scholarly journals Characterization of proteinase K-resistant N- and C-terminally truncated PrP in Nor98 atypical scrapie

2006 ◽  
Vol 87 (6) ◽  
pp. 1751-1760 ◽  
Author(s):  
Mikael Klingeborn ◽  
Lotta Wik ◽  
Magnus Simonsson ◽  
Lena H. M. Renström ◽  
Therese Ottinger ◽  
...  
Keyword(s):  
Prion Protein ◽  
Structural Gene ◽  
Proteinase K ◽  
Atypical Scrapie ◽  
Antigenic Composition ◽  
Electrophoretic Profile ◽  
Brain Extracts ◽  
Overlapping Region

An increasing number of scrapie cases with atypical characteristics, designated Nor98, have recently been recognized. Here, the proteinase K (PK)-resistant prion protein (PrP) fragments from two Swedish cases of Nor98 atypical scrapie have been characterized. The prominent, fast-migrating band in the distinct Nor98 Western immunoblot electrophoretic profile was determined to be of 7 kDa in size and was accordingly designated Nor98-PrP7. The antigenic composition of Nor98-PrP7, as assayed by a panel of anti-PrP antibodies, revealed that this fragment comprised a mid-region of PrP from around aa 85 to 148. N- and C-terminally truncated fragments spanning the mid-region of PrP have only been observed in the genetic prion disorder Gerstmann–Sträussler–Scheinker disease. It is shown here that the long-term PK resistance of Nor98-PrP7 is reduced compared with that of PrPres in classical scrapie. Enzymic deglycosylation did not change the distinct electrophoretic profile of Nor98-PrP7. A previously unidentified, PK-resistant, C-terminal PrP fragment of around 24 kDa was detected and its PK resistance was investigated. After deglycosylation, this fragment migrated as a 14 kDa polypeptide and was designated PrP-CTF14. Antigenic determination and the size of 14 kDa suggested a fragment spanning approximately aa 120–233. The existence of two PK-resistant PrP fragments, Nor98-PrP7 and PrP-CTF14, that share an overlapping region suggests that at least two distinct PrP conformers with different PK-resistant cores are present in brain extracts from Nor98-affected sheep. The structural gene of PrP in three Nor98-affected sheep was analysed, but no mutations were found that could be correlated to the aberrant PK-resistant profile observed.

scholarly journals Immunological characterization of abnormal prion protein from atypical scrapie cases in sheep using a panel of monoclonal antibodies

2006 ◽  
Vol 87 (12) ◽  
pp. 3715-3722 ◽  
Author(s):  
Anja Gretzschel ◽  
Anne Buschmann ◽  
Jan Langeveld ◽  
Martin H. Groschup
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Proteinase K ◽  
Atypical Scrapie ◽  
Surveillance Programme ◽  
Rapid Tests ◽  
The Brain ◽  
Sheep Scrapie ◽  
Scrapie Case

After the implementation of an active surveillance programme for scrapie in sheep in the EU, the number of diagnosed classical scrapie cases rose sharply and a novel kind of so-called atypical scrapie case was discovered. These atypical scrapie cases display unusual features concerning the distribution of the abnormal prion protein (PrPSc) in the brain, a distinct electrophoretic profile of PrPSc and an inconsistent reaction pattern in the currently used rapid tests. In this report, PrPSc of two German atypical sheep scrapie cases was characterized by epitope mapping using a panel of 18 monoclonal antibodies that were directed against epitopes located throughout the prion protein. This analysis suggests that PrPSc derived from atypical scrapie cases and treated with proteinase K is largely composed of an 11 kDa fragment (previously referred to as the 12 kDa band) and of polymeric fragments thereof. The 11 kDa band corresponds to a prion protein fragment spanning approximately aa 90–153 and may therefore represent a novel PrPSc type.

scholarly journals Five Novel PRNP Gene Polymorphisms and Their Potential Effect on Scrapie Susceptibility in Three Native Ethiopian Sheep Breeds

2020 ◽  
Author(s):  
Eden Yitna Teferedegn ◽  
Yalcin Yaman ◽  
Cemal Un
Keyword(s):  
Prion Protein ◽  
Prnp Gene ◽  
Potential Effect ◽  
Classical Scrapie ◽  
Proteinase K ◽  
The Novel ◽  
Atypical Scrapie ◽  
Sheep Breeds ◽  
Novel Variants ◽  
Scrapie Susceptibility

Abstract Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at codon 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.

scholarly journals Five Novel PRNP Gene Polymorphisms and Their Potential Effect on Scrapie Susceptibility in Three Native Ethiopian Sheep Breeds

