The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Conformational change, aggregation and fibril formation induced by detergent treatments of cellular prion protein

The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.     

Highly promiscuous nature of prion polymerization

The primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.     

Identification of cellular proteins binding to the scrapie prion protein

The scrapie prion protein (PrPSc) is an abnormal isoform of the cellular protein PrPc. PrPSc is found only in animals with scrapie or other prion diseases. The invariable association of PrPSc with infectivity suggests that PrPSc is a component of the infectious particle. In this study, we report the identification of two proteins from hamster brain of 45 and 110 kDa (denoted PrP ligands Pli 45 and Pli 110) which were able to bind to PrP 27-30, the protease-resistant core of PrPSc on ligand blots. Pli 45 and Pli 110 also bound PrPC. Both Pli's had isoelectric points of approximately 5. The dissociation rate constant of the Pli 45/PrP 27-30 complex was 3 x 10(-6) s-1. Amino acid and protein sequence analyses were performed on purified Pli 45. Both the composition and the sequence were almost identical with those predicted for mouse glial fibrillary acidic protein (GFAP). Furthermore, antibodies to Pli 45 reacted with recombinant GFAP. The identification of proteins which interact with the PrP isoforms in normal and diseased brain may provide new insights into the function of PrPC and into the molecular mechanisms underlying prion diseases.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.     

Formation of protease-resistant prion protein in cell-free systems

In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.     

Efficient conversion of normal prion protein (PrP) by abnormal hamster PrP is determined by homology at amino acid residue 155

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.     

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.     

In vitro conversion of mammalian prion protein into amyloid fibrils displays unusual features

The "protein only" hypothesis of prion propagation postulates that the abnormal isoform of the prion protein, PrP(Sc), acts as a causative and transmissible agent of prion disease. In attempt to reconstitute prion infectivity in vitro, we previously developed a cell-free conversion protocol for generating amyloid fibrils from a recombinant prion protein encompassing residues 89-231 (rPrP 89-230) [Baskakov et al. (2002) J. Biol. Chem. 277, 21140]. When inoculated into transgenic mice, these amyloid fibrils induced prion disease, which can be efficiently transmitted to both wild-type and transgenic mice [Legname et al. (2004) Science 305, 673]. Here we show that the polymerization of rPrPs into the fibrils displays a number of distinctive kinetic features that are not typical for polymerization by other amyloidogenic polypeptides. Specifically, the lag phase of polymerization showed only modest dependence on protein concentration, and the conversion reaction displayed a dramatic volume-dependent threshold effect. To explain these unique kinetic features, we proposed that the conversion reaction is regulated by the dynamics between the rates of multiplication and deactivation of self-propagating fibrillar isoforms. Our further studies demonstrated that surface-dependent sorption of fibrillar isoforms is responsible for their deactivation in vitro, while fibril fragmentation seems to account for the multiplication of the active centers of polymerization. Our findings support the hypothesis that development of prion disease is controlled by a fine dynamic balance between self-propagation and clearance/deactivation of PrP(Sc).     

Methionine oxidation interferes with conversion of the prion protein into the fibrillar proteinase K-resistant conformation

In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease.     

Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.     

Phosphorylated human tau associates with mouse prion protein amyloid in scrapie-infected mice but does not increase progression of clinical disease

Tauopathies are a family of neurodegenerative diseases in which fibrils of human hyperphosphorylated tau (P-tau) are believed to cause neuropathology. In Alzheimer disease, P-tau associates with A-beta amyloid and contributes to disease pathogenesis. In familial human prion diseases and variant CJD, P-tau often co-associates with prion protein amyloid, and might also accelerate disease progression. To test this latter possibility, here we compared progression of amyloid prion disease in vivo after scrapie infection of mice with and without expression of human tau. The mice used expressed both anchorless prion protein (PrP) and membrane-anchored PrP, that generate disease associated amyloid and non-amyloid PrP (PrPSc) after scrapie infection. Human P-tau induced by scrapie infection was only rarely associated with non-amyloid PrPSc, but abundant human P-tau was detected at extracellular, perivascular and axonal deposits associated with amyloid PrPSc. This pathology was quite similar to that seen in familial prion diseases. However, association of human and mouse P-tau with amyloid PrPSc did not diminish survival time following prion infection in these mice. By analogy, human P-tau may not affect prion disease progression in humans. Alternatively, these results might be due to other factors, including rapidity of disease, blocking effects by mouse tau, or low toxicity of human P-tau in this model.     

The dominant-negative effect of the Q218K variant of the prion protein does not require protein X

Previous studies identified several single-point mutants of the prion protein that displayed dominant-negative effects on prion replication. The dominant-negative effect was assumed to be mediated by protein X, an as-yet-unknown cellular cofactor that is believed to be essential for prion replication. To gain insight into the mechanism that underlies the dominant-negative phenomena, we evaluated the effect of the Q218K variant of full-length recombinant prion protein (Q218K rPrP), one of the dominant-negative mutants, on cell-free polymerization of wild-type rPrP into amyloid fibrils. We found that both Q218K and wild-type (WT) rPrPs were incorporated into fibrils when incubated as a mixture; however, the yield of polymerization was substantially decreased in the presence of Q218K rPrP. Furthermore, in contrast to fibrils produced from WT rPrP, the fibrils generated in the mixture of WT and Q218K rPrPs did not acquire the proteinase K-resistant core of 16 kDa that was shown previously to encompass residues 97-230 and was similar to that of PrP(Sc). Our studies demonstrate that the Q218K variant exhibits the dominant-negative effect in cell-free conversion in the absence of protein X, and that this effect is, presumably, mediated by physical interaction between Q218K and WT rPrP during the polymerization process.     

