The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrP^Sc, a misfolded and aggregated form of the cellular prion protein PrP^C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP^C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP^C and PrP^Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP^C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP^C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP^Sc. Other antibodies immunoprecipitate PrP^C, but not PrP^Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP^C and PrP^Sc. Amino-proximal antibodies were found to react with repetitive PrP^C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

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.     

The Extent of Protease Resistance of Misfolded Prion Protein Is Highly Dependent on the Salt Concentration

Transmissible spongiform encephalopathies are neurodegenerative diseases caused by prions in mammals. An aberrantly folded protein (PrP^Sc) is the main component of these proteinaceous infectious particles. Prions exhibit strong resistance to protease digestion, which is typically exploited for biochemical discrimination from its native cellular form (PrP^C). This classical feature has been partially challenged by the isolation of sizeable amounts of protease-sensitive PrP^Sc isoforms that self-propagate in vivo. Here, we report that the degree of PrP^Sc protease resistance is highly dependent on the concentration of salt in the solution. Similar changes were observed in PrP^Sc obtained from different strains and species. Strikingly, the effect of salt is reversible and is associated with changes on the size of PrP^Sc particles, but surprisingly, the more protease-sensitive species consists of a larger size. These findings shed light on the mechanistic aspects of prion proteolysis and should be considered when assessing samples of biomedical relevance.     

Overexpression of Nonconvertible PrPcΔ114–121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to trans-Dominant Inhibition of Wild-Type PrPSc Accumulation

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP^Sc of a normal cellular glycoprotein, PrP^c, which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP^c molecule, should play a role in promoting the conversion into PrP^Sc. By using persistently scrapie-infected mouse neuroblastoma (Sc⁺-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP^c that are capable of blocking the conversion of endogenous, wild-type PrP^c into PrP^Sc. We constructed a deletion mutant (PrP^cΔ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP^c or PrP^cΔ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP^c versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc⁺-MNB cells revealed that PrP^cΔ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP^Sc derived from endogenous PrP^c. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP^c plays an important role in the conversion process of PrP^c into PrP^Sc and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP^Sc accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.     

Immunopurification of Pathological Prion Protein Aggregates

Background

Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP^C) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP^Sc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP^C molecules. A great deal of effort has been devoted to developing protocols for purifying PrP^Sc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP^Sc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Principal Findings

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP^Sc and mutant PrP aggregates at electrophoretic homogeneity. PrP^Sc purified from prion-infected mice was able to seed misfolding of PrP^C in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

Significance

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.     

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrP^Sc) that occur throughout the brain. Formation of synthetic or in vitro PrP^Sc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrP^Sc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrP^c (spanning residues 90–231). We then obtained two-dimensional solution NMR spectra of the PrP^c monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (¹H–¹⁵N) studies revealed that, in comparison to monomeric PrP^c, the oligomer has intense amide peak signals in the N-terminal (residues 90–114) and C-terminal regions (residues 226–231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrP^c monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.     

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrP^Sc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation

We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrP^Sc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrP^Sc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions

A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

     

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP^C) into a prion (PrP^Sc). The information necessary for this conversion is contained in the conformation of PrP^Sc. Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10^?18 mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP^Sc and GPI-anchorless PrP^Sc. It has also been used to study the quaternary structure of the PrP^Sc multimer. Small molecule reagents react differently with the same lysine in the PrP^C conformation than in the PrP^Sc conformation. Such differences can be detected by Western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrP^Sc without the need for proteinase K pretreatment and can be used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions. Furthermore these tools are or can be applied to the study of the other protein misfolding diseases such as Alzheimer Disease, Parkinson Disease, or ALS.     

Rapid, high-throughput detection of PrPSc by 96-well immunoassay

Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C. Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP^Sc. This assay has PrP^Sc detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.Key words: prion disease, immunodetection, PrP^Sc     

Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Str?ussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy

Prion diseases are classically characterized by the accumulation of pathological prion protein (PrP^Sc) with the protease resistant C-terminal fragment (PrP^res) of 27–30 kDa. However, in both humans and animals, prion diseases with atypical biochemical features, characterized by PK-resistant PrP internal fragments (PrP^res) cleaved at both the N and C termini, have been described. In this study we performed a detailed comparison of the biochemical features of PrP^Sc from atypical prion diseases including human Gerstmann-Sträussler-Scheinker disease (GSS) and variably protease-sensitive prionopathy (VPSPr) and in small ruminant Nor98 or atypical scrapie. The kinetics of PrP^res production and its cleavage sites after PK digestion were analyzed, along with the PrP^Sc conformational stability, using a new method able to characterize both protease-resistant and protease-sensitive PrP^Sc components. All these PrP^Sc types shared common and distinctive biochemical features compared to PrP^Sc from classical prion diseases such as sporadic Creutzfeldt-Jakob disease and scrapie. Notwithstanding, distinct biochemical signatures based on PrP^res cleavage sites and PrP^Sc conformational stability were identified in GSS A117V, GSS F198S, GSS P102L and VPSPr, which allowed their specific identification. Importantly, the biochemical properties of PrP^Sc from Nor98 and GSS P102L largely overlapped, but were distinct from the other human prions investigated. Finally, our study paves the way towards more refined comparative approaches to the characterization of prions at the animal–human interface.     

PK-sensitive PrPSc Is Infectious and Shares Basic Structural Features with PK-resistant PrPSc

One of the main characteristics of the transmissible isoform of the prion protein (PrP^Sc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP^Sc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP^Sc that is sensitive to PK hydrolysis (sPrP^Sc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP^Sc versus the PK-resistant (rPrP^Sc) fractions of PrP^Sc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP^Sc and rPrP^Sc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP^Sc to the rPrP^Sc forms of PrP^Sc. Although the sPrP^Sc and rPrP^Sc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.     

Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1

The abnormal prion protein (scrapie-associated prion protein, PrP^Sc) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP^Sc is highly resistant to normal sterilization procedures, the decontamination of PrP^Sc is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP^Sc. Although PrP^Sc is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP^Sc under extreme conditions. The level of PrP^Sc in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP^Sc at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP^Sc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP^Sc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP^Sc degradation.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrP^Sc of the host-encoded prion protein (PrP^c). A profound conformational change of PrP^c underlies formation of PrP^Sc and prion propagation involves conversion of PrP^c substrate by direct interaction with PrP^Sc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrP^Sc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.     

Scrapie prion protein structural constraints obtained by limited proteolysis and mass spectrometry

Elucidation of the structure of scrapie prion protein (PrP^Sc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrP^Sc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrP^Sc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrP^Sc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrP^Sc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrP^Sc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrP^Sc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.     

Using small molecule reagents to selectively modify epitopes based on their conformation

PrP^Sc is an infectious protein. The only experimentally verified difference between PrP^Sc and its normal cellular isoform (PrP^C) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrP^Sc isoform. The N-hydroxysuccinimide esters of acetic acid and 4-trimethylammoniumbutyric acid were synthesized and reacted with detergent-solubilized brain extracts from Me7-infected mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by western blots probed with the antibodies 3F4, 6D11, 7D9, AG4, AH6, GE8 or MAB5424. The 3F4, 6D11, AH6, and GE8 antibodies recognize an epitope that is encrypted in the PrP^Sc isoform, but exposed in the PrP^C isoform. These reagents permit the detection of prion infected brain extracts without the need for proteinase K digestion. In addition they can be used, with an appropriate antibody, to determine which amino acids of PrP^Sc are exposed on the surface and which are encrypted, thus providing useful structural information. This approach was used to distinguish between the 263K and drowsy strains of hamster-adapted scrapie without the use of proteinase K.     

Using small molecule reagents to selectively modify epitopes based on their conformation

PrP^Sc is an infectious protein. The only experimentally verified difference between PrP^Sc and its normal cellular isoform (PrP^C) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrP^Sc isoform. The N-hydroxysuccinimide esters of acetic acid and 4-trimethylammoniumbutyric acid were synthesized and reacted with detergent-solubilized brain extracts from Me7-infected mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by western blots probed with the antibodies 3F4, 6D11, 7D9, AG4, AH6, GE8 or MAB5424. The 3F4, 6D11, AH6, and GE8 antibodies recognize an epitope that is encrypted in the PrP^Sc isoform, but exposed in the PrP^C isoform. These reagents permit the detection of prion infected brain extracts without the need for proteinase K digestion. In addition they can be used, with an appropriate antibody, to determine which amino acids of PrP^Sc are exposed on the surface and which are encrypted, thus providing useful structural information. This approach was used to distinguish between the 263K and drowsy strains of hamster-adapted scrapie without the use of proteinase K.