Sheep Prion Protein Research Articles

Comparing the Extent of Methionine Oxidation in the Prion and Native Conformations of PrP.

Scrapie is a prion disease of sheep and goats. Prions (PrPSc) replicate by inducing a natively expressed protein (PrPC) to refold into the prion conformation. PrPC and PrPSc contain a disproportionately large number of methionines. Surface exposed methionines are more prone to chemical oxidation. Chemical oxidation is a means of measuring the surface exposure of the methionines in a prion, as these covalent changes are retained after an oxidized prion is denatured prior to analysis. Scrapie prions and recombinant sheep prion protein were oxidized in 0, 10, 20, or 50 mM solutions of hydrogen peroxide. The samples were digested with trypsin or trypsin followed by chymotrypsin to yield a set of peptides (TNMK, MLGSAMSR, ENMYR, IMER, VVEQMCITQYQR) containing the methionines present in sheep PrP. The mass spectrometry based multiple reaction monitoring (MRM) method was used to analyze these peptides. Analysis of the rPrP samples showed that surface exposed methionines (132, 137, and 157) were more oxidized than those less surface exposed (209 and 216). The extent of methionine oxidation in sheep scrapie PrPSc is 216 > 137 > 132 > 157 > 209 > 112. These results demonstrate that this approach can be used to map the surface exposure of the methionines in order to distinguish among PrP conformations and effect a kind of conformational sequence.

PRNP Sequences of Tibetan Antelope, Blue Sheep, and Plateau Pika from the Qinghai-Tibet Plateau and Reactivity of PrP Proteins to Rodent-Adapted Scrapie Strains in RT-QuIC and PMCA

Background: Tibetan antelope (Rhinopithecus), blue sheep (Pseudois nayauris), and plateau pika (Ochotona curzoniae) are wild animals living on the Qinghai-Tibet Plateau. There have been no reports of naturally-occurring transmissible spongioform encephalopathies (TSEs) involving these animals. Furthermore, the PRNP genes have not been described in the literature. Methods: The PRNP genes from 21 Tibetan antelopes, 4 blue sheep, and 3 plateau pikas were obtained and sequenced. The recombinant proteins were then prepared. Using scrapie strains (263K, 139A, ME7, and S15) as the seeds, the reactivity of the PrP proteins from sheep (rSheepPrP25-234) and pika (rPikaPrP23-230) were tested using real-time quaking-induced conversion (RT-QuIC). Protein misfolding cyclic amplification (PMCA) tests of the brain homogenates from domestic sheep and rabbits were performed with the seeds of strains 263K and ME7. Results: The PRNP genes of bovids were 771 bp long and encoded 256 amino acids (aa), showing 100% homology with the wild-type sheep prion protein (PrP) aa sequence. The PRNP gene of pika was 759 bp long and encoded 252 amino acids, showing 92.1% homology with the aa sequence of domestic rabbits. The sheep and pika proteins revealed positive reactions in 10-5 diluted seeds. Only rPikaPrP23-230 produced positive curves in 10-7 diluted seeds. The PMCA tests failed to produce proteinase K (PK)-resistant PrP (PrPres). Conclusion: This is the first description of PRNP genes and PrP aa sequences of Tibetan antelope, blue sheep, and plateau pike from the Qinghai-Tibet Plateau. In the presence of rodent prions, the PrPs of sheep and pika efficiently induce fibrillation in RT-QuIC, but do not generate PrPres in PMCA. Our results indicate that pika, as one of the important links in the Qinghai-Tibet Plateau biological chain, may play an important role in the prion circulation. Pika PrP deserves further analysis for its potential application value in assays for human prion disease.

Excessive replacement changes drive evolution of global sheep prion protein (PRNP) sequences.

