Purification and Partial Characterization of the Normal Cellular Homologue of the Scrapie Agent Protein

Abstract

The scrapie agent protein (Sp33-37) is a degradation-resistant protein that aggregates into fibrils and amyloid plaques. This protein is derived from a normal cellular protein (Cp33-37). Understanding the mechanism responsible for the conversion of Cp33-37 to Sp33-37 may explain scrapie agent replication. Cp33-37 was extracted from normal hamster brain and purified 2700-fold by an immunoaffinity method. Both Cp33-37 purified from normal hamster brain and Sp33-37 purified from scrapie-affected hamster brain had apparent masses of 33-37 kilodaltons and displayed microheterogeneity characteristic of glycoproteins. Cp33-37 was completely digested by proteinase K under conditions that resulted in conversion of Sp33-37 to the protease-resistant fragment PrP27-30. Cp33-37 did not cause scrapie when inoculated intracerebrally into hamsters. Fractions containing purified Sp33-37 had average titers of greater than 10(11) LD50 of the scrapie agent/mg of protein; these titers were not diminished by proteinase K. These results indicate that altered sensitivity to proteolysis in vitro reflects an intrinsic difference between Sp33-37 and Cp33-37.

Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method

“…On this basis Bendheim et al (1988) estimate the total amount of TSE-specific amyloid in a scrapie-affected hamster brain to be about 6(~160lag. In a recent publication Rubenstein et aI.…”

“…This amyloid protein is derived from a highly conserved host-encoded precursor, a cellular glycoprotein (tool. mass 33-35 kDa, also designated PrP c) (Oesch et al, 1985;Robakis et al, 1986;Locht et al, 1986;Basler et al, 1986;Bolton et al, 1987;Bendheim et al, 1988) located mainly at the surface of nerve cells (Stahl et al, 1987(Stahl et al, , 1990a. During the course of a TSE infection, an as yet unknown post-translational event (Borchelt et al, 1990) converts this cellular protein into a disease-specific pathological isoform (also termedpathological isoforms of the protein differ in their sensitivity towards proteolytic activity.…”

Copurification of Sp33-37 and Scrapie Agent from Hamster Brain Prior to Detectable Histopathology and Clinical Disease

“…An abnormal host glycoprotein is the only macromolecule that has been shown to be specifically associated with these diseases and to purify with the agents (Bolton et al, 1982(Bolton et al, , 1987aMcKinley et al 1983;Prusiner et al, 1982a;Prusiner & McKinley, 1987;Gabizon et al, 1987;Carp et al, 1989). This glycoprotein, Sp33-37, differs from its normal cellular form, Cp33-37, in that it aggregates to form high molecular mass structures and is remarkably resistant to protease degradation (Bolton et al, 1982(Bolton et al, , 1987aMcKinley et al, 1983;Meyer et al, 1986;Bendheim et al, 1988b;Prusiner et al, 1983;Barry et al, 1985). [This protein has been referred to variously as PrP, PrP-27-30, SAF protein, PrP 33-35 sc, PrP so, PrP 35-38 and Sp33-37 (McKinley et al, 1983;Bolton et al, 1985Bolton et al, , 1987aKascsak et al, 1986;Meyer et al, 1986;Gabizon et al, 1987;Bendheim & Bolton, 1986).…”

“…Sp33-37 accumulates in the brain and spleen during disease, with the maximum concentration being attained in the brain near the time of death. Sp33-37 has not been detected in normal brain at any time (Bolton et al, 1987a, b;Meyer et al, 1986;Bendheim et al, 1988b) and was not found in brains of grey tremor mice, a mutant strain that spontaneously develops a disease with similar pathology (Bendheim et al, 1988a). Cp33-37 is synthesized in the brain throughout life, beginning from the early neonatal period .…”

Prion distribution in hamster lung and brain following intraperitoneal inoculation.

“…Thirty-four Syrian hamsters (virus-free weanling females) were inoculated in the peritoneal cavity with 50 µl 263K scrapie prions (1 : 100 dilution), purified as described previously (Bolton et al, 1987 ;Bendheim et al, 1988). Three hamsters were humanely killed by CO # asphyxiation on each of the days indicated in Fig.…”