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Interactions and conversions of prion protein isoforms
Abstract
The properties of amyloidogenic protease-resistant form (PrP-res) have failed attempts to determine its high resolution structure. Nonetheless, low resolution analyses have shown that PrP-res retains significant α-helical content, but has much higher β-sheet content than PrPC. Biochemical analyses indicate that within its ordered aggregate, PrPSc molecules have, at least, three distinguishable folding domains. Under physiologically compatible conditions, PrP-res can also induce the conversion of PrP-sen to a protease resistant state reminiscent of PrPSc. This conversion reaction is highly species- and strain-specific, as is PrP-res formation in vivo. Heterologous PrP-sen molecules from one species can bind to PrP-res of another and interfere with the conversion of homologous PrP-sen molecules. It is not known whether the cell-free conversion reaction, or any other in vitro manipulation of PrP-sen, generates infectivity.
... 20 No. 22 pp. 6236±6245, 2001 Caughey et al., 2001 ...
... ). Certainly conformational variation exists (reviewed in Caughey et al., 2001; Collinge, 2001), but whether it is the molecular basis of strains is unproven. [PSI + ] strains are classi®ed by the extent of their translational termination defects and by how stable they are. ...
... By comparison, conversion by the mammalian PrP is generally much less ef®cient than conversion by Sup35 PSI+ . It is usually limited to sub-stoichiometric yields in vitro (Caughey, 2000; Wong et al., 2001), although the yield can be substantially improved with repeated cycles of sonication (Saborio et al., 2001). This difference appears to be biologically relevant since yeast can mitotically divide in as little as 90 min, whereas neuronal cells are not mitotic. ...
Yeast prions are protein-based genetic elements that produce phenotypes through self-perpetuating changes in protein conformation. For the prion [PSI(+)] this protein is Sup35, which is comprised of a prion-determining region (NM) fused to a translational termination region. [PSI(+)] strains (variants) with different heritable translational termination defects (weak or strong) can exist in the same genetic background. [PSI(+)] variants are reminiscent of mammalian prion strains, which can be passaged in the same mouse strain yet have different disease latencies and brain pathologies. We found that [PSI(+)] variants contain different ratios of Sup35 in the prion and non-prion state that correlate with different translation termination efficiencies. Indeed, the partially purified prion form of Sup35 from a strong [PSI(+)] variant converted purified NM much more efficiently than that of several weak variants. However, this difference was lost in a second round of conversion in vitro. Thus, [PSI(+)] variants result from differences in the efficiency of prion-mediated conversion, and the maintenance of [PSI(+)] variants involves more than nucleated conformational conversion (templating) to NM alone.
... Although TSE diseases may be sporadic, genetic, or infectious in origin, it is commonly argued that, in each circumstance, the disease propagates itself through a self-catalytic mechanism involving existing multimers of PrP-res. This idea is supported by experiments that show that PrP-res can induce the conversion of native PrP into PrP-res-like aggregates in vitro (9,10). However, the precise molecular mechanism of the conversion between the normal and pathogenic forms of prion proteins remains poorly understood. ...
... Cell-free Conversion Reaction-PrP-res from 263K scrapie-infected hamsters was prepared using a revision of the Bolton et al. method (25) and was stored in 0.5% sulfobetaine (Calbiochem, La Jolla, CA, Zwittergent [3][4][5][6][7][8][9][10][11][12][13][14] in phosphate-buffered saline as described previously (9,20). Crude normal and 263K-infected hamster brain microsomes were prepared as described previously (26). ...
... Because the above conversion conditions (especially the presence of GdnHCl) may have blocked the ionic interactions necessary for the presence of the helix 1 salt bridges, conversion reactions were performed without detergents or denaturants as described recently (10). This method uses a crude scrapie brain microsome fraction containing PrP-res to drive conversion. ...
A key event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of PrP-sen to PrP-res. Morrissey and Shakhnovich (Morrissey, M. P., and Shakhnovich, E. I. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11293-11298) proposed that the conversion mechanism involves critical interactions at helix 1 (residues 144-153) and that the helix is stabilized on PrP-sen by intra-helix salt bridges between two aspartic acid-arginine ion pairs at positions 144 and 148 and at 147 and 151, respectively. Mutants of the hamster prion protein were constructed by replacing the aspartic acids with either asparagines or alanines to destabilize the proposed helix 1 salt bridges. Thermal and chemical denaturation experiments using circular dichroism spectroscopy indicated the overall structures of the mutants are not substantially destabilized but appear to unfold differently. Cell-free conversion reactions performed using ionic denaturants, detergents, and salts (conditions unfavorable to salt bridge formation) showed no significant differences between conversion efficiencies of mutant and wild type proteins. Using conditions more favorable to salt bridge formation, the mutant proteins converted with up to 4-fold higher efficiency than the wild type protein. Thus, although spectroscopic data indicate the salt bridges do not substantially stabilize PrP-sen, the cell-free conversion data suggest that Asp-144 and Asp-147 and their respective salt bridges stabilize PrP-sen from converting to PrP-res.
... PrP C and PrP Sc do not appear to differ systematically in covalent structure (Baldwin 2001); however, they can be discriminated in several other ways (reviewed in Caughey et al. 2001). For instance, PrP C is fully digested by proteinase K (PK), whereas PK removes roughly the N-terminal third of the ∼210 total residues from most types of PrP-res molecules (Hope et al. 1986, Oesch et al. 1985, Hope et al. 1988). ...
... PrP C is soluble in mild detergents, whereas PrP-res is much less soluble and is found in amorphous aggregates, amyloid fibril-like structures, or bead-like clusters in vitro (Merz et al. 1981, Prusiner et al. 1983, Diringer et al. 1983, Wille et al. 2002). While PrP C is highly alpha-helical, PrP Sc has a preponderance of β-sheet (reviewed in Wuthrich & Riek 2001 and Caughey et al. 2001). PrP-res is generally associated with TSE infectivity, while PrP C is not. ...
... can be formed from a single PrP polypeptide sequence, as evidenced by a variety of TSE strain–dependent spectral, biochemical, and immunological differences in PrP-res types isolated from a single host species (reviewed in Caughey et al. 2001). Striking strain-dependent differences in the spatial distribution of PrP-res accumulation in brain tissue are also observed, which, in turn, often coincide with differences in the pattern of neuropathological lesions (Bruce et al. 1989, DeArmond et al. 1993, Jeffrey et al. 2001 ). ...
Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.
... This process has been modeled in cell-free reactions (Kocisko et al., 1994; Horiuchi et al., 1999; Saborio et al., 2001). However, no in vitro-generated PrP-res has been shown to be infectious (Hill et al., 1999; Caughey et al., 2001). Although the mechanism by which PrP-sen is converted to PrP-res in TSE-infected cells and in vivo is not clear, data from cell-free reactions suggest this process is akin to autocatalytic polymerization (reviewed in Caughey et al., 2001). ...
... However, no in vitro-generated PrP-res has been shown to be infectious (Hill et al., 1999; Caughey et al., 2001). Although the mechanism by which PrP-sen is converted to PrP-res in TSE-infected cells and in vivo is not clear, data from cell-free reactions suggest this process is akin to autocatalytic polymerization (reviewed in Caughey et al., 2001). Some of our knowledge of the conversion process is derived from studies of neuronal cell lines persistently infected with scrapie (ScN2a cells). ...
Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6--7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells.
... Although they are highly heterogeneous, all these diseases are associated with deposition and accumulation of a pathological and infectious scrapie prion protein termed prion or PrP Sc in the central nervous system [1]. PrP Sc derives from its cellular conformer PrP C through a conformational transition from a α-helix structure into a β-sheet structure [2,3]. The structural transition of PrP C confers altered physicochemical properties to PrP Sc with partial resistance to proteinase K (PK) digestion, insolubility in detergents and propensity to form aggregates. Indeed, comparing PrP C and ...