Sheep prion protein (PRNP) is the major host genetic factor responsible for susceptibility to scrapie. We aimed to understand the evolutionary history of sheep PRNP, and primarily focused on breeds from Turkey and Ethiopia, representing genome-wise ancient sheep populations. Population molecular genetic analyses are extended to European, South Asian, and East Asian populations, and for the first time to scrapie associated haplotypes. 1178 PRNP coding region nucleotide sequences were analyzed. High levels of nucleotide diversity driven by extensive low-frequency replacement changes are observed in all populations. Interspecific analyses were conducted using mouflon and domestic goat as outgroup species. Despite an abundance of silent and replacement changes, lack of silent or replacement fixations was observed. All scrapie-associated haplotype analyses from all populations also showed extensive low-frequency replacement changes. Neutrality tests did not indicate positive (directional), balancing or strong negative selection or population contraction for any of the haplotypes in any population. A simple negative selection history driven by prion disease susceptibility is not supported by the population and haplotype based analyses. Molecular function, biological process enrichment, and protein-protein interaction analyses suggested functioning of PRNP protein in multiple pathways, and possible other functional constraint selections. In conclusion, a complex selection history favoring excessive replacement changes together with weak purifying selection possibly driven by frequency-dependent selection is driving PRNP sequence evolution. Our results is not unique only to the Turkish and Ethiopian samples, but can be generalized to global sheep populations.

Canine D163-PrP polymorphic variant does not provide complete protection against prion infection in small ruminant PrP context

E/D163 polymorphism of dog prion protein (PrP) has been recently proposed as the variant responsible for canid prion resistance. To further investigate the protective role of this variant against prion replication, the transgenic mouse model OvPrP-Tg532 expressing sheep/goat PrP carrying the substitution D162 (equivalent to D163 position of dog PrP) was generated and intracranially inoculated with a broad collection of small ruminant prion strains. OvPrP-Tg532 mice showed resistance to classical bovine spongiform encephalopathy (BSE) from sheep and some classical scrapie isolates from sheep and goat but were susceptible to ovine atypical L-BSE and numerous classical scrapie isolates. Strikingly, some of these classical scrapie isolates showed a shift in their prion strain properties. These results suggest that other PrP residues apart from E/D163 variant of dog PrP or factors distinct than PrP may participate in prion resistance of canids and that different factors may be required for D162 sheep PrP to provide effective protection to sheep against ruminant prions.

A comparative analysis between PrPc and PrPsc to determine the structure‐function relationship of protein misfolding in Scrapie prion disease

The Minnetonka MSOE Center for BioMolecular Modeling SMART Team used 3D modeling and printing technology to examine structure‐function relationships of PrPsc. Scrapies is caused by prions, misfolded proteins in animals and humans, resulting in the development of fatal neurodegenerative encephalitis. Scrapies was one of the first prion diseases to be isolated and studied, and was found to be caused by the misfolding of the PrP protein into the PrPsc prion. We will do this by creating, annotating, and comparing two models, one of the original PrP protein, and one of the PrPsc prion. Scrapies disease is caused when the alpha‐helix rich prion protein (PrP) is converted into into a beta‐structure‐rich insoluble conformer (PrPsc) and is thought to be highly infectious. These structural changes in PrPsc contribute to its build up in the brain of sheep and goats PrP, leading to a deadly swelling of the brain. The buildup is caused by the inability of proteinase K (PK) to fully degrade PrPsc. PK has the ability to fully degrade PrP, but, interacting with PrPsc, can only create N terminally truncated PrPsc, resulting in a PK resistant core. There is currently no cure for Scrapies; however, further research is being done to discover how to reverse the functional effects of prion diseases. Our goal is to present which structural changes occur in the original protein PrP that cause the defective protein PrPsc, as well as determine how this misfolding occurs and what effect it has on the protein's function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genotyping of prion protein in black merino sheep from the Iberian Peninsula

