Conversion of a-helices into ,f-sheets features in the formation of the scrapie prion proteins

Prions are composed largely, if not entirely, of prion protein (PrPsc in the case of scrapie). Although the formation of PrPs from the cellular prion protein (PrPc) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPsc synthesis. To assess this possibility, we purified both PrPC and PrPsc by using nondenaturing procedures and determined the secondary structure ofeach. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high a-helix content (42%) and no (3sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the -sheet content of PrPSc was 43% and the a-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPsc derived by limited proteolysis, designated PrP 27-30, has an even higher -sheet content (54%) and a lower a-helix content (21%). Neither PrPC nor PrPsc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing rmdings argue that the conversion of a-helices into 1-sheets underlies the formation of PrPsc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSC initiates this process. Since PrPsc seems to be the only component of the "infectious" prion particle, it is Ihkely that this conformational transition is a fundamental event in the propagation of prions. Prions are proteinaceous infectious particles that are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) designated, in the case of scrapie, PrPSc (1). Prions cause four neurodegenerative diseases of humans and six of animals, including scrapie of sheep and bovine spongiform encephalopathy. That the human prion diseases are manifest as infectious, familial, and sporadic disorders posed an enigma until it was discovered that mutations in the PrP gene are genetically linked to development of neurodegeneration (2). PrPsC is synthesized from the normal cellular isoform PrPC by a post-translational process that probably occurs in endosomes (3-6). Attempts to identify a post-translational chemical modification that features in the conversion of PrpC into PrPSc have been unsuccessful (7). These findings raised the possibility that PrPSc differs from PrPC only with respect to conformation. Many properties of PrPSc differ from those of PrPC: (i) PrPSc is insoluble in detergents, while PrPC is readily solubilized under nondenaturing conditions (8); (ii) PrPSc is partially hydrolyzed by proteases to form a fragment designated PrP 27-30, while PrPC is completely degraded under the same conditions (9); (iii) PrPSc accumulates, whereas PrPC turns over rapidly (3); and (iv) the patterns of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. PrPSc accumulation in brain are distinct from the distribution of NPc (10). The protease-resistant core of PrPSc designated PrP 27-30 polymerizes into rod-shaped structures which are indistinguishable from many purified amyloids both ultrastructurally and tinctorially (11). In the brains of some, but not all, animals and humans that have died of prion diseases, amyloid plaques are found which contain PrP, as determined by immunostaining and Edman protein sequencing studies (12-14). That PrP 27-30 polymerizes into amyloid suggests that it might have a ,(pleated sheet structure (11), since all amyloids studied, to date, have been found to have this structure (15). About 50%o of the secondary structure of PrP 27-30 is (3sheet, as measured by Fourier-transform infrared (FTIR) spectroscopy (16, 17), a percentage that is much higher than that predicted from the amino acid sequence (18). Two-thirds of the p-sheet content of PrP 27-30 is low-frequency (LF) p-sheet, which often reflects intermolecular associations that are a characteristic of amyloids (17). The LF p-sheet content ofPrP 27-30 declined upon denaturation under conditions that diminished scrapie infectivity (17). Disruption of the amyloid polymers composed of PrP 27-30 by dispersion into liposomes altered neither the LF p-sheet content nor prion infectivity (19). Computational analyses of a family of homologous PrP sequences suggested that PrPC might be folded into a fourhelix bundle (20). When the four putative a-helices were synthesized as peptides, three of the four polymerized into amyloid fibrils with -70% of the secondary structure LF p-sheet (20). Other investigators have also demonstrated polymerization of synthetic PrP peptides into amyloid (21-23). To examine the possibility that formation ofPrPSc involves the conversion of a-helices in prPC into p-sheets, we developed a PrPC purification protocol utilizing nondenaturing procedures. As reported here, we determined the secondary structures of both prPC and PrPSC. Our findings suggest that the fundamental event in the formation of PrPSC as well as propagation of prion infectivity is the conversion of a-helices in PrPC into p-sheets. MATERIALS AND METHODS All chemicals were of the highest grade commercially available. 2H20 was purchased from Aldrich; nondenaturing detergents, including Zwittergent 3-12 (ZW), were from Calbiochem; SDS was from BDH; proteinase K was from Beckman; Abbreviations: PrP, prion protein; PrPC, cellular PrP; PrPSc, scrapie PrP; PrP 27-30, protease-resistant fragment of PrPsc; FTIR, Fouriertransform infrared; LF, low-frequency; mAb, monoclonal antibody; ZW, Zwittergent 3-12; WGA, wheat germ agglutinin; EM, electron microscopy. tPresent address: Departmento Bioquimica, Universidade Complutense, 28040 Madrid, Spain. 'To whom reprint requests should be addressed at: Department of Neurology, HSE-781, University of California, San Francisco, CA 94143-0518.