Prion is an infectious protein (PrPSc) that is derived from a cellular glycoprotein (PrPC) through a conformational transition and associated with a group of prion diseases in animals and humans. Characterization of proteinase K (PK)-resistant PrPSc by western blotting has been critical to diagnosis and understanding of prion diseases including Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease in humans. However, formation as well as biochemical and biological properties of the glycoform-selective PrPSc in variably protease-sensitive prionopathy (VPSPr) remain poorly understood. Here we reveal that formation of the ladder-like PrPSc in VPSPr is a PK-dependent two-step process, which is enhanced by basic pH. Two sets of PrPSc fragments can be identified with antibodies directed against an intermediate or a C-terminal domain of the protein. Moreover, antibodies directed against specific PrP glycoforms reveal faster electrophoretic migrations of PrP fragments mono-glycosylated at residue 181 and 197 in VPSPr than those in sporadic CJD (sCJD). Finally, RT-QuIC assay indicates that PrPSc-seeding activity is lower and its lag time is longer in VPSPr than in sCJD. Our results suggest that the glycoform-selective PrPSc in VPSPr is associated with altered glycosylation, resulting in different PK-truncation and aggregation seeding activity compared to PrPSc in sCJD.
... 122 Other studies show that GPI-anchor on PrP Sc allows the formation of smaller oligomeric assemblies that have an enhanced seeding activity, neurotoxicity, and infectivity while limiting the formation of large and stable aggregates, as amyloid plaques. 123,124 More recently, Marshall and coworkers have shown that GPI anchoring and the localization of PrP C to lipid rafts are crucial to the ability of PrP C to propagate as a prion. 125 However, when PrP C is targeted to lipid rafts, its GPI anchor is able to gain information about its subcellular trafficking, indeed, directs it to lipid rafts where it misfolds and an efficient formation of scrapie prion occurs. ...
Cellular prion protein (PrPC) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The precise function of PrPC remains elusive but may depend upon its cellular localization. PrPC misfolds to a pathogenic isoform PrPSc, the causative agent of neurodegenerative prion diseases. Nonetheless some forms of prion disease develop in the apparent absence of infectious PrPSc, suggesting that molecular species of PrP distinct from PrPSc may represent the primary neurotoxic culprits. Indeed, in some inherited cases of human prion disease, the predominant form of PrP detectable in the brain is not PrPSc but rather CtmPrP, a transmembrane form of the protein. The relationship between the neurodegeneration occurring in prion diseases involving PrPSc and that associated with CtmPrP remains unclear. However, the different membrane topology of the PrP mutants, as well as the presence of the GPI anchor, could influence both the function and the intracellular localization and trafficking of the protein, all being potentially very important in the pathophysiological mechanism that ultimately causes the disease.
... (puffer fish, Takifugu rubripes) (Oidtmann et al., 2003, Rivera-Milla et al., 2003, Suzuki et al., 2002, (Atlantic salmon, Salmo salar) (Oidtmann et al., 2003), (Tetraodon nigroviridis) (Premzl et al., 2004), (Japanese sea bass, Lateolabrax japonicus) (Liao et al., 2005), zebrafish (Danio rerio) (Cotto et al., 2005), (Japanese flounder, Paralichthys olivaceus) (Liao et al., 2005) (Caughey et al., 2001;Prusiner et al., 1990), PrP homology . PrP , (Rivera-Milla et al., 2003). ...
Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are a threat to food safety and to human and animal health. Variant Creutzfeldt-Jakob disease (vCJD) in humans is caused by the consumption of meat contaminated with bovine spongiform encephalopathy (BSE, mad cow disease). The BSE epidemic in the United Kingdom was shown to be related with the extensive use of BSE-contaminated meat-and-bone meal (MBM) and bovine offal. Many countries worldwide use MBM, as well as meat from cows, for aquaculture feed. This raises concerns about the safety of farmed fish, a major protein source for humans. The present work reviews recent studies on fish prion protein and the transmissibility of mammalian prion agents to fish, providing insights into the future direction of fish prion research.
... 1.1 entsprechen, konnten PrP-Aggregate produziert werden, die ein wesentliches Hauptcharakeristikum von PrP Sc teilen. Sie sind im Vergleich zu PrP C wesentlich resistenter gegenüber [Prusiner, 1991, Eigen, 1996 der Verdauung mit Proteinase K. Es konnte aber keine Entstehung neuer Infektiösität belegt werden [Caughey et al., 2001]. ...
Diese Arbeit befasst sich mit der Kinetik der Fehlfaltung und Aggregation von Proteinen. Anhand dreier Beispiele, der Phosphoglyceratkinase (PGK) aus Hefe, einer Variante von Barstar und des Prion-Proteins des Syrischen Hamsters (SHaPrP(90-232)) wurde insbesondere die Kinetik der Bildung von Amyloidfibrillen und deren kinetischer Vorläuferstrukturen mittels dynamischer und statischer Lichtstreuung, Circulardichroismus, Infrarotspektroskopie, Elektronenmikroskopie und teilweise analytischer Chromatographie untersucht. Die Kinetiken wurden mit Konzepten der Aggregationstheorie von Kolloiden und der chemischen Kinetik beschrieben. Die Modellierung der Kinetiken weist ausgehend von der monomeren PGK bei pH 2 und 190 mM NaCl auf eine zweistufige Reaktionskaskade, bestehend aus irreversiblen, bimolekularen Elementarschritten hin. Während der ersten Stufe wird ein engverteiltes Ensemble von Oligomeren mit einer mittleren Masse von 10 Monomeren und wesentlichen Anteilen an beta-Faltblattstrukturen gebildet. Die Protofibrillen entstehen durch die Vereinigung der strukturell polaren Oligomere, die durch die erste Reaktionsstufe bereitgestellt werden und als kritische Oligomere bezeichnet werden. Die gefundene Kopplung des Wachstums der intermediären Zustände und die Zunahme der beta-Faltblattstruktur kann innerhalb eines verallgemeinerten Diffusions-Kollisions-Modells interpretiert werden, bei dem die beta-Stränge durch intermolekulare Wechselwirkungen stabilisiert werden. Die Fehlfaltung und Aggregation des SHaPrP(90-232) bei pH 4.2 und 1 M GuHCl und geeigneten Zusätzen an Salz zeigt einen augenscheinlichen Zweizustandsübergang mit hoher Reaktionsordnung ( >2.5) zwischen dem monomeren, alpha-helikalen Ausgangszustand und einem beta-faltblattreichen, ringförmigen Oktamer. Die Progresskurven der Umwandlung der Sekundärstruktur lassen sich mit dem Zeitverlauf einer bimolekularen Reaktion anpassen. Das Oktamer bildet bei hohen eingesetzten Proteinkonzentrationen Multimere. Auf sehr langen Zeitskalen setzt die Bildung von Protofibrillen ein. Das kritische Oktamer stellt die Vorstufe der nachgeschalteten Wachstumsphänomene dar. Unter geeigneten Umgebungsbedingungen kann der nicht-nativ, partiell gefaltete Zustand von Barstar bei niedrigem pH (A-Zustand) in einem zweistufigen Prozess erst in Protofibrillen und anschlie"send in reife Amyloidfibrillen konvertiert werden. Zur Aktivierung der Konversion des oligomeren A-Zustandes (mittlere Masse von 16 Monomeren) sind moderate Ionenstärken ([NaCl]>0) und erhöhte Temperaturen (T>50°C) notwendig. Die Bildung der Protofibrillen ist unabhängig von der eingesetzten Proteinkonzentration. Bei Raumtemperatur und entsprechender Ionenstärke bilden sich amorphe Aggregate. Dagegen führt die Erhöhung der Temperatur in Abwesenheit von Salz zur Dissoziation des oligomeren A-Zustandes. Alle drei Proteine müssen zur Ausbildung protofibrillärer Strukturen und gegebenenfalls reifer Fibrillen oligomere Zustände mit partiell gefalteter Konformation einnehmen. Diese kritischen Oligomere sind langlebige Intermediate, die den Dreh- und Angelpunkt für die Bildung nachgeordneter Strukturen darstellen. Die Bildung von Amyloidfibrillen ist somit ein mehrstufiger hierarchischer Strukturbildungsprozess. Die in der Literatur bekannten Modelle der nukleierten Polymerisierung und der nukleierten Konformationskonversion werden dem höchstens in gewissen Teilaspekten gerecht. Die Annahme einer universellen Kinetik der Amyloidbildung kann im Lichte der Ergebnisse dieser Arbeit nicht aufrechterhalten werden. Dagegen scheinen die Zustände des kritischen Oligomers und der Protofibrille als Hierarchiestufen der Amyloidbildung generische Bestandteile des Prozesses zu sein. Die Kinetik der Bildung der verschiedenen Hierarchiestufen weist keine nennenswerten Gemeinsamkeiten zwischen den drei untersuchten Proteinen auf.
