The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).

Abstract

Scrapie‐associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt‐Jakob disease. A major component of hamster fibrils has been described as a protease‐resistant glycoprotein with an apparent mol. wt of 27,000‐30,000 (PrP27‐30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co‐purifying with hamster SAF have mol. wts of 33,000‐35,000 (PrP33‐35) and 26,000‐29,000 (PrP26‐29). We find a Lys‐Lys‐Arg‐Pro‐Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27‐30, and which has recently been predicted to be the N‐terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33‐35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two‐dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33‐35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid‐like SAF or into association with a non‐protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.

Discovery and characterization of a mammalian amyloid disaggregation activity

“…Mammalian amyloid disaggregase and proteolysis activities are sensitive to low and high pH pretreatment. Dialysis of HEK 293 PNS (1 mg/mL total protein) for 16 h at 4 C into buffers at low pH (and to a lesser extent high pH) decreases its ability (at a final total PNS concentration of 10 lg/mL) to disaggregate Ab fibrils (65 lg/mL) and to degrade Ab 1-40 . resistance of amyloid to proteolysis, it seems likely that the mammalian disaggregase activity precedes the proteolysis activity, [39][40][41] and the proteolysis activity is crucial for preventing reaggregation. 35 The discovery and partial characterization of the mammalian amyloid disaggregase activity(ies) are a step towards identification of its molecular underpinnings.…”

“…The tight linkage of disaggregation and proteolysis may be due to a physical linkage of proteins or protein complexes that carry out these activities. Additionally, because amyloid is known to be protease resistant, [39][40][41] it is likely that disaggregation is required for proteolysis, and this relationship would necessitate a linear correlation between disaggregation and proteolysis (Fig. 7).…”

“…39 The kinetic disaggregation assay was conducted in aggregation buffer (50 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.02% NaN 3 ) containing ThT (40 lM). Mammalian tissue PNS (3-45 lg/mL total protein, as specified) or human cell PNS (3-45 lg/mL total protein, as specified) was added to the specified concentration of sonicated Ab 1-40 fibrils or sonicated 8 kDa gelsolin fibrils in Corning 96-well clear bottom plates (final volume 100 lL/well).…”

Molecular biology of transmissible spongiform encephalopathies

“…Seed formation is kinetically controlled and extremely slow; once a seed is present, monomer addition can ensue at a rapid rate [58,59]. [16,21,29]. Pulse-chase experiments in scrapie-infected neuroblastoma cells suggest that PrP c is converted to PrP sc either at the cell surface or following endocytosis [30].…”

Atypical Scrapie in a Swiss Goat and Implications for Transmissible Spongiform Encephalopathy Surveillance

“…30 It is characterized by progressive neurodegeneration, including neuronal death, gliosis, spongiform changes, and deposition of partially proteinase K-resistant prion protein (PrP sc ), an abnormal isoform of the physiological host-encoded prion protein (PrP c ), in the central nervous system (CNS). 21 In contrast to bovine spongiform encephalopathy (BSE), 35 there is no evidence that scrapie is harmful to humans. However, it has been shown repeatedly that BSE is transmissible to both sheep and goats 15,16 under experimental conditions.…”