2020 ◽  
Author(s):  
Eden Yitna Teferedegn ◽  
Yalcin Yaman ◽  
Cemal Un
Keyword(s):  
Prion Protein ◽  
Prnp Gene ◽  
Potential Effect ◽  
Classical Scrapie ◽  
Proteinase K ◽  
The Novel ◽  
Atypical Scrapie ◽  
Sheep Breeds ◽  
Novel Variants ◽  
Scrapie Susceptibility

Abstract Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene ( PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at position 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA) and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231 and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie. Keywords: Ethiopian sheep; Novel Variations; Polymorphism; Prion gene; Scrapie Susceptibility

scholarly journals Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc

Pathogens ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1060
Author(s):  
Joo-Hee Wälzlein ◽  
Karla A. Schwenke ◽  
Michael Beekes
Keyword(s):  
Prion Protein ◽  
In Vitro Propagation ◽  
Cell Model ◽  
Animal Experiments ◽  
Cationic Lipid ◽  
Brain Homogenate ◽  
Proteinase K ◽  
Lipid Mixture

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.

scholarly journals Five Novel PRNP Gene Polymorphisms and Their Potential Effect on Scrapie Susceptibility in Three Native Ethiopian Sheep Breeds

2020 ◽  
Author(s):  
Eden Yitna Teferedegn ◽  
Yalcin Yaman ◽  
Cemal Un
Keyword(s):  
Prion Protein ◽  
Prnp Gene ◽  
Potential Effect ◽  
Classical Scrapie ◽  
Proteinase K ◽  
The Novel ◽  
Atypical Scrapie ◽  
Sheep Breeds ◽  
Novel Variants ◽  
Scrapie Susceptibility

Abstract Background: Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at position 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results : Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion: Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.

scholarly journals Isolation and characterization of a polymerized prion protein

2002 ◽  
Vol 364 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Bao-Yuan LU ◽  
Jui-Yoa CHANG
Keyword(s):  
Prion Protein ◽  
Cd Spectroscopy ◽  
Reversed Phase ◽  
Size Exclusion ◽  
Proteinase K ◽  
Guanidinium Chloride ◽  
Isolation And Characterization ◽  
Size Exclusion Hplc ◽  
Β Sheet

A polymerized form of recombinant mouse prion protein (mPrP) domain 23–231 [mPrP-(23–231)], designated mPrP-z, was generated at acidic pH (pH 2–5) in the presence of selected concentrations of denaturant (2M guanidinium chloride or 5M urea). This isoform of mPrP is stable in acidic solution after removal of denaturant. It can be isolated and purified using reversed-phase HPLC or size-exclusion HPLC. mPrP-z bears structural properties that partially resemble those of scrapie prion. Unlike the native mPrP-(23–231) (mPrP-N), mPrP-z exhibits a high content of β-sheet structure, as shown by CD spectroscopy, and exists as an oligomer with an approximate molecular mass of 340000Da, as measured by light scattering. However, similarly to mPrP-N, mPrP-z contains the intact disulphide bond and is sensitive to digestion by proteinase K.

scholarly journals Characterization of Prion Disease Associated with a Two-Octapeptide Repeat Insertion

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1794
Author(s):  
Nicholas Brennecke ◽  
Ignazio Cali ◽  
Tze Mok ◽  
Helen Speedy ◽  
Laszlo Hosszu ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Genetic Variant ◽  
Molecular Subtype ◽  
Case Series ◽  
Low Risk ◽  
Proteinase K ◽  
Jakob Disease ◽  
Insight Into

Genetic prion disease accounts for 10–15% of prion disease. While insertion of four or more octapeptide repeats are clearly pathogenic, smaller repeat insertions have an unclear pathogenicity. The goal of this case series was to provide an insight into the characteristics of the 2-octapeptide repeat genetic variant and to provide insight into the risk for Creutzfeldt–Jakob disease in asymptomatic carriers. 2-octapeptide repeat insertion prion disease cases were collected from the National Prion Disease Pathology Surveillance Center (US), the National Prion Clinic (UK), and the National Creutzfeldt–Jakob Disease Registry (Australia). Three largescale population genetic databases were queried for the 2-octapeptide repeat insertion allele. Eight cases of 2-octapeptide repeat insertion were identified. The cases were indistinguishable from the sporadic Creutzfeldt–Jakob cases of the same molecular subtype. Western blot characterization of the prion protein in the absence of enzymatic digestion with proteinase K revealed that 2-octapeptide repeat insertion and sporadic Creutzfeldt–Jakob disease have distinct prion protein profiles. Interrogation of large-scale population datasets suggested the variant is of very low penetrance. The 2-octapeptide repeat insertion is at most a low-risk genetic variant. Predictive genetic testing for asymptomatic blood relatives is not likely to be justified given the low risk.

scholarly journals Five Novel PRNP Gene Polymorphisms and Their Potential Effect on Scrapie Susceptibility in Three Native Ethiopian Sheep Breeds