... The prion disease mechanism has been considered by mimicking the structural transition and associated fibrillization of PrP in solution using a number of techniques, 3−7,10−21 such as fluorescence, 1,14,15 transmission electron microscopy (TEM), 5,6 atomic force microscopy (AFM), 4,5,7 attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), 8,13,18,19 circular dichroism (CD), 20,21 and so forth. Although these studies have substantially improved the understanding of the pathways of protein aggregation, the molecular mechanism of this remarkable conformational transition still remains elusive so far. ...
Protein aggregation is associated with many "protein deposition diseases". A precise molecular detail of the conformation transitions of such membrane-associated protein structure is critical to understanding disease mechanism and developing effective treatments. One potential model peptide for studying the mechanism of protein deposition diseases is prion protein fragment [118-135] (PrP118-135), which shares homology with the C-terminal domain of the Alzheimer's β-amyloid peptide. In this study, sum frequency generation vibrational spectroscopy (SFG-VS) has been applied to characterize interactions between PrP118-135 and 1-palmitoyl-2-oleoyl-sn- glycero-3- phospho- (1'-rac-glycerol) (POPG) lipid bilayer in situ. The conformation change and orientation of PrP118-135 in lipid bilayer have been determined using SFG spectra with different polarization combination. It was found that low-concentration PrP118-135 adopts predominantly α-helical structure but with tiny β-sheet structure. With PrP118-135 concentration increasing, the molecular number ratio of parallel β-sheet structure increases and reaches 44 % at the concentration of 0.10 mg/mL, indicating the formation of abnormally folded scrapie isoforms. The α-helical structure inserts into lipid bilayer with a tilt angle of 32(o) versus the surface normal, while the β-sheet structure lies down on the lipid bilayer with the tilt and twist angle both of 90(o). The 3300 cm(-1) N-H stretching signals in psp spectra arises from α-helical structure at low PrP concentration and from the β-sheet structure at high PrP concentration. Results from this study will provide an in-depth insight into the early events in the aggregation of PrP in cell membrane.
... The detected lag phase corresponds to the formation of the initial nuclei on which the polymerization or fibril growth would further spontaneously proceed. Seeded protein polymerization is a well-established mechanism for in vivo amyloid fibril formation and underlies prion propagation323334. InFigure 4 , it is shown, the effect of the presence of preformed amyloid Sup35-NM and Ure2p fibrils on the kinetics of fibril formation. ...
Background
Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively.
Results
Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold.
Conclusions
An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.
... During prion propagation, the increase in the level of infectivity is associated in most cases with the accumulation of the misfolded PrP Sc protein, believed to convert PrP C through a pathological conformational rearrangement [13]. Although the mechanism explaining this transconformation remains unclear, two different models have been proposed: nucleation-polymerization and template-assisted conversion [14,15]. Both involve the recruitment of normal prion protein PrP C . ...
So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD), thought to result from the Bovine Spongiform Encephalopathy (BSE) prion agent, have shown Methionine-Methionine (M/M) homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion.
We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs) from three blood donor populations with Methionine-Methionine (M/M), Valine-Valine (V/V) and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism.
These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.
... This process has been modeled in cell-free reactions (Horiuchi et al., 1999Horiuchi et al., , 2001 Kocisko et al., 1994; Saborio et al., 2001 ). The sequenceand strain-specificities of these reactions suggest that such interactions between the normal and abnormal isoforms play important roles in the propagation and pathogenesis of TSE diseases (Caughey et al., 2001). It has previously been reported (Li et al., 2000) that binding of Mabs to the N-terminus of recombinant PrP (in the region of aa 23–145) could block the recognition of another antibody that normally binds to a conformational epitope located within the C terminus. ...
We have characterized the antibody-antigen binding events of the prion protein (PrP) utilizing three new PrP-specific monoclonal antibodies (Mabs). The degree of immunoreactivity was dependent on the denaturation treatment with the combination of heat and SDS resulting in the highest levels of epitope accessibility and antibody binding. Interestingly however, this harsh denaturation treatment was not sufficient to completely and irreversibly abolish protein conformation. The Mabs differed in their PrP epitopes with Mab 08-1/11F12 binding in the region of PrP(93-122), Mab 08-1/8E9 reacting to PrP(155-200) and Mab 08-1/5D6 directed to an undefined conformational epitope. Using normal and infected brains from hamsters, sheep and deer, we demonstrate that the binding of PrP to one Mab triggers PrP epitope unmasking, which enhances the binding of a second Mab. This phenomenon, termed positive immunocooperativity, is specific regarding epitope and the sequence of binding events. Positive immunocooperativity will likely increase immunoassay sensitivity since assay conditions for PrP(Sc) detection does not require protease digestion.
... It leads to an increase in -sheet structure, insolubility, and partial resistance against digestion with proteinase K (3)(4)(5)(6). This conversion has been investigated in vitro, predominantly by using recombinant PrP (recPrP), expressed in Escherichia coli, (7)(8)(9)(10). However, the eucaryotic PrP C is posttranslationally modified, carrying two N-glycosylations and a GPI anchor. ...
Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular β-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity, is thought to be a crucial event in the development of prion diseases.
• FTIR
• membrane anchoring
• prion protein
• protein aggregation
• secondary structure
... Numerous efforts have made to recapitulate the conversion of PrP-sen to PrP-res by exposing recombinant PrP-sen molecules to various treatments that can affect protein conformation (reviewed in Caughey et al. 2001;Glockshuber 2001). High b-sheet, aggregation-prone forms of PrP have been induced by low pH, reduction of the disul®de bond (Mehlhorn et al. 1996;Swietnicki et al. 1997;Hornemann and Glockshuber 1998;Jackson et al. 1999), manganese (Brown et al. 2000), and nucleic acids (Nandi and Leclerc 1999). ...
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.
... If PrP-res is the infectious agent, strain-specific information must be encoded within the PrP-res molecule either structurally or via post-translational modification. Interestingly, TSE strains can be defined by differences in the conformation, glycosylation, protease resistance, and aggregation state of PrP-res (7,(12)(13)(14)(15)(16). Several lines of evidence demonstrate that PrP-res molecules associated with different TSE strains have distinct protein conformations. ...
Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.
... Consistent with this proposal, previous studies have localized residues clustered near the Cterminus in the NMR structure of PrP-sen as potentially contributing to the PrP-res binding site (8,24,(49)(50)(51). When PrP-sen is modeled in a GPI-anchor-directed membrane-bound state (11), it is conceivable that the PrP-res binding region is not readily accessible for interaction with large aggregates of exogenous PrP-res. ...
Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant
membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive
PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted
into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI+) and GPI anchor-deficient (GPI−) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not
detectably altered by glycosylation and was markedly reduced by deletion of residues 34–94. The SCRL-associated PrP molecules
were not removed by treatments with either high salt or carbonate buffer. However, only GPI+ PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one
of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found
to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res
as contained in microsomes. SCRL-bound GPI+ PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated
with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI− PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by
PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.
