The role of electrostatic interaction in triggering the unraveling of stable helix 1 in normal prion protein. A molecular dynamics simulation investigation

The conversion of normal prion protein (PrPC) into scrapie isoform (PrPSc) is a key event in the pathogenesis of prion diseases. However, the conversion mechanism has given rise to much controversy. For instance, there is much debate on the behavior of helix 1 (H1) in the conversion. A series of experiments demonstrated that H1 in isolated state was very stable under a variety of conditions. But, other experiments indicated that helices 2 and 3 rather than H1 were retained in PrPSc. In this paper, molecular dynamics (MD) simulation is employed to investigate the dynamic behavior of H1. It is revealed that although the helix 1 of Human PrPC (HuPrPC) is very stable in the isolated state, it becomes unstable when incorporated into native HuPrPC, which likely results from the long-range electrostatic interaction between Asp147 and Arg208 located in the helices 1 and 3, respectively. This explanation is supported by experimental evaluation and MD simulation on D147N mutant of HuPrPC that the mutant becomes a little more stable than the wild type HuPrPC. This finding not only help to reconcile the existing debate on the role of helix 1 in the PrPC-->PrPSc transition, but also reveals a possible mechanism for triggering the PrPC-->PrPSc conversion.     

Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPC): Single Molecule Discrimination and PrPC Kinetics

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP^C) in the central nervous system into the infectious isoform (PrP^Sc). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP^Sc. A number of pathogenic PrP^C mutants exist that are characterized by an increased propensity for conversion into PrP^Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP^C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP^C, (a PrP^C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP^C capture event. Moreover, we present a four-state model to describe wild-type PrP^C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP^C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

Pressure-assisted dissociation and degradation of “proteinase K-resistant” fibrils prepared by seeding with scrapie-infected hamster prion protein

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP^C, into a fibrous pathogenic form, PrP^Sc, which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP^Sc into PrP^C is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP^Sc from PrP^C eventually in vivo. Here we show that, by applying pressures at kbar range, the “proteinase K-resistant” fibrils (rHaPrP^res) prepared from hamster prion protein (rHaPrP [23–231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP^res fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP^Sc from PrP^C is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP^Sc from various systems of pathogenic concern.     

Lipopolysaccharide induced conversion of recombinant prion protein

The conformational conversion of the cellular prion protein (PrP^C) to the β-rich infectious isoform PrP^Sc is considered a critical and central feature in prion pathology. Although PrP^Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP^C to proteinase K resistant PrP^Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP^C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP^C to PrP^Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).     

Novel amplification mechanism of prions through disrupting sortilin-mediated trafficking

Conformational conversion of the cellular prion protein, PrP^C, into the abnormally folded isoform of prion protein, PrP^Sc, which leads to marked accumulation of PrP^Sc in brains, is a key pathogenic event in prion diseases, a group of fatal neurodegenerative disorders caused by prions. However, the exact mechanism of PrP^Sc accumulation in prion-infected neurons remains unknown. We recently reported a novel cellular mechanism to support PrP^Sc accumulation in prion-infected neurons, in which PrP^Sc itself promotes its accumulation by evading the cellular inhibitory mechanism, which is newly identified in our recent study. We showed that the VPS10P sorting receptor sortilin negatively regulates PrP^Sc accumulation in prion-infected neurons, by interacting with PrP^C and PrP^Sc and trafficking them to lysosomes for degradation. However, PrP^Sc stimulated lysosomal degradation of sortilin, disrupting the sortilin-mediated degradation of PrP^C and PrP^Sc and eventually evoking further accumulation of PrP^Sc in prion-infected neurons. These findings suggest a positive feedback amplification mechanism for PrP^Sc accumulation in prion-infected neurons.     

Sialylation of Prion Protein Controls the Rate of Prion Amplification,the Cross-Species Barrier,the Ratio of PrPSc Glycoform and Prion Infectivity

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP^C) into the disease-associated, transmissible form (PrP^Sc). PrP^C is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP^C and the strain-specific structure of PrP^Sc. The current work highlights the previously unappreciated role of sialylation of PrP^C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP^Sc glycoform ratio. The current study demonstrates that undersialylated PrP^C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP^C. As a result, PMCAb-derived PrP^Sc was less sialylated than brain-derived PrP^Sc. A decrease in PrP^Sc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP^C using sialidase was found to increase the rate of PrP^Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP^C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP^C was also found to control PrP^Sc glycoform ratio. A decrease in PrP^C sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP^Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP^C differed from that of spleen-derived PrP^C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP^C, suggesting that Neu1 is not responsible for desialylation of PrP^C. The current work highlights previously unappreciated role of PrP^C sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.     

PrPST,a Soluble,Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

While the conversion of PrP^C into PrP^Sc in the transmissible form of prion disease requires a preexisting PrP^Sc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP^ST), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP^ST as in TgMHu2ME199K mice, and “classical” PrP^Sc as in infectious prion diseases, coincide in the patient''s post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.     