2020 ◽  
Author(s):  
Eden Yitna Teferedegn ◽  
Yalcin Yaman ◽  
Cemal Un
Keyword(s):  
Prion Protein ◽  
Prnp Gene ◽  
Potential Effect ◽  
Classical Scrapie ◽  
Proteinase K ◽  
The Novel ◽  
Atypical Scrapie ◽  
Sheep Breeds ◽  
Novel Variants ◽  
Scrapie Susceptibility

Abstract Background Classical scrapie susceptibility in sheep has been linked to three polymorphisms at codon 136, 154, and 171 in the prion protein gene (PRNP) whereas atypical scrapie susceptibility is related to polymorphisms at codon 141. Many other variants over the length of the PRNP have been reported. Some of the variants may play crucial roles in fighting against the emergence of a new form of scrapie disease. Scrapie surveillance, scrapie associated genotyping and PRNP characterization studies have been conducted across the globe. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breed’s PRNP gene has socioeconomic and scientific merits. This study aimed to identify PRNP variants in three native Ethiopian sheep breeds and their potential effect on scrapie susceptibility. Results Five novel variants were identified in the PRNP gene of three native Ethiopian sheep breeds. Four non-synonymous heterozygous substitutions i.e. H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) were detected. In addition to the novel variants, polymorphisms at codon 126,127,138,142,146,231, and 237 were also identified. The haplotype ARR was observed in Menz and Afar breeds at frequencies of 0.02 and 0.05 respectively. Neither ARR/ARR nor VRQ/VRQ genotypes were identified in the population under study. Conclusion Two of the novel variants at codon 99 and 103 that are placed closer to the proteinase K cleavage site and the variant at codon 116 in the palindrome region along with variants at codon 127 in glycine repeat domain may influence the conformational flexibility of prion protein. The rarity of ARR haplotype and the abundance of 141L variant demonstrated that the present study population was less resistant to classical scrapie and less predisposed to genotype associated atypical scrapie. This study provides a valuable dataset that can be potentially integrated into selective breeding strategies during interbreeding, crossbreeding and help to take precautionary measures against scrapie.

scholarly journals Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin

2008 ◽  
Vol 416 (2) ◽  
pp. 297-305 ◽  
Author(s):  
Sabrina Cronier ◽  
Nathalie Gros ◽  
M. Howard Tattum ◽  
Graham S. Jackson ◽  
Anthony R. Clarke ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Rocky Mountain ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Prion Strains ◽  
Human Counterpart ◽  
Jakob Disease ◽  
The Brain

Disease-related PrPSc [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrPC(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrPSc using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrPSc in its full-length form. In the present study, we show that thermolysin can degrade PrPC while preserving both PK-sensitive and PK-resistant isoforms of disease-related PrP in both rodent and human prion strains. For mouse RML (Rocky Mountain Laboratory) prions, the majority of PK-sensitive disease-related PrP isoforms do not appear to contribute significantly to infectivity. In vCJD (variant Creutzfeldt–Jakob disease), the human counterpart of BSE (bovine spongiform encephalopathy), up to 90% of total PrP present in the brain resists degradation with thermolysin, whereas only ∼15% of this material resists digestion by PK. Detection of PK-sensitive isoforms of disease-related PrP using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases.

scholarly journals Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice

2010 ◽  
Vol 91 (8) ◽  
pp. 2132-2138 ◽  
Author(s):  
Peter C. Griffiths ◽  
John Spiropoulos ◽  
Richard Lockey ◽  
Anna C. Tout ◽  
Dhanushka Jayasena ◽  
...  
Keyword(s):  
Great Britain ◽  
Western Blot ◽  
Prion Protein ◽  
Brain Lesion ◽  
Distribution Patterns ◽  
Atypical Scrapie ◽  
Wild Type ◽  
Survival Times ◽  
Incubation Periods

Twenty-four atypical scrapie cases from sheep with different prion protein genotypes from Great Britain were transmitted to transgenic tg338 and/or TgshpXI mice expressing sheep PrP alleles, but failed to transmit to wild-type mice. Mean incubation periods were 200–300 days in tg338 mice and 300–500 days in TgshpXI mice. Survival times in C57BL/6 and VM/Dk mice were >700 days. Western blot analysis of mouse brain samples revealed similar multi-band, protease-resistant prion protein (PrPres) profiles, including an unglycosylated band at ∼8–11 kDa, which was shown by antibody mapping to correspond to the ∼93–148 aa portion of the PrP molecule. In transgenic mice, the incubation periods, Western blot PrPres profiles, brain lesion profiles and abnormal PrP (PrPSc) distribution patterns produced by the Great Britain atypical scrapie isolates were similar and compatible with the biological characteristics of other European atypical scrapie or Nor98 cases.

Sign in / Sign up

Export Citation Format

Share Document