... The molecular mechanism of the self-propagating PrP C 3 PrP Sc conversion remains enigmatic. Nonetheless, certain clues regarding this mechanism have been provided by the cell-free conversion experiments of Caughey and co-workers (7,8,20,21) and recent studies on conformational transitions of the recombinant prion protein (22)(23)(24). One intriguing finding of these studies is that the conversion of the recombinant PrP to the oligomeric -sheet-rich structure (PrP Sc -like form) is strongly promoted by the presence of common salts (23), suggesting the importance of electrostatic interactions in prion protein folding and stability. ...
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.
... As for familial prion diseases, mutations within the prion protein gene would favor PrP folding in a pathogenic conformation. An important question still to be answered is: are all PrP C isoforms equal substrates for conversion [103,104]? ...
Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.
... Thus, few individuals have (thus far) developed vCJD through the dietary consumption of BSE-infected beef, implying a low infectivity or a long incubation time. However, once infection has crossed the species barrier, the subsequent passage of infection between individuals within a species may then occur readily and evolve more rapidly (Bruce et al, 1994;Caughey et al, 2001). The second barrier applies to the route through which infection enters the body. ...
... PrP C is rich in a-helical structure, monomeric, and susceptible to enzyme digestion, whereas PrP Sc has a large content of b-sheet, forms highly insoluble aggregates , and is partially resistant to proteolytic digestion [2] [3]. It is apparent that a major refolding event underlying the conversion of PrP C to PrP Sc plays a key role in the pathogenesis of prion diseases [4]. However, the molecular details of this conformational transition are not clearly understood. ...
A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.
... The most widely accepted hypothesis is that PrPsc, or a precursor of it, is the infective agent (29); introduced into the organism, the abnormal conformer is thought to recruit and convert the cellular PrP into a likeness of itself. However, the molecular details of PrP conversion, including possible cofactor requirements, are unclear (8). At the cellular level, too, a number of uncertainties remain. ...
During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational
isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is
proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial
cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further
show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that
the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list
of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data
raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further
support for a crucial role of PrP at the cell surface in prion infection of target cells.
... Several continuous lines of cell cultures have been developed that are chronically infected with TSE agents (usually scrapie), and are used to screen the efficacy of drugs and antibodies to reduce the burden of animal-based infectivity studies. Such ex vivo studies are sometimes run in parallel with in vitro tests of normal-to-misfolded protein conversion inhibition (Caughey et al. 2001). ...
The family of illnesses called transmissible spongiform encephalopathies (TSEs), or "prion" diseases, is composed of a small number of human and animal neurodegenerative diseases caused by unique pathogenic agents that are still not fully defined. They are best considered as "protein-misfolding diseases" (together with Alzheimer's disease, Parkinson's disease, and a few other rare examples) resulting from the conversion of a normal body protein into a misfolded amyloid multimer. The pathogenic agents display a unique resistance to conventional disinfection methods and an extraordinary environmental durability, which has led the US Department of Agriculture to designate the causative agent of bovine spongiform encephalopathy as a bioterrorism security threat. In this review, precautions and regulations concerning the handling of TSE agents are discussed in relation to personnel and environmental biosafety.
... The conversion of the PrP C to the PrP Sc is a remarkable molecular event, involving a major refolding of a-helix and random coil in PrP C to b-sheet structure in PrP Sc [8]. Sporadic cases of prion diseases are associated with this extremely rare event of erratic folding of PrP C , whereas in the infectious cases prion conversion appears to occur via a template-assisted mechanism, in which PrP C is refolded by the interaction with PrP Sc [51]. Previous studies have shown that recombinant PrP can be refolded as an a-helical conformation (a-PrP), representing PrP C , which under a variety of conditions can be converted to aggregated and fibrillar forms rich in b-sheet, with physical properties resembling those of PrP Sc [19,20,52]. ...
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.
Prion diseases are progressive neurodegenerative maladies that are characterised by changes in the physical properties and turnover of a glycosylphosphatidyl-inositol (GPI)-linked membrane glycoprotein (cellular prion protein, PrPC). The conversion of the normal, detergent-soluble, proteinase K (PK)- sensitive PrPSc to the abnormal, detergent-insoluble, partially PK-resistant isoform, PrPse is a common feature of all these transmissible encephalophies including scrapie in sheep, Creutzfeldt-Jakob disease (CJD) of humans and bovine spongiform encephalopathy (BSE) of cattle [1]. Direct interaction between PrPC and PrPSc is implicated by in vivo studies and features in current ideas of molecular pathogenesis and transmissibility such as the heterodimer (template-assisted) and nucleation (seed)-dependent conversion models (Fig. 1). In 1994, Caughey and co-workers introduced a new tool for investigating this process by showing that PrPSc isolated from the brains of scrapieaffected animals could induce the conversion of radiolabelled recombinant PrP (rPrP) into a PK-resistant isoform, PrPres, in vitro [1-5].
Fig. 1
Models of PrPSc replication.In the heterodimer (tem-plate-assisted) model, PrPSc exists as a monomer that binds to PrPC, forming a heterodimer and catalysing the conversion of PrPC to PrPSc. The homodimer than splits to give two PrPSc seeds for further conversion of PrPc. In the nucleated (seed)-polymeri-sation model, the conver¬sion of PrPc to PrPSc is reversible and PrPSc is stabilised by aggregation.
Syrian hamster nervous tissue was investigated by FTIR microspectroscopy with conventional and synchrotron infrared light sources. Various tissue structures from the cerebellum and medulla oblongata of scrapie-infected and control hamsters were investigated at a spatial resolution of 50 mum. Single neurons in dorsal root ganglia of scrapie-infected hamsters were analyzed by raster scan mapping at 6 mum spatial resolution. These measurements enabled us to (i) scrutinize structural differences between infected and non-infected tissue and (ii) analyze for the first time the distribution of different protein structures in situ within single nerve cells. Single nerve cells exhibited areas of increased beta-sheet content, which co-localized consistently with accumulations of the pathological prion protein (PrPSc). Spectral data were also obtained from purified, partly proteinase K digested PrPSc isolated from scrapie-infected nervous tissue of hamsters to elucidate similarities/dissimilarities between prion structure in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP90-232, whose in vitro transition from the predominantly a-helical isoform to beta-sheet rich oligomeric structures was also investigated by FTIR spectroscopy.
Prion diseases are fatal neurodegenerative diseases of mammals. They are characterized by the conversion of normal prion protein (PrP) to a misfolded conformational state that accumulates as plaques in the brain. The diagnosis of prion diseases relies on the ability to differentiate between normal PrP and its misfolded, infectious form. This is difficult to accomplish by traditional testing methods, since it requires discerning between conformational states of a protein that is present in both normal and diseased tissue, rather than identifying the appearance of a new protein associated with infection. We wish to design a reporter PrP substrate that may be monitored by fluorescence spectroscopy. After the conversion of normal-PrP to its infectious state, some amino acid residues of PrP will undergo a change in their local solvent environment. We propose to identify these residues by monitoring the fluorescence emission spectrum of a series of mutant 7-AzaTrp-substituted PrP proteins. The 7-AzaTrp fluorescence emission spectrum is both unique compared with normal Trp and exquisitely sensitive to its local environment. This could lead to the development of a rapid, sensitive, and inexpensive technique to detect infectious PrP, based on its ability to bind 7-AzaTrp-substituted PrP, and convert it to the misfolded form.