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans^9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP^C) and the infectious protein (PrP^Sc), an abnormally-folded isoform of PrP^{C 8}.One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation¹³. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP^Sc are poorly characterized due to their unusual conformation and aggregation states.PrP^Sc can seed the conversion of PrP^C to PrP^Scin vitro¹⁴. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP^Sc, at an accelerated rate, from a system containing excess amounts of PrP^C and minute amounts of the PrP^Sc seed¹⁹. This technique has proven to effectively recapitulate the species and strain specificity of PrP^Sc conversion from PrP^C, to emulate prion strain interference, and to amplify very low levels of PrP^Sc from infected tissues, fluids, and environmental samples^6,7,16,23 .This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.     

Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations

Background

Prion diseases are associated with a conformational switch for PrP from PrP^C to PrP^Sc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.

Scope of review

MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2-H2 loop to a 3₁₀ helix.

Major conclusions

Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP^C to PrP^Sc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP^Sc assemblies.     

Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

In prion diseases, the cellular form of the prion protein, PrP^C, undergoes a conformational conversion to the infectious isoform, PrP^Sc. PrP^C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP^C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP^C. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP^C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP^C from rafts, promoting its endocytosis. Glypican-1 and PrP^C colocalised on the cell surface and both PrP^C and PrP^Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP^Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP^C on the β-secretase cleavage of the Alzheimer''s amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP^C and PrP^Sc in lipid rafts.     

Parallel In-register Intermolecular β-Sheet Architectures for Prion-seeded Prion Protein (PrP) Amyloids

Structures of the infectious form of prion protein (e.g. PrP^Sc or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP^Sc retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP^C), but evidence is accumulating that these helices are absent in PrP^Sc amyloid. Moreover, recombinant PrP^C can form amyloid fibrils in vitro that have parallel in-register intermolecular β-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily β-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90–231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP^Sc fibrils, can have stable parallel in-register β-sheet cores. These simulations revealed that the C-terminal residues ∼124–227 more readily adopt stable tightly packed structures than the N-terminal residues ∼90–123 in the absence of cofactors. Variations in the placement of turns and loops that link the β-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures.     

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into disease-related isoforms (PrP^Sc). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation was examined using a cell painting technique. PrP^Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP^C. In contrast, PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc. Furthermore, the presence of desialylated PrP^C inhibited the production of PrP^Sc within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP^C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP^C. Desialylated PrP^C was less sensitive to cholesterol depletion than PrP^C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP^C in neurons caused the dissociation of cytoplasmic phospholipase A₂ from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A₂. These findings show that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.     

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP^C molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP^C is pre-incubated with poly(A) RNA and PrP^Sc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP^Sc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP^Sc molecules, most likely through an epitope containing residue 172.     

Identifying critical sites of PrPc-PrPSc interaction in prion-infected cells by dominant-negative inhibition

A direct physical interaction of the prion protein isoforms is a key element in prion conversion. Which sites interact first and which parts of PrP^c are converted subsequently is presently not known in detail. We hypothesized that structural changes induced by PrP^Sc interaction occur in more than one interface and subsequently propagate within the PrP^C substrate, like epicenters of structural changes. To identify potential interfaces we created a series of systematically-designed mutant PrPs and tested them in prion-infected cells for dominant-negative inhibition (DNI) effects. This showed that mutant PrPs with deletions in the region between first and second α-helix are involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. Although some PrPs did not reach the plasma membrane, they had access to the locales of prion conversion and PrP^Sc recycling using autophagy pathways. Using other series of mutant PrPs we already have identified additional sites which constitute potential interaction interfaces. Our approach has the potential to characterize PrP-PrP interaction sites in the context of prion-infected cells. Besides providing further insights into the molecular mechanisms of prion conversion, this data may help to further elucidate how prion strain diversity is maintained.     

A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP^C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP^C nor for the formation of a stable prion structure.     

Intracellular accumulation of a 46 kDa species of mouse prion protein as a result of loss of glycosylation in cultured mammalian cells

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of an abnormal isoform (PrP^Sc) of the normal cellular prion protein (PrP^C) in the brain. Reportedly, abnormal N-linked glycosylation patterns in PrP^C are associated with disease susceptibility; thus, we compared the glycosylation status of normal and several mutant forms of the murine prion protein (MuPrP) in cultured mammalian cells. Substitution of the N-terminal signal sequence of normal MuPrP with a heterologous signal peptide did not alter glycosylation. When expressed without the C-terminal glycophosphatidylinositol anchor signal, the majority of MuPrP remained intracellular and unglycosylated, and a 46 kDa species (p46) of the unglycosylated PrP^C was detected on reducing gels. p46 was also observed when wild-type MuPrP was expressed in the presence of tunicamycin or enzymatically deglycosylated in vitro. A rabbit polyclonal anti-serum raised against dimeric MuPrP cross-reacted with p46 and localized the signal within the Golgi apparatus. We propose that the 46 kDa signal is a dimeric form of MuPrP and in the light of recent studies, it can be argued that a relatively stable, non-glycosylated, cytoplasmic PrP^C dimer, produced as a result of compromised glycosylation is an intermediate in initiating conversion of PrP^C to PrP^Sc in sporadic transmissible spongiform encephalopathies.