The black merino variety is an emerging fine-wool merino breed variety of sheep. Their flocks have been reared in Portugal and Spain under different criteria regarding the selection for scrapie resistance haplotypes. This study was aimed to report for the first time in the black merino the prion protein (PRNP) haplotype frequency distribution using sequencing. A sample of 120 sheep belonging to four flocks was analyzed in this study. Three unselected flocks from Portugal and one selected for ARR/ARR genotypes from Spain. The number of polymorphism sites was extended here from three positions (136, 154 and 171) to five by adding the positions 141 and 143 due to both to show its current intermediate frequency in Portuguese sheep and to study the genetic relationship among haplotypes using networks analysis. In this way susceptibility to classical and atypical scrapie can be elucidated. Eight haplotypes could be reported in the black merino as follows: ALHRR, ALHRQ, ALRRQ, ALHHQ, AFHRQ, ALHRH, VLHRQ and T112ALHRQ but the last was a singleton haplotype. The results showed high genetic differentiation among flocks due to an unbalanced frequency distribution of haplotypes suggesting scarce genetic flow among them in Portugal. After collapse haplotypes to three positions (136, 154 and 171) the black merino from Portugal showed similar haplotype frequencies than in previous study except for the VRQ haplotypes with higher frequency in this study. The finding of AFHRQ was interesting because it was associated to atypical scrapie being the only cases declared in Portugal of this disease. In conclusion, we reported high richness in haplotypes in this sheep, but its distribution critically depends on the studied flock. This genetic richness enables to analyze different scenarios before implemented breeding programmes. In this way the application of a breeding programme to control scrapie appears to be a less challenging task.

Polymorphisms Modulate Sheep Prion Protein Susceptibility to Misfolding by Altering the Residue Network of Interactions

The autocatalytic misfolding of the cellular form of the prion protein, PrPC, to the infectious form, PrPSc, is critical to the development of prion diseases. Though the mechanism for this misfolding is not well understood, previous research recognizes the role of genetic mutations in varying susceptibilities to prion diseases in sheep. Using structural bioinformatics techniques, our study examines the residue interactions in three different sheep PrPC structures. These structures contain different point mutations and have different levels of susceptibility to scrapie prion disease. We will discuss our results in relation to the role of mutations on networks of interaction, and propose a mechanism for the primary misfolding of the a-helical rich PrPC into the b-sheet rich PrPSc. This work was made possible partly by grants from the National Institute for General Medical Science (NIGMS) (5P20GM103427), a component of the National Institutes of Health (NIH), and its contents are the sole responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Re-transmissibility of mouse-adapted ME7 scrapie strain to ovine PrP transgenic mice.

Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A136 R154 Q171/ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrPSc) deposition in the affected brains. PrPSc deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded.

Identification of prion protein genotype in sheep: 11 years of proficiency tests in Italy

Scrapie of sheep belongs to a group of fatal neurodegenerative diseases known as prion diseases. Scrapie is a naturally transmissible disease and sheep susceptibility is highly dependent on the host prion protein (PrP) genotype. On this basis, the European Union has established management strategies of scrapie in sheep population, to be achieved by an ad hoc breeding programme to increase the genetic resistance of the ovine population and by selective culling, both based upon the PrP genotyping. The Istituto Superiore di Sanita, appointed by the Italian Ministry of Health as the Reference Laboratory for PrP genotyping, has since 2005 organised proficiency tests (PTs) for sheep PrP genotype identification. The PT aims at assessing the accuracy of the genetic analysis performed by the laboratories involved in official control at national level. From 2005 to 2016, 12 laboratories on average participated to nine PT rounds. The overall analytical performance was satisfactory with few laboratories reporting errors in PrP genotype identification. A total of 22 errors including sixteen analytical and six formal errors were identified out of 2650 results reported (0.8 %). The number of laboratories reporting 100 % correct results increased over the years although no statistical trend could be observed. The low number of laboratories failing the PT revealed an overall good technical capability and skills of laboratories and technicians in performing the genetic analysis. This is highly important since the availability of an efficient laboratory network is crucial for the implementation and management of scrapie control plan at national level.

Effect of Urea, Arginine, and Ethanol Concentration on Aggregation of 179CVNITV184 Fragment of Sheep Prion Protein.