Basic Properties of Bacterial, Adhesive Surface OrganellesStructure and Function of Pilus ChaperonesStructure and Folding of Pilus SubunitsStructure and Function of Pilus UshersConclusions and OutlookExperimental Protocols
Originally published in: Protein Folding Handbook. Part II. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2
The sections in this article are
Introduction
Prions and the “Protein‐Only” Hypothesis
Models of Pr P Sc Propagation
Properties of Pr P C and Pr P Sc
Three‐dimensional Structure and Folding of Recombinant Pr P
Expression of the Recombinant Prion Protein for Structural and Biophysical Studies
Three‐dimensional Structures of Recombinant Prion Proteins from Different Species and Their Implications for the Species Barrier of Prion Transmission
Solution Structure of Murine Pr P
Comparison of Mammalian Prion Protein Structures and the Species Barrier of Prion Transmission
Biophysical Characterization of the Recombinant Prion Protein
Folding and Stability of Recombinant Pr P
Role of the Disulfide Bond in Pr P
Influence of Point Mutations Linked With Inherited TSE s on the Stability of Recombinant Pr P
Generation of Infectious Prions in vitro : Principal Difficulties in Proving the Protein‐Only Hypothesis
Understanding the Strain Phenomenon in the Context of the Protein‐Only Hypothesis: Are Prions Crystals?
Conclusions
The process by which transmissible spongiform encephalopathy (TSE) agents, or prions, infect cells is unknown. There are also
no effective treatments available for TSE diseases. Studies of cultured cells persistently infected with TSE agents have greatly
contributed to understanding these and many other aspects of TSE disease. New cell lines have been developed to increase the
repertoire of TSE strains that can be investigated in ex vivo models. Candidates for TSE therapeutics have been identified.
Initial events involving the internalization and trafficking of TSE agents and the effect of membranes on the infection process
have been examined. Recent progress in these areas is discussed below, which together illustrate the value of cell culture
models in the study of prion diseases.
Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No
accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 μM VPA. Higher
VPA concentrations (240 and 600 μM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.
Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in beta-sheet.
Prions are infectious proteins, without the need for an accompanying nucleic acid. Nonetheless, there are connections of prions with translation and RNA, which we explore here. Most prions are based on self‐propagating amyloids. The yeast [PSI+] prion is an amyloid of Sup35p, a subunit of the translation termination factor. The normal function of the Sup35p prion domain is in shortening the 3′ polyA of mRNAs and thus in mRNA turnover. The [ ISP ⁺ ] prion is so named because it produces antisuppression, the opposite of the effect of [ PSI ⁺ ]. Another connection of prions with translation is the influence on prion propagation and generation of ribosome‐associated chaperones, the Ssbs, and a chaperone activity intrinsic to the 60S ribosomal subunits. Copyright © 2010 John Wiley & Sons, Ltd.
This article is categorized under: Translation > Translation Mechanisms
Translation > Translation Regulation
Cellular PrP is actively cycled between the cell surface and the endosomal pathway. The exact site and mechanism of conversion from PrP(C) to PrP(Sc) remain unknown. We have previously used recombinant antibodies containing grafts of PrP sequence to identify three regions of PrP(C) (aa23-27, 98-110, and 136-158) that react with PrP(Sc) at neutral pH. To determine if any regions of PrP(C) react with PrP(Sc) at an acidic pH similar to that of an endosomal compartment, we tested our panel of grafted antibodies for the ability to precipitate PrP(Sc) in a range of pH conditions. At pH near or lower than 6, PrP-grafted antibodies representing the octapeptide repeat react strongly with PrP(Sc) but not PrP(C). Modified grafts in which the histidines of the octarepeat were replaced with alanines did not react with PrP(Sc). PrP(Sc) precipitated by the octapeptide at pH 5.7 was able to seed conversion of normal PrP to PrP(Sc) in vitro. However, modified PrP containing histidine to alanine substitutions within the octapeptide repeats was still converted to PrP(Sc) in N2a cells. These results suggest that once PrP has entered the endosomal pathway, the acidic environment facilitates the binding of PrP(Sc) to the octarepeat of PrP(C) by the change in charge of the histidines within the octarepeat.
Fungal prions are fascinating protein-based genetic elements. They alter cellular phenotypes through self-perpetuating changes in protein conformation and are cytoplasmically partitioned from mother cell to daughter. The four prions of Saccharomyces cerevisiae and Podospora anserina affect diverse biological processes: translational termination, nitrogen regulation, inducibility of other prions, and heterokaryon incompatibility. They share many attributes, including unusual genetic behaviors, that establish criteria to identify new prions. Indeed, other fungal traits that baffled microbiologists meet some of these criteria and might be caused by prions. Recent research has provided notable insight about how prions are induced and propagated and their many biological roles. The ability to become a prion appears to be evolutionarily conserved in two cases. [PSI(+)] provides a mechanism for genetic variation and phenotypic diversity in response to changing environments. All available evidence suggests that prions epigenetically modulate a wide variety of fundamental biological processes, and many await discovery.
The conversion of the cellular prion protein into the beta-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C-terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined. Our results show that all these fragments, excepted that strictly encompassing helix 1, have a very high propensity to form structured aggregates with a dominant content of beta-sheet structures.
Prion diseases are associated with a major refolding event of the normal cellular prion protein, PrP(C), where the predominantly alpha-helical and random coil structure of PrP(C) is converted into a beta-sheet-rich aggregated form, PrP(Sc). Under normal physiological conditions PrP(C) is attached to the outer leaflet of the plasma membrane via a GPI anchor, and it is plausible that an interaction between PrP and lipid membranes could be involved in the conversion of PrP(C) into PrP(Sc). Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of beta-PrP to model lipid membranes and compares the structural changes in alpha- and beta-PrP induced upon membrane binding. beta-PrP binds to negatively charged POPG membranes and to raft membranes composed of DPPC, cholesterol, and sphingomyelin. Binding of beta-PrP to raft membranes results in substantial unfolding of beta-PrP. This membrane-associated largely unfolded state of PrP is slowly converted into fibrils. In contrast, beta-PrP and alpha-PrP gain structure with POPG membranes, which instead leads to amorphous aggregates. Furthermore, binding of beta-PrP to POPG has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents, whereas raft vesicles are not destabilized upon binding of beta-PrP.
Tg(PG14) mice express a prion protein (PrP) with a nine-octapeptide insertion associated with a human familial prion disease.
These animals spontaneously develop a fatal neurodegenerative disorder characterized by ataxia, neuronal apoptosis, and accumulation
in the brain of an aggregated and weakly protease-resistant form of mutant PrP (designated PG14spon). Brain homogenates from Tg(PG14) mice fail to transmit disease after intracerebral inoculation into recipient mice, indicating
that PG14spon, although pathogenic, is distinct from PrPSc, the infectious form of PrP. In contrast, inoculation of Tg(PG14) mice with exogenous prions of the RML strain induces accumulation
of PG14RML, a PrPSc form of the mutant protein that is infectious and highly protease resistant. Like PrPSc, both PG14spon and PG14RML display conformationally masked epitopes in the central and octapeptide repeat regions. However, these two forms differ profoundly
in their oligomeric states, with PG14RML aggregates being much larger and more resistant to dissociation. Our analysis provides new molecular insight into an emerging
puzzle in prion biology, the discrepancy between the infectious and neurotoxic properties of PrP.
The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K-resistant form of the prion protein, which has been converted from the endogenous, proteinase K-sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high-throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and the protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a nonisotopic labeling strategy for large-scale screens.
Scrapie is a transmissible spongiform encephalopathy (TSE) and its spread across the intestine of sheep is linked to the biology of intestinal Peyer's patches (PPs). Specialized epithelial cells, M cells, would appear to be the portal of entry for the scrapie agent, PrP(Sc), while lymphoid nodules of PPs become major sites of accumulation of PrP(Sc) as the infection becomes established. Furthermore, evidence suggests that the enteric nervous system supplying the PPs is important for neuroinvasion. The gut-associated lymphoid tissue (GALT) of ruminants shows morphological and functional differences to the GALT of mice and humans. Recent investigations of aging scrapie-affected sheep revealed a substantial network of nerve fibres in the lymphoid nodules of PPs, contradicting the widely held notion that lymphoid nodules are poorly innervated. Advances in the understanding of the pathogenesis of scrapie may be achieved by a deeper appreciation of the development, morphology and function of GALT in small ruminants.
Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment.
Prion diseases, a group of fatal neurodegenerative disorders, are characterized by the presence of the abnormal scrapie isoform of prion protein (PrP(Sc)) in affected brains. A conformational change is believed to convert the normal cellular prion protein into PrP(Sc). Detection of PrP(Sc) for diagnosis and prophylaxis is impaired because available Abs recognizing epitopes on PrP fail to distinguish between PrP(Sc) and normal cellular prion protein. Here, we report that an anti-DNA Ab, OCD4, as well as gene 5 protein, a well established DNA-binding protein, capture PrP from brains affected by prion diseases in both humans and animals but not from unaffected controls. OCD4 appears to immunoreact with DNA (or a DNA-associated molecule) that forms a conformation-dependent complex with PrP in prion diseases. Whereas PrP immunocaptured by OCD4 is largely protease-resistant, a fraction of it remains protease-sensitive. Moreover, OCD4 detects disease-associated PrP >10 times more efficiently than a widely used Ab to PrP. Our finding that anti-DNA Abs and gene 5 protein specifically target disease-associated DNA-PrP complexes in a wide variety of species and disease phenotypes opens new avenues in the study and diagnosis of prion diseases.
Prion diseases are among the most intriguing infectious diseases and are associated with unconventional proteinaceous infectious agents known as prions. Prions seem to lack nucleic acid and propagate by transmission of protein misfolding. The nature of prions and their unique mode of transmission present challenges for early diagnosis of prion diseases. In this article, state-of-the-art prion diagnostic techniques, together with the new strategies that are being used to develop sensitive, early and non-invasive diagnoses for these diseases are reviewed.
The transmissible spongiform encephalopathies could represent a new mode of transmission for infectious diseases--a process more akin to crystallization than to microbial replication. The prion hypothesis proposes that the normal isoform of the prion protein is converted to a disease-specific species by template-directed misfolding. Therapeutic and prophylactic strategies to combat these diseases have emerged from immunological and chemotherapeutic approaches. The lessons learned in treating prion disease will almost certainly have an impact on other diseases that are characterized by the pathological accumulation of misfolded proteins.
Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrP(C)) into a protease-resistant isoform (PrP(res)). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrP(C) constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrP(res) was detected by immunoblotting with the mAb 6H4, which recognizes residues 144-152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109-112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrP(C) was converted into a proteinase-resistant form of PrP(res) in T98G cells.
We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able
to purge PrPSc, the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal
of PrPSc by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of
ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrPSc to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure–activity analysis
revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrPSc effectively from cells. The removal of PrPSc by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with
branched polyamines at acidic pH rendered the PrPSc susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be
useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.
Prion protein consists of an ensemble of glycosylated variants or
glycoforms. The enzymes that direct oligosaccharide processing, and
hence control the glycan profile for any given glycoprotein, are often
exquisitely sensitive to other events taking place within the cell in
which the glycoprotein is expressed. Alterations in the populations of
sugars attached to proteins can reflect changes caused, for example, by
developmental processes or by disease. Here we report that normal
(PrPC) and pathogenic (PrPSc) prion proteins
(PrP) from Syrian hamsters contain the same set of at least 52 bi-,
tri-, and tetraantennary N-linked oligosaccharides, although the
relative proportions of individual glycans differ. This conservation of
structure suggests that the conversion of PrPC into
PrPSc is not confined to a subset of PrPs that contain
specific sugars. Compared with PrPC, PrPSc
contains decreased levels of glycans with bisecting GlcNAc residues and
increased levels of tri- and tetraantennary sugars. This change is
consistent with a decrease in the activity of
N-acetylglucosaminyltransferase III (GnTIII) toward
PrPC in cells where PrPSc is formed and argues
that, in at least some cells forming PrPSc, the
glycosylation machinery has been perturbed. The reduction in GnTIII
activity is intriguing both with respect to the pathogenesis of the
prion disease and the replication pathway for prions.
Prion diseases can be infectious, sporadic and genetic. The infectious forms of these diseases, including bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, are usually characterized by the accumulation in the brain of the transmissible pathogen, an abnormally folded isoform of the prion protein (PrP) termed PrPSc. However, certain inherited PrP mutations appear to cause neurodegeneration in the absence of PrPSc, working instead by favoured synthesis of CtmPrP, a transmembrane form of PrP. The relationship between the neurodegeneration seen in transmissible prion diseases involving PrPSc and that associated with ctmPrP has remained unclear. Here we find that the effectiveness of accumulated PrPSc in causing neurodegenerative disease depends upon the predilection of host-encoded PrP to be made in the ctmPrP form. Furthermore, the time course of PrPSc accumulation in transmissible prion disease is followed closely by increased generation of CtmPrP. Thus, the accumulation of PrPSc appears to modulate in trans the events involved in generating or metabolising CtmPrP. Together, these data suggest that the events of CtmPrP-mediated neurodegeneration may represent a common step in the pathogenesis of genetic and infectious prion diseases.
Infectious scrapie prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) which is designated PrPSc. A chromosomal gene encodes both the cellular prion protein (PrPC) as well as PrPSc. Pulse-chase experiments with scrapie-infected cultured cells indicate that PrPSc is formed by a post-translational process. PrP is translated in the endoplasmic reticulum, modified as it passes through the Golgi, and is transported to the cell surface. Release of nascent PrP from the cell surface by phosphatidylinositol-specific phospholipase C or hydrolysis with dispase prevented PrPSc synthesis. At 18 degrees C, the synthesis of PrPSc was inhibited under conditions that other investigators report a blockage of endosomal fusion with lysosomes. Our results suggest that PrPSc synthesis occurs after PrP transits from the cell surface. Whether all of the PrP molecules have an equal likelihood to be converted into PrPSc or only a distinct subset is eligible for conversion remains to be established. Identifying the subcellular compartment(s) of PrPSc synthesis should be of considerable importance in defining the molecular changes that distinguish PrPSc from PrPC.
Gerstmann-Sträussler-Scheinker disease (GSS) is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. In GSS, the amyloid is immunoreactive to antisera raised against the prion protein (PrP) 27-30, a proteinase K-resistant peptide of 27-30 kDa that is derived by limited proteolysis from an abnormal isoform of a neuronal sialoglycoprotein of 33-35 kDa designated PrPSc. Polyclonal antibodies raised against synthetic peptides homologous to residues 15-40 (P2), 90-102 (P1), and 220-232 (P3) of the amino acid sequence deduced from hamster PrP cDNA were used to investigate immunohistochemically the distribution of PrP and PrP fragments in the brains of two patients from the Indiana kindred of GSS. Two types of anti-PrP-immunoreactive deposits were found: (i) amyloid deposits, which were exclusively labeled by anti-P1 antiserum to residues 90-102 of PrP, and (ii) preamyloid deposits, which were labeled by all anti-PrP antisera but did not exhibit the tinctorial and optical properties of amyloid. The latter appeared as diffuse immunostaining of the neuropil that targeted to areas in which amyloid deposits were most abundant. They were partially resistant to proteinase K digestion and consisted ultrastructurally of amorphous, flaky, electron-dense material. These findings substantiate our previous observation that the major amyloid component in the GSS Indiana kindred is an internal fragment of PrP and indicate that full-length abnormal isoforms of PrP and/or large PrP fragments accumulate in brain regions most affected by amyloid deposition. These findings support the view that in the GSS Indiana kindred a stepwise degradation of PrP occurs in situ in the process of amyloid fibril formation.
The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.
