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

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.

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

Journal of General Virology ◽

10.1099/vir.0.81618-0 ◽

2006 ◽

Vol 87 (6) ◽

pp. 1751-1760 ◽

Cited By ~ 47

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.

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

Biochemical Journal ◽

10.1042/bj20081235 ◽

2008 ◽

Vol 416 (2) ◽

pp. 297-305 ◽

Cited By ~ 99

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.

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Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

10.7287/peerj.preprints.694 ◽

2014 ◽

Author(s):

Alessandro Didonna ◽

Anja Colja Venturini ◽

Katrina Hartman ◽

Tanja Vranac ◽

Vladka Curin Serbec ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Prion Protein ◽

Biochemical Characterization ◽

Prion Diseases ◽

Physiological Role ◽

Prion Infection ◽

Promising Tool ◽

C Terminus ◽

N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

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Characterization of PrP binding proteins

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1994.0042 ◽

1994 ◽

Vol 343 (1306) ◽

pp. 443-445 ◽

Cited By ~ 8

Keyword(s):

Membrane Proteins ◽

Prion Protein ◽

Binding Proteins ◽

Mammalian Species ◽

Integral Membrane Proteins ◽

Infectious Particle ◽

Spongiform Degeneration ◽

The Brain ◽

Total Membrane

Prions cause spongiform degeneration in various mammalian species. The scrapie prion protein (PrP Sc ) is part of the infectious particle and may mediate infection and spreading of the disease in the brain. It was therefore of interest to purify and analyse PrP ligands (Plis). Plis were identified on ligand blots using either intact PrP or peptides corresponding to the central portion of PrP. Here, characterization of a 110 and a 125 kDa Pli is reported. Both Plis were found in total membrane fractions and could be extracted with carbonate indicating that they are not integral membrane proteins. On sucrose gradients both PrP ligands sedimented with high density particles.

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

10.21203/rs.2.19308/v4 ◽

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.

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

10.21203/rs.2.19308/v2 ◽

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

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

10.21203/rs.2.19308/v3 ◽

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.

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Production and Characterization of Monoclonal Antibodies Specific for Prion Protein

Prions ◽

10.1007/4-431-29402-3_25 ◽

2006 ◽

pp. 195-196

Author(s):

Masanori Morita ◽

Akimasa Ohmizu ◽

Hideki Maeno ◽

Takato Matsuo ◽

Yoichi Ogata ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Prion Protein

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Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

Biochemical Journal ◽

10.1042/bj20061264 ◽

2006 ◽

Vol 401 (2) ◽

pp. 475-483 ◽

Cited By ~ 64

Author(s):

Alana M. Thackray ◽

Lee Hopkins ◽

Raymond Bujdoso

Keyword(s):

Prion Protein ◽

Phosphotungstic Acid ◽

Limited Proteolysis ◽

Proteinase K ◽

Genotypic Differences ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Natural Scrapie ◽

Proteinase K Treatment ◽

Sheep Scrapie

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27–30 kDa (PrP27–30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27–30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

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Isolation and characterization of a polymerized prion protein

Biochemical Journal ◽

10.1042/bj3640081 ◽

2002 ◽

Vol 364 (1) ◽

pp. 81-87 ◽

Cited By ~ 24

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.

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