Understanding protein aggregation is of utmost importance as it is responsible for causing several neurodegenerative diseases and one of the serious impediments in large-scale biopharmaceutical production. The prion protein is responsible for pathological states in fatal transmissible spongiform conditions, such as Creutzfeldt–Jakob disease and bovine spongiform encephalopathy. The peptide fragment 178–191 of Syrian hamster prion protein is known to be amyloidogenic. Here, we identified the fragment 179CVNITV184 as an aggregation-prone fragment in sheep prion protein. This fragment is conserved sequence among sheep and Syrian hamster prion protein and also falls in the previously identified amyloidogenic sequence. The mechanistic details of the aggregation behavior are analyzed in three different concentrations of urea, arginine, and ethanol. Urea and arginine are found to be aggregation suppressors, but ethanol enhances the protein aggregation through β-sheet formation. We have also analyzed the influence of these osmolyte on water dynamics in the presence of the octamer of this aggregation-prone fragment and correlated this water dynamics with the aggregation behavior of the octamer.

In silico investigation of the prion protein glycosylation profiles in relation to scrapie disease resistance in domestic sheep (Ovis aries)

The prion protein is a membrane-bound glycoprotein which consists mainly α-helix structure. In contrast, the infectious prion protein shows the beta-sheet structure. The prion-associated diseases are all lethal neurodegenerative abnormalities, called transmissible spongiform encephalopathies. Scrapie is the most common type of these illnesses affecting sheep, goats, and moufflon. The VRQ, AHQ, ARR and N146S polymorphisms in the sheep prion gene have been found to be associated with resistance to scrapie disease. So far, the relationship of polymorphisms to three-dimensional protein structures, post-translational modifications, and scrapie resistance has not been studied. In this study, the potential N- and O-glycosylation positions of sheep prion protein polymorphisms were analyzed, the secondary and three-dimensional protein structure models were predicted, three-dimensional glycoprotein models were constructed and the role of glycosylation positions in protein interactions was investigated. Here, we found that protein secondary and three-dimensional structures vary among polymorphisms. Moreover, we found wild-type prion and all polymorphic variants show N-glycosylation at Asn184 and Asn200 positions, while O-glycosylation profiles are variant-specific. We also found that structural changes among prion polymorphisms leads to the formation of variant spesific O-glycosylation profiles and these positions are associated with protein interactions. Based on these findings, we suggest that O-glycosylation may be effective on resistance/susceptibility of sheep prion polymorphisms to scrapie disease.

The association between 171 K polymorphism and resistance against scrapie affection in Greek dairy sheep

Polymorphisms at codon 171 of the sheep prion protein gene (PRNP) are known to be genetically associated with scrapie (Hunter et al., 1993, 1997). Glutamine (Q) and histidine (H) at codon 171 contribute to scrapie susceptibility, while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 171 (171 K), but its effect on scrapie resistance has not been fully determined yet. To investigate the role of 171 K in dairy sheep in Greece, we initially analyzed blood samples from 2368 sheep with no clinical signs of the disease, from thirteen scrapie-affected flocks that were then monitored for 5 years for scrapie clinical signs. During this period 177 sheep resulted positive in classical scrapie, after obex testing. The 177 scrapie-affected brain samples PRNP polymorphisms were analyzed using a Real Time-PCR protocol while the 2368 sheep blood samples were analyzed by a micro-array method based on primer extension (Homogenous Mass Extend Assay – SEQUENOM, Genaissance Pharmaceuticals) in MALDI-TOF-MS. Single Nucleotide Polymorphisms (SNPs) present at PRNP codons 136A/V (Alanine/Valine), 154R/H and 171Q/R/H/K were detected. The ARQ haplotype as well as the ARQ/ARQ genotype predominated. In the studied population, ARK haplotype was found at the highest frequency reported so far (6.98%) whereas, ARK/-genotypes had a total frequency of 12.81%. Relative risk (RR) and the percent relative effect were calculated to assess the association between ARK/-genotypes and scrapie affection. The results showed that carriers of K allele at codon 171 compared to Q and H alleles had ca. 0.25 and 0.40 times the risk of scrapie affection, respectively, indicating a possible association of K allele at codon 171 with scrapie resistance.

