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.

The X-ray crystallographic structures of the anti-Syrian hamster prion protein (SHaPrP) monoclonal Fab 3F4 alone, as well as the complex with its cognate peptide epitope (SHaPrP 104-113), have been determined to atomic resolution. The conformation of the decapeptide is an Omega-loop. There are substantial alterations in the antibody combining region upon epitope binding. The peptide binds in a U-shaped groove on the Fab surface, with the two specificity determinants, Met109 and Met112, penetrating deeply into separate hydrophobic cavities formed by the heavy and light chain complementarity-determining regions. In addition to the numerous contacts between the Fab and the peptide, two intrapeptide hydrogen bonds are observed, perhaps indicating the structure bound to the Fab exists transiently in solution. This provides the first structural information on a portion of the PrP N-terminal region observed to be flexible in the NMR studies of SHPrP 90-231, SHaPrP 29-231 and mouse PrP 23-231. Antibody characterization of the antigenic surfaces of PrPC and PrPSc identifies this flexible region as a component of the conformational rearrangement that is an essential feature of prion disease.

Conversion of the cellular isoform of prion protein (PrPC) into the scrapie isoform (PrPSc) involves an increase in the beta-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPC and PrPSc form a complex during PrPSc formation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPC to determine whether its properties were altered. Peptides encompassing two alpha-helical domains of PrP when mixed with PrPC produced a complex that displayed many properties of PrPSc. The PrPC-peptide complex formed fibrous aggregates and up to 65% of complexed PrPC sedimented at 100,000 x g for 1 h, whereas PrPC alone did not. These complexes were resistant to proteolytic digestion and displayed a high beta-sheet content. Unexpectedly, the peptide in a beta-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPC sensitive to protease digestion. While the pathogenic A117V mutation increased the efficacy of complex formation, anti-PrP monoclonal antibody prevented interaction between PrPC and peptides. Our findings in concert with transgenetic investigations argue that PrPC interacts with PrPSc through a domain that contains the first two putative alpha-helices. Whether PrPC-peptide complexes possess prion infectivity as determined by bioassays remains to be established.

Although no chemical modifications have been found to distinguish the cellular prion protein PrPC from its infectious analogue PrPSc, spectroscopic methods such as Fourier transform infrared (FTIR) spectroscopy reveal a major conformational difference. PrPC is rich in alpha-helix but is devoid of beta-sheet, whereas PrPSc is high in beta-sheet. N-terminal truncation of PrPSc by limited proteolysis does not destroy infectivity but it increases the beta-sheet content and shifts the FTIR absorption to lower frequencies, typical of the cross beta-pleated sheets of amyloids. Thus the formation of PrPSc from PrPC involves a conformational transition in which one or more alpha-helical regions of the protein is converted to beta-sheet. This transition is mimicked by synthetic peptides, allowing predictions of domains of PrP involved in prion diseases.

Prions are composed largely, if not entirely, of prion protein (PrP(Sc) in the case of scrapie). Although the formation of PrP(Sc) from the cellular prion protein (PrP(C)) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrP(Sc) synthesis. To assess this possibility, we purified both PrP(C) and PrP(Sc) by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrP(C) has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrP(Sc) was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrP(Sc) derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrP(C) nor PrP(Sc) formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrP(Sc), we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrP(Sc) initiates this process. Since PrP(Sc) seems to be the only component of the ''infectious'' prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.

The cellular prion protein (PrPC) is encoded by a chromosomal gene, and its scrapie isoform (PrPSc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrPC has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrPC forms designated PrPC-I and -II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu2+ ion affinity chromatography. PrPC-II appears to be generated from PrPC-I by limited proteolysis of the N-terminus. Fractions enriched for PrPC-I were purified further by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Greater than 90% of the final product migrated as a broad band of M(r) 33-35 kDa as judged by silver staining after SDS-PAGE. Digestion of PrPC-I with peptide-N-glycosidase (PNGase) compressed the band and shifted its mobility giving an M(r) of 27 kDa. The protocol described should be amenable to large-scale preparation of PrPC, enabling physical comparisons of PrPC and PrPSc.

Scrapie prions are composed largely, if not entirely, of PrPSc molecules. The prion isolates Sc237 and 139H exhibit markedly different incubation times in Syrian, Armenian, and Chinese hamsters, as well as in transgenic (Tg) 81 mice expressing Syrian hamster PrP (SHaPrP). Repassage of prions from transgenic mice or Chinese hamsters into Syrian hamsters revealed that the original properties of the prion isolates are retained. When Syrian hamsters were first inoculated with 139H prions and subsequently challenged with Sc237 prions, the incubation period was determined by the faster Sc237 isolate. Regional mapping studies demonstrated different kinetics and patterns of PrPSc accumulation for Sc237 and 139H prions in the brains of Syrian hamsters as well as Tg(SHaPrP)7 mice. That distinct prion isolates induce different region-specific accumulations of PrPSc in brain suggests a novel mechanism for propagation of isolates whereby they replicate in particular sets of neurons. The prion isolates could be targeted to specific CNS cells by differing conformations of PrPSc, post-translational modifications of PrPSc such as Asn-linked glycosylation, or an as yet undetected macromolecule complexed with PrPSc in the prion.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.