonly referred to as scrapie prion protein or PrP Sc (Prusiner 1998). The PrP C to PrP Sc conversion results in the formation of distinct types of PrP Sc , also referred to as PrP Sc strains (Bruce et al. 1992; Carp et al. 1994). In experimental prion diseases in animals, each PrP Sc strain has unique biological properties, such as incubation time, host range, clinical symptoms, profile of the lesion it induces upon passage from host to host and biochemical features (Bessen and Marsh 1992, 1994; Bruce et al. 1992; Carp et al. 1994). In human prion Received July 1, 2004; revised manuscript received August 26, 2004; accepted September 2, 2004. Address correspondence and reprint requests to Man-Sun Sy at Institute of Pathology, Biomedical Research Building Room 933,10900 Euclid Ave, Cleveland, OH 44120, USA. E-mail: mxs92@po.cwru.edu Abbreviations used: PNGase F, N-glycosidase F; PrP, prion protein; PrP C , cellular prion protein; PrP Sc , scrapie prion protein; sCJD, sporadic Creutzfeld-Jakob disease; sFI, sporadic fatal insomnia; vCJD, variant Creutzfeld-Jakob disease. Journal of Neurochemistry , 2005, 92, 132–142 doi:10.1111/j.1471-4159.2004.02859.x 132 Ó 2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 132–142