Spontaneous formation of nanofilms under interaction of 4th generation pyrydylphenylene dendrimer with proteins

The use of materials based on protein-polymer compositions is a promising method for solving a number of problems in biotechnology and medicine. In our work, we produced multilayer nanofilms based on 4th generation (G4) pyridylphenylene dendrimers with fully pyridine-based periphery containing a protein component. For dairy beta- and kappa-caseins, alpha-lactalbumin, recombinant sheep prion protein, lysozyme, trypsin, and alpha-chymotrypsin the ability to integrate into self-organizing structures about 230–710 nm thick, measured by atomic force microscopy, was found. The formation of nanofilms is dependent on the protein/dendrimer ratio in the initial solution. At the same time, the nanofilms are resistant to pH changes, and the action of detergents. We demonstrated that trypsin and chymotrypsin incorporated into the nanofilms still conserved proteolytic activities and were stable for at least 3 weeks. We have also conducted in silico molecular modeling of the G4 dendrimer interactions with lysozyme. Spatial structure of the dendrimer-protein complex was predicted and most probable variant remained almost intact under 100 ns molecular dynamics simulation conditions. We believe that dendrimer-protein systems are promising tools for possible applications in the development of new stable biomaterials and biosensors.

Current understanding of PrnP Genetics: A tool for Molecular Assisted Selection in Sheep Populations (A review)

Scrapie is a neurodegenerative prion disease of sheep, goats and mouflons, belonging to the group of transmissible spongiform encephalopathies (TSEs), which affects humans as well. Even though classical scrapie has been known for over 250 years, the 1985 BSE crisis related to the advent of new forms of the Creutzfeldt-Jakob disease (vCJD) in humans imposed the implementation of rapid coercive legal measures of prevention, control and eradication of TSEs. According to the prion hypothesis, the transmissible agent is the pathological isoform (PrP Sc ) of cellular prion protein (PrP C ). Specific polymorphisms of the gene that encodes cell prion protein (PrnP) in sheep have been associated with resistance / natural susceptibility to the development and progression of the disease. Combinations of alleles at three adjacent codons (136 [A/V], 154 [H/R], 171 [H/Q/R]) underpin the classification of 15 possible genotypes in risk classes, applicable in selection schemes where the maximum resistance is conferred by ARR allele, and the minimum by the VRQ allele. Although, after applying these programmes, the genetic structure of sheep populations has changed favourably, genotype association studies showed that no genotype is completely resistant to the infection, including homozygote ARR / ARR. With the discovery of atypical scrapie (Nor98), it became evident that the connection between the genetics of prion protein gene polymorphisms and susceptibility to the disease must be re-evaluated individually for each breed. In scrapie monitoring and control programmes, three diagnostic categories of the disease are observed: classical scrapie, atypical scrapie and BSE scrapie in small ruminant. This review shows the chronology of progress in the fight for the eradication of TSEs in sheep, 30 years after the BSE epidemic outburst, focusing especially on the link between the molecular diagnostic forms and the genetics of the disease. Â

Genetics of Prion Disease in Cattle.

Bovine spongiform encephalopathy (BSE) is a prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. As a transmissible disease of livestock, it has impacted food safety, production practices, global trade, and profitability. Genetic polymorphisms that alter the prion protein in humans and sheep are associated with transmissible spongiform encephalopathy susceptibility or resistance. In contrast, there is no strong evidence that nonsynonymous mutations in the bovine prion gene (PRNP) are associated with classical BSE (C-BSE) disease susceptibility, though two bovine PRNP insertion/deletion polymorphisms, in the putative region, are associated with susceptibility to C-BSE. However, these associations do not explain the full extent of BSE susceptibility, and loci outside of PRNP appear to be associated with disease incidence in some cattle populations. This article provides a review of the current state of genetic knowledge regarding prion diseases in cattle.

Influence of Breed and Genotype on the Onset and Distribution of Infectivity and Disease-associated Prion Protein in Sheep Following Oral Infection with the Bovine Spongiform Encephalopathy Agent