Atypical effect of salts on the thermodynamic stability of human prion protein

Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below approximately 50 mm), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90-231 and huPrP122-231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144-Arg148 and/or Asp147-Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.     

Factors affecting interactions between prion protein isoforms

Interactions between normal, protease-sensitive prion protein (PrP-sen or PrP(C)) and its protease-resistant isoform (PrP-res or PrP(Sc)) are critical in transmissible spongiform encephalopathy (TSE) diseases. To investigate the propagation of PrP-res between cells we tested whether PrP-res in scrapie brain microsomes can induce the conversion of PrP-sen to PrP-res if the PrP-sen is bound to uninfected raft membranes. Surprisingly, no conversion was observed unless the microsomal and raft membranes were fused or PrP-sen was released from raft membranes. These results suggest that the propagation of infection between cells requires transfer of PrP-res into the membranes of the recipient cell. To assess potential cofactors in PrP conversion, we used cell-free PrP conversion assays to show that heparan sulphate can stimulate PrP-res formation, supporting the idea that endogenous sulphated glycosaminoglycans can act as important cofactors or modulators of PrP-res formation in vivo. In an effort to develop therapeutics, the antimalarial drug quinacrine was identified as an inhibitor of PrP-res formation in scrapie-infected cell cultures. Confirmation of the latter result by others has led to the initiation of human clinical trials as a treatment for Creutzfeldt-Jakob disease. PrP-res formation can also be inhibited using a variety of other types of small molecule, specific synthetic PrP peptides, and an antiserum directed at the C-terminus of PrP-sen. The latter results help to localize the sites of interaction between PrP-sen and PrP-res. Disruption of those interactions with antibodies or peptidomimetic drugs may be an attractive therapeutic strategy. The likelihood that PrP-res inhibitors can rid TSE-infected tissues of PrP-res would presumably be enhanced if PrP-res formation were reversible. However, our attempts to measure dissociation of PrP-sen from PrP-res have failed under non-denaturing conditions. Finally, we have attempted to induce the spontaneous conversion of PrP-sen into PrP-res using low concentrations of detergents. A conformational conversion from alpha-helical monomers into high-beta-sheet aggregates and fibrils was induced by low concentrations of the detergent sarkosyl; however, the aggregates had neither infectivity nor the characteristic protease-resistance ofPrP-res.     

Copper(II) inhibits in vitro conversion of prion protein into amyloid fibrils

In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu(2+) on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu(2+) inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for Cu(2+), and less so for Zn(2+), while Mn(2+) had no effect on the conversion. Cu(2+)-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu(2+)-binding capacity. Using rPrP 89-230, we found that Cu(2+)-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu(2+) inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu(2+) also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu(2+) induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu(2+) induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu(2+) in the pathogenesis of prion diseases is complex. Because Cu(2+) may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.     

Branched chain mechanism of polymerization and ultrastructure of prion protein amyloid fibrils

The discovery of prion disease and the establishment of the protein only hypothesis of prion propagation raised substantial interest in the class of maladies referred to as conformational diseases. Although significant progress has been made in elucidating the mechanisms of polymerization for several amyloidogenic proteins and peptides linked to conformational disorders and solving their fibrillar 3D structures, studies of prion protein amyloid fibrils and their polymerization mechanism have proven to be very difficult. The present minireview introduces the mechanism of branched-chain reaction for describing the peculiar kinetics of prion polymerization and summarizes our current knowledge about the substructure of prion protein amyloid fibrils.     

Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state.

In the transmissible spongiform encephalopathies, normal prion protein (PrP-sen) is converted to a protease-resistant isoform, PrP-res, by an apparent self-propagating activity of the latter. Here we describe new, more physiological cell-free systems for analyzing the initial binding and subsequent conversion reactions between PrP-sen and PrP-res. These systems allowed the use of antibodies to map the sites of interaction between PrP-sen and PrP-res. Binding of antibodies (alpha219-232) to hamster PrP-sen residues 219-232 inhibited the binding of PrP-sen to PrP-res and the subsequent generation of PK-resistant PrP. However, antibodies to several other parts of PrP-sen did not inhibit. The alpha219-232 epitope itself was not required for PrP-res binding; thus, inhibition by alpha219-232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP-sen structure. The selectivity of the binding reaction was tested by incubating PrP-res with cell lysates or culture supernatants. Only PrP-sen was observed to bind PrP-res. This highly selective binding to PrP-res and the localized nature of the binding site on PrP-sen support the idea that PrP-sen serves as a critical ligand and/or receptor for PrP-res in the course of PrP-res propagation and pathogenesis in vivo.     

Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.