Detection of Scrapie-associated Fibril (SAF) Proteins Using Anti-SAF Antibody in Non-purified Tissue Preparations

Abstract

SUMMARY Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

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).

“…Affinity-purified immunoglobulins from animals immunised with PrP27 -30 detect a form of this protein in extracts of scrapie-infected brain and uninfected brain which has an apparent mol. wt of 33 000-35 000, significantly larger than (Oesch et al, 1985;Manuelidis et al, 1985;Rubenstein et al, 1986). This PrP 33-35 protein in homogenates of uninfected hamster brain is completely degraded by proteinase K (Oesch et al, 1985;Rubenstein et al, 1986) and neither fibrils nor protein are found in fractions of uninfected brain prepared by the methods developed to isolate SAF.…”

“…wt of 33 000-35 000, significantly larger than (Oesch et al, 1985;Manuelidis et al, 1985;Rubenstein et al, 1986). This PrP 33-35 protein in homogenates of uninfected hamster brain is completely degraded by proteinase K (Oesch et al, 1985;Rubenstein et al, 1986) and neither fibrils nor protein are found in fractions of uninfected brain prepared by the methods developed to isolate SAF. This evidence can be used to support a model for the pathogenesis of SAF in which scrapie infection induces the proteolysis of PrP33 -35 to PrP27 -30 and that it is this latter, abnormal by-product of infection which then polymerises into the characteristic disease-specific fibrils ( Figure 4A).…”

Immunohistochemical Detection of Prion Protein in Sheep with Scrapie

“…One antibody was generated by injecting a rabbit with antigen derived from sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified PrP-res from brains of mice inoculated with the ME7 strain of scrapie. 17,31 The other antibody (R27) was to a synthetic peptide corresponding to residues 89-103 of the complete mouse PrP amino acid sequence. 28 Slides were incubated in primary antibody overnight at 4 C, rinsed 3 times in Automation buffer, then held in the buffer at 37 C for 30 min.…”

The Role of Helix 1 Aspartates and Salt Bridges in the Stability and Conversion of Prion Protein

“…PrP-res forms insoluble aggregates and is partially resistant to proteinase K (PK), which removes ϳ67 amino acid residues (6 -7 kDa) from the N terminus (1,(3)(4)(5). These differences in biophysical properties most likely reflect different conformations of the two protein isoforms.…”