The onset and distribution of infectivity and disease-specific prion protein (PrP(d)) accumulation was studied in Romney and Suffolk sheep of the ARQ/ARQ, ARQ/ARR and ARR/ARR prion protein gene (Prnp) genotypes (where A stands for alanine, R for arginine and Q for glutamine at codons 136, 154 and 171 of PrP), following experimental oral infection with cattle-derived bovine spongiform encephalopathy (BSE) agent. Groups of sheep were killed at regular intervals and a wide range of tissues taken for mouse bioassay or immunohistochemistry (IHC), or both. Bioassay results for infectivity were mostly coincident with those of PrP(d) detection by IHC both in terms of tissues and time post infection. Neither PrP(d) nor infectivity was detected in any tissues of BSE-dosed ARQ/ARR or ARR/ARR sheep or of undosed controls. Moreover, four ARQ/ARQ Suffolk sheep, which were methionine (M)/threonine heterozygous at codon 112 of the Prnp gene, did not show any biological or immunohistochemical evidence of infection, while those homozygous for methionine (MARQ/MARQ) did. In MARQ/MARQ sheep of both breeds, initial PrP(d) accumulation was identified in lymphoreticular system (LRS) tissues followed by the central nervous system (CNS) and enteric nervous system (ENS) and finally by the autonomic nervous system and peripheral nervous system and other organs. Detection of infectivity closely mimicked this sequence. No PrP(d) was observed in the ENS prior to its accumulation in the CNS, suggesting that ENS involvement occurred simultaneously to that of, or followed centrifugal spread from, the CNS. The distribution of PrP(d) within the ENS further suggested a progressive spread from the ileal plexus to other ENS segments via neuronal connections of the gut wall. Differences between the two breeds were noted in terms of involvement of LRS and ENS tissues, with Romney sheep showing a more delayed and less consistent PrP(d) accumulation than Suffolk sheep in such tissues. Whether this accounted for the slight delay (∼5 months) in the appearance of clinical signs in Romney sheep is debatable since by the last scheduled kill before animals reached clinical end point, both breeds showed widespread accumulation and similar magnitudes of PrP(d) accumulation in the brain.

In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

BackgroundTransmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown.ResultsWe acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R154Q171 genotype). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R154Q171 genotype sheep.ConclusionsOur results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Antigenic mimicry-mediated anti-prion effects induced by bacterial enzyme succinylarginine dihydrolase in mice

Prions, the causative agents of prion diseases, are immunologically tolerated because their major component, prion protein (PrP), is a host-encoded molecule. Therefore, no effective prion vaccines have been developed. We previously showed that heterologous bovine and sheep PrP immunizations of mice overcame tolerance by an antigenic mimicry mechanism to efficiently induce anti-PrP auto-antibodies (Abs), significantly prolonging incubation times in mice subsequently infected with the mouse-adapted Fukuoka-1 prion. These results prompted us to investigate if non-mammal derived molecules able to antigenically mimic anti-prion epitopes, could act as prion vaccines. We show here that immunization of mice with recombinant succinylarginine dihydrolase, a bacterial enzyme with a peptide sequence similar to an anti-prion epitope, induced anti-PrP auto-Abs with anti-prion activity and significantly retarded survival times of the mice subsequently infected with Fukuoka-1 prions. These results might open a way for development of a new type of antigenic mimicry-based prion vaccine.

Microsecond Unfolding Kinetics of Sheep Prion Protein Reveals an Intermediate that Correlates with Susceptibility to Classical Scrapie

The microsecond folding and unfolding kinetics of ovine prion proteins (ovPrP) were measured under various solution conditions. A fragment comprising residues 94–233 of the full-length ovPrP was studied for four variants with differing susceptibilities to classical scrapie in sheep. The observed biexponential unfolding kinetics of ovPrP provides evidence for an intermediate species. However, in contrast to previous results for human PrP, there is no evidence for an intermediate under refolding conditions. Global analysis of the kinetic data, based on a sequential three-state mechanism, quantitatively accounts for all folding and unfolding data as a function of denaturant concentration. The simulations predict that an intermediate accumulates under both folding and unfolding conditions, but is observable only in unfolding experiments because the intermediate is optically indistinguishable from the native state. The relative population of intermediates in two ovPrP variants, both transiently and under destabilizing equilibrium conditions, correlates with their propensities for classical scrapie. The variant susceptible to classical scrapie has a larger population of the intermediate state than the resistant variant. Thus, the susceptible variant should be favored to undergo the PrP C to PrP Sc conversion and oligomerization.