A common feature of scrapie and related transmissible spongiform encephalopathies is the accumulation of an abnormal protease-resistant form of PrP which may be the major component of the infectious agent. While it is known that both the normal (protease-sensitive) PrP and protease-resistant PrP are encoded by the same endogenous gene, the nature of the disease-associated modification of PrP is not understood. To study the cellular events leading to the formation of protease-resistant PrP, we have compared its biosynthesis to that of its normal isoform in scrapie-infected mouse neuroblastoma cells. In pulse-chase labeling experiments, the protease-resistant PrP was synthesized and degraded much more slowly than the normal PrP, suggesting that protease-resistant PrP is made from a protease-sensitive precursor. More significantly, we found that the precursor of protease-resistant PrP was eliminated from intact cells by treatments with phosphatidylinositol-specific phospholipase C and trypsin. This demonstrated that, unlike the protease-resistant PrP itself, the precursor is phospholipase- and protease-sensitive and at least transiently found on the cell surface. By these criteria, the precursor of protease-resistant PrP is indistinguishable from the normal PrP isoform. These results indicate that the conversion of PrP to the protease- and phospholipase-resistant state is a post-translational event that occurs after the precursor reaches the cell surface.
Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.
Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
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-associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt-Jakob disease. A major component of hamster fibrils has been described as a protease-resistant glycoprotein with an apparent mol. wt of 27,000-30,000 (PrP27-30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co-purifying with hamster SAF have mol. wts of 33,000-35,000 (PrP33-35) and 26,000-29,000 (PrP26-29). We find a Lys-Lys-Arg-Pro-Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27-30, and which has recently been predicted to be the N-terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33-35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two-dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33-35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid-like SAF or into association with a non-protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.
During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.
Treatment of scrapie-infected mice with dextran sulphate (DS) 500 resulted in considerably reduced spleen titres over a long period of time. Subsequently, the central nervous system disease was delayed or even prevented during the 350-day period of observation. Both effects increased after multiple injections of the compound. The potency of DS 500 to protect against scrapie was greatest when treatment and infection were carried out simultaneously. Under these conditions the lethality of 500 to 1000 LD50 was reduced to almost zero. Treatment as early as 10 weeks before infection still prolonged the incubation periods. Of several other polyions tested, dextran sulphate 5 and pentosan polysulphate also impaired scrapie pathogenesis.
Prion diseases are transmissible neurodegenerative conditions characterized by the accumulation of protease-resistant forms of the prion protein (PrP), termed PrPres, in the brain. Insoluble PrPres tends to aggregate into amyloid fibrils. The anthracycline 4′-iodo-4′-deoxy-doxorubicin (IDX) binds to amyloid fibrils and induces amyloid resorption in patients with systemic amyloidosis. To test IDX in an experimental model of prion disease, Syrian hamsters were inoculated intracerebrally either with scrapie-infected brain homogenate or with infected homogenate coincubated with IDX. In IDX-treated hamsters, clinical signs of disease were delayed and survival time was prolonged. Neuropathological examination showed a parallel delay in the appearance of brain changes and in the accumulation of PrPres and PrP amyloid.
Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e, removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization, Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP, Within one minute the or-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers, Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.
Prions are unprecedented infectious pathogens that cause a group of
invariably fatal neurodegenerative diseases by an entirely novel
mechanism. Prion diseases may present as genetic, infectious, or
sporadic disorders, all of which involve modification of the prion
protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of
sheep, and Creutzfeldt–Jakob disease (CJD) of humans are among the
most notable prion diseases. Prions are transmissible particles that
are devoid of nucleic acid and seem to be composed exclusively of a
modified protein (PrPSc). The normal, cellular PrP
(PrPC) is converted into PrPSc through a
posttranslational process during which it acquires a high β-sheet
content. The species of a particular prion is encoded by the sequence
of the chromosomal PrP gene of the mammals in which it last replicated.
In contrast to pathogens carrying a nucleic acid genome, prions appear
to encipher strain-specific properties in the tertiary structure of
PrPSc. Transgenetic studies argue that PrPSc
acts as a template upon which PrPC is refolded into a
nascent PrPSc molecule through a process facilitated by
another protein. Miniprions generated in transgenic mice expressing
PrP, in which nearly half of the residues were deleted, exhibit unique
biological properties and should facilitate structural studies of
PrPSc. While knowledge about prions has profound
implications for studies of the structural plasticity of proteins,
investigations of prion diseases suggest that new strategies for the
prevention and treatment of these disorders may also find application
in the more common degenerative diseases.
A group of brain diseases, including BSE (or mad cow disease) and Creutzfeldt-Jacob disease of humans, are transmitted by "prions." In his Perspective, Cheseboro outlines the evidence that prions are composed only of protein (an idea advanced by recent Nobel Prize winner Stanley Prusiner) and the evidence against that hypothesis. He cautions against complacency regarding the obstacles remaining in the field, since, he believes, the central core of the problem remains unresolved.
Epidemiological and clinicopathological studies, allied with pathological prion protein (PrPSc) analysis, strongly support the hypothesis that the human prion disease new variant Creutzfeldt-Jakob disease (vCJD) is causally related to bovine spongiform encephalopathy (BSE)1,2, but considerable controversy remains. Distinct prion strains are distinguished by their biological properties on transmission to laboratory animals and by physical and chemical differences in PrPSc strains. We now find that the biological and molecular transmission characteristics of vCJD are consistent with it being the human counterpart of BSE.
A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrPC) to a protease-resistant form (PrP-res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.
The mechanism of protein-only prion replication is controversial. A detailed mathematical model of prion replication by nucleated polymerisation is developed, and its parameters are estimated from published data. PrP-res decay is around two orders of magnitude slower than PrP-sen decay, a plausible ratio of two parameters estimated from very different experiments. By varying the polymer breakage rate, we reveal that systems of short polymers grow the fastest. Drugs which break polymers could therefore accelerate disease progression. Growth in PrP-res seems slower than growth in infectious titre. This can be explained either by a novel hypothesis concerning inoculum clearance from a newly infected brain, or by the faster growth of compartments containing smaller polymers. The existence of compartments can also explain why prion growth sometimes reaches a plateau. Published kinetic data are all compatible with our mathematical model, so the nucleated polymerisation hypothesis cannot be ruled out on dynamic grounds.
Complexes of the Syrian hamster cellular prion protein (PrPC) and synthetic Syrian hamster PrP peptides were found to mimic many of the characteristics of the scrapie PrP isoform (PrPSc). Either PrPC expressed in chinese hamster ovary (CHO) cells or a C-terminal fragment of 142 residues of recombinant PrP protein (rPrP) produced in Escherichia coli was mixed with an excess of a synthetic 56 amino acid peptide, denoted PrP(90-145). Complex formation required PrPC or rPrP to be destabilized by guanidine hydrochloride (GdnHCl) or urea and PrP(90-145) to be in a coil conformation; it was enhanced by an acidic environment, salt and detergent. If PrP(90-145) was in a β-sheet conformation, then no complexes were formed. While complex formation was rapid, acquisition of protease resistance was a slow process. Amorphous aggregates with a PrPC/PrP(90-145) ratio of 1:1 were formed in phosphate buffer, whereas fibrils with a diameter of ∼10 nm and a PrPC/PrP(90-145) ratio of 1:5 were formed in Tris buffer. The complexes were stable only in the presence of excess peptide in either the coil or β-sheet conformation; they dissociated rapidly after centrifugation and resuspension in buffer without peptide. Neither a peptide having a similar hydrophobicity profile/charge distribution to PrP(90-145) nor a scrambled version, denoted hPrP(90-145) and sPrP(90-145), respectively, were able to induce complex formation. Although hPrP(90-145) could stabilize the PrPC/PrP(90-145) complexes, sPrP(90-145) could not. Studies of PrPC/peptide complexes may provide insights into how PrPC interacts with PrPSc during the formation of a nascent PrPSc molecule and into the process by which PrPC is converted into PrPSc.
The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the β-form of this peptide developed signs of central nervous system dysfunction at ∼360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Sträussler-Scheinker disease. By contrast, eight of eight mice receiving a non-β-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.
