Purification and structural studies of a major scrapie prion protein

Scrapie is a degenerative, neurological disorder caused by a slow infectious agent or prion. Extensively purified preparations of prions were denatured by boiling in sodium dodecyl sulfate and the major protein component (PrP 27-30) was isolated by preparative HPLC size exclusion chromatography after proteinase K digestion. The purified PrP 27-30 molecules were not infectious. Ultraviolet absorption spectra of purified PrP 27-30 demonstrated the absence of covalently linked polynucleotides. Amino acid composition studies showed that PrP 27-30 contains at least 17 naturally occurring amino acids. A single N-terminal amino acid sequence for PrP 27-30 was obtained; the sequence is N-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys and it does not share homology with any known proteins. The same amino acid sequence was found when an extensively purified preparation of prions aggregated into rods and containing approximately 10(9.5) ID50 U/ml was sequenced directly. Knowledge of the amino acid sequence should permit determination of the genetic origin and replication mechanism of prions.     

Characterization of prion proteins with monospecific antisera to synthetic peptides

The prion protein (PrP) 27-30 is the major macromolecular component in highly purified preparations of prions derived from scrapie-infected hamster brain. Immunoblotting studies demonstrated that this protein is generated by partial protease digestion of a larger precursor (PrPSc) with an apparent Mr of 33 to 35 kDa, and that a protease-sensitive cellular PrP isoform, designated PrPC, is present in normal hamster brain. To characterize the relationships among these proteins, ELISA and immunoblotting studies were undertaken with rabbit antisera raised against three synthetic PrP peptides. All three antisera were found to specifically react with the prion proteins, and failed to identify other lower or higher m.w. PrP proteins. Our results provide evidence that the primary structures of PrP 27-30, PrPSc, and PrPC are related; this conclusion supports molecular cloning studies indicating that these proteins are encoded by the same chromosomal gene.     

Antisera to scrapie-associated fibril protein and prion protein decorate scrapie-associated fibrils.

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.     

A cellular gene encodes scrapie PrP 27-30 protein

A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.     

Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins.

Scrapie and Creutzfeldt-Jakob disease are transmissible, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrPSc, encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrPC. We established clonal cell lines which support the replication of mouse scrapie or Creutzfeldt-Jakob disease prions. Mouse neuroblastoma N2a cells were exposed to mouse scrapie prions and subsequently cloned. After limited proteinase K digestion, three PrP-immunoreactive proteins with apparent molecular masses ranging between 20 and 30 kilodaltons were detected in extracts of scrapie-infected N2a cells by Western (immuno-) blotting. The authenticity of these PrPSc molecules was established by using monospecific antiserum raised against a synthetic peptide corresponding to a portion of the prion protein. Those clones synthesizing PrPSc molecules possessed scrapie prion infectivity as measured by bioassay; clones without PrPSc failed to demonstrate infectivity. Detection of PrPSc molecules in scrapie-infected N2a cells supports the contention that PrPSc is a component of the infectious scrapie particle and opens new approaches to the study of prion diseases.     

Properties of scrapie prion protein liposomes

Purified scrapie prions contain one identifiable macromolecule, PrP 27-30, which polymerizes into rod-shaped amyloids. The rods can be dissociated with retention of scrapie infectivity upon incorporation of PrP 27-30 into detergent-lipid-protein complexes (DLPC) as well as liposomes. As measured by end-point titration, scrapie infectivity was increased greater than 100-fold upon dissociating the rods into liposomes. The incorporation of PrP 27-30 into liposomes was demonstrated by immunoelectron microscopy using colloidal gold. Detergent extraction of prion liposomes followed by chloroform/methanol extraction resulted in the reappearance of rods, indicating that this process is reversible. Scrapie prion infectivity in rods and liposomes was equally resistant to inactivation by irradiation at 254 nm and was unaltered by exposure to nucleases. A variety of lipids used for producing DLPC and liposomes did not alter infectivity. Fluorescently labeled PrP 27-30 in liposomes was used to study its entry into cultured cells. Unlike the rods which remained as large fluorescent extracellular masses, the PrP 27-30 in liposomes rapidly entered the cells and was seen widely distributed within the interior of the cell. PrP 27-30 is derived by limited proteolysis from a larger protein designated PrP(Sc) which is membrane bound. PrP(Sc) in membrane fractions was solubilized by incorporation in DLPC, thus preventing its aggregation into amyloid rods. The functional solubilization of scrapie prion proteins in DLPC and liposomes offers new approaches to the study of prion structure and the mechanism by which they cause brain degeneration.     

Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity.

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.     

Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.     

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.     

Molecular characteristics of the major scrapie prion protein

A major protein was identified that purifies with the scrapie agent extracted from infected hamster brains. The protein, designated PrP 27-30, was differentiated from other proteins in purified fractions containing the scrapie agent by its microheterogeneity (Mr 27000-30000) and its unusual resistance to protease digestion. PrP 27-30 was found in all fractions enriched for scrapie prions by discontinuous sucrose gradient sedimentation or sodium dodecyl sarcosinate-agarose gel electrophoresis. It is unlikely that PrP 27-30 is a pathologic product because it was found in fractions isolated from the brains of hamsters sacrificed prior to the appearance of histopathology. If PrP 27-30 is present in normal brain, its concentration must be 100-fold lower than that found in equivalent fractions from scrapie-infected hamsters. Three protease-resistant proteins similar to PrP 27-30 were found in fractions obtained by discontinuous sucrose gradient sedimentation of scrapie-infected mouse brain. These proteins were not evident in corresponding fractions prepared from normal mouse brain. One-dimensional peptide maps comparing PrP 27-30 and normal hamster brain proteins of similar molecular weight demonstrated that PrP 27-30 has a primary structure which is distinct from these normal proteins. Heating substantially purified scrapie fractions to 100 degrees C in sodium dodecyl sulfate inactivated the prion and rendered PrP 27-30 susceptible to protease digestion. Though the scrapie agent appears to be hydrophobic, PrP 27-30 remained in the aqueous phase after extraction with organic solvents, indicating that it is probably not a proteolipid. PrP 27-30 is the first structural component of the scrapie prion to be identified.