A polysaccharide consisting of mainly 1,4-linked glucose units was found associated with prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein (PrP 27-30) exhibiting the ultrastructural and tinctorial properties of amyloid. The polysaccharide differs in composition from the Asn-linked oligosaccharides and the GPI-anchor of the prion protein. Prion rods were prepared from scrapie-infected hamster brains using two different purification protocols. Prolonged digestion of rods with proteinase K reduced PrP by a factor of at least 500, leaving about 10% (w/w) of the sample as an insoluble remnant. Only glucose was obtained by acid hydrolysis of the remnant and methylation analysis showed 80% 1,4-, 15% 1,6- and 5% 1,4,6-linked glucose units. The physical and chemical properties as well as the absence of terminal glucose units indicate a very high molecular mass of the polysaccharide. No evidence was found for covalent bonds between PrP and the polysaccharide. The polysaccharide certainly contributes to the unusual chemical and physical stability of prion rods, acting like a scaffold. A potential structural and/or functional relevance of the polysaccharide scaffold is discussed.
The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.
Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.
Transmissible spongiform encephalopathies (prion diseases), Alzheimer's disease, and other amyloidoses result in the accumulation of certain abnormally stable proteins that are thought by many to play central roles in disease pathogenesis. Using scrapie-infected neuroblastoma cells as a model system, we found that Congo red, an amyloid-binding dye, potently inhibits the accumulation of the scrapie-associated, protease-resistant isoform of protein PrP without affecting the metabolism of the normal isoform. Growth of the cells with submicromolar concentrations of Congo red for 5 days reduced the amount of protease-resistant PrP detected in the cultures by greater than 90%. This activity of Congo red suggests that it selectively disrupts the conversion of PrP to the protease-resistant isoform or destabilizes this isoform once it is made. Potential therapeutic applications of Congo red are discussed.
Prions cause transmissible and genetic neurodegenerative diseases, including scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases of humans. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein, which is encoded by a chromosomal gene. A posttranslational process, as yet unidentified, converts the cellular prion protein into an abnormal isoform. Scrapie incubation times, neuropathology, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neuro-degeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Understanding prion diseases may advance investigations of other neurodegenerative disorders and of the processes by which neurons differentiate, function for decades, and then grow senescent.
The human prion diseases, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler syndrome (GSS), are neurodegenerative diseases that are unique in being both infectious and genetic. Transmission of both diseases and the animal spongiform encephalopathies (for example, scrapie and bovine spongiform encephalopathy) to experimental animals by intracerebral inoculation with brain homogenates is well documented. Despite their experimental transmissibility, missense and insertional mutations in the prion protein gene are associated with both GSS and familial CJD, demonstrating that the human familial cases are autosomal dominant diseases. More than 80% of CJD cases occur sporadically, however, and are not known to be associated with mutations. Here we report that 21 of 22 sporadic CJD cases and a further 19 of 23 suspected sporadic CJD cases are homozygous at the polymorphic amino-acid residue 129; 51% of the normal population are heterozygous at this site. We argue that homozygosity predisposes towards sporadic CJD and that this directly supports the hypothesis that interaction between prion protein molecules underlies the disease process.
A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.
Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.
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.
The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent.
The uniqueness of individuals within any species is perpetuated through genetically programmed characteristics. This controlling influence on phenotypic traits is clearly important in multicellular and single celled organisms down to the simplest infectious agents, viruses and viroids. This phenomenon extends even to the infectious agents of scrapie and the other unconventional slow infections. Indeed, as documented by Bruce and Fraser in this volume, a variety of biological parameters are influenced by the inherent properties of scrapie agents independent of the host species. Additional information on the genetic interaction between host and scrapie agent as well as agents of the other unconventional slow infections has been detailed in several recent reviews (Carp et al. 1989b, c). With regard to those parameters under genetic control, our published work has concentrated on analyzing five of the mouse-adapted scrapie strains; ME7, 22A, 22L, 139A and 87V (Carp et al. 1984, 1985a, 1987; Carp and Callahan 1986; Kascsak et al. 1985, 1986, 1987; Rubenstein et al. 1986; Kim et al. 1987a, b) and three hamster adapted strains, 263K, 139-H and 22C-H (Kascsak et al. 1985, 1986; Carp et al. 1990).
Strain variation in scrapie was first recognised about 30 years ago, when experimental sheep scrapie was transmitted to and serially passaged in goats. Two separate passage lines were maintained which produced strikingly different clinical signs in unselected goats from the same herd, either a “drowsy” or a “scratching” syndrome (Pattison and Millson 1961). This difference between the two isolates remained constant on repeated goat-to-goat passage. Since then, overwhelming evidence for scrapie strain variation has accumulated, mainly from the long-term studies conducted in Edinburgh by Dickinson and coworkers (including ourselves), who have serially passaged scrapie from a wide range of natural and experimental sources in inbred mice (Dickinson 1976). About 20 strains of scrapie have been identified so far in mice, each with its own distinct and repeatable set of disease characteristics.
There is now very persuasive evidence that the transmissible agent for spongiform encephalopathies such as scrapie, consists of a modified form of the normal host protein PrPc, devoid of any nucleic acid. On the other hand, because there are many different strains of scrapie agent with distinct phenotypes which can be propagated in animals homozygous for the PrPc gene, it has been suggested that a nucleic acid must be a component of the agent. Can the two views be reconciled?
PrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher Mr forms (Mr 34,000 and 29,000) of PrP can be reduced in apparent Mr to a lower Mr form (Mr 25,000) with Peptide-N-glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher Mr forms whereas others bind more strongly to the lower Mr form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in Mr. The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.
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.
A single intraperitoneal injection of 250 micrograms dextran sulphate 500 (DS500) reduced the susceptibility of mice to scrapie given by the same route. A lower dose (25 micrograms) was less effective but still produced significant incubation period lengthening, while a high dose (2.5 mg) further increased the degree of prolongation. This reduced susceptibility occurred with DS500 administered up to at least 4 weeks prior to intraperitoneal scrapie inoculation and up to at least 2 weeks after scrapie inoculation. A reduced average effect, but more variable between mice, was obtained with DS500 given 1 month or 2 months after scrapie. The effective scrapie titre was reduced by 90% when DS500 was injected either 72 h before or 7 h after ME7 scrapie. Using a relatively lower but normally still fatal dose of the 22A strain of scrapie approximately 50% of the treated mice survived. The effective 90% loss of titre was consistent with either of these strains of scrapie in 11 different inbred strains of mice (BALB/c, BSC, BRVR, C3H, C57BL, IM, LM, MM, RIII, VL and VM). No significant increase in the prolongation effect was obtained using multiple DS500 doses in two different time combinations. DS500 causes long-term interference in both the early processing and the replication of scrapie agent, unlike those immunomodulators which increase susceptibility.
Studies of polyanions that suppress scrapie have been done to pinpoint the cell types in the lymphoreticular system which
are important in pathogenesis and to suggest possible prophylactic or therapeutic strategies for the unconventional slow viruses.
A regime of three daily injections of the inorganic heteropolyanion HPA-23 reduced the effective scrapie dose by more than
99%; i.e., some mice survived peripherally injected doses of 100 50% lethal dose units. The effect was greatest when the first
dose of HPA-23 was given 4 h after injecting scrapie, but it declined rapidly as this interval was increased, and there was
virtually no effect 2 days after infection. A single dose of high-molecular-weight organic polyanions such as carrageenan
or dextran sulfate (DS-500) greatly reduced (i.e., greater than 99%) the efficiency of scrapie infection. In contrast to HPA-23,
DS-500 was equally effective whether given 4 days before or 8 h after the time of infection. The antiscrapie effect of DS-500
appeared to be independent of its activity as a B-cell mitogen and of its ability to produce a cytotoxic blockade of phagocytic
cells. DS-500 probably caused the aggregation and loss from blood of scrapie inoculum which was present immediately after
injection, but it had additional effects on scrapie at later times.