scholarly journals Mapping the Prion Protein Using Recombinant Antibodies

1998 ◽  
Vol 72 (11) ◽  
pp. 9413-9418 ◽  
Author(s):  
R. Anthony Williamson ◽  
David Peretz ◽  
Clemencia Pinilla ◽  
Hadyn Ball ◽  
Raiza B. Bastidas ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Prion Protein ◽  
Disease Process ◽  
Cellular Prion Protein ◽  
Epitope Region ◽  
Molecular Events ◽  
C Terminus ◽  
Phage Display Libraries ◽  
Recombinant Molecule

ABSTRACT The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPCbut absent from PrPSc.

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

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.

scholarly journals Driving Forces Stabilizing Cellular Prion Protein (PrpC) Monomers and Dimers on the Cell Surface

2019 ◽  
Vol 116 (3) ◽  
pp. 516a
Author(s):  
Frances Tiffany Morden ◽  
India Claflin ◽  
Patricia Soto
Keyword(s):  
Cell Surface ◽  
Prion Protein ◽  
Driving Forces ◽  
Cellular Prion Protein

scholarly journals A Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
N. C. Ferreira ◽  
L. M. Ascari ◽  
A. G. Hughson ◽  
G. R. Cavalheiro ◽  
C. F. Góes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Prion Protein ◽  
Molecular Mechanisms ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Globular Domain ◽  
Mrna Levels ◽  
Spongiform Encephalopathies

ABSTRACTThe search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identifiedin vitroto be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPCmRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23–231) as the substrate and PrPScseeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90–231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPCglobular domain. J8 also reduced the fibrillation of mouse rPrP23–231seeded within vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPCon the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPCcontent on the cell surface.

scholarly journals The structural transformation comparison of the human Prion protein mutants V176G, E196A, and I215V by using molecular dynamics simulation

2021 ◽  
Author(s):  
Ashraf Fadhil Jomah ◽  
Sepideh Parvizpour ◽  
Jafar Razmara ◽  
Mohd Shahir Shamsir
Keyword(s):  
Prion Protein ◽  
Aqueous Media ◽  
Point Mutations ◽  
Cellular Prion Protein ◽  
Dynamics Simulation ◽  
Globular Domain ◽  
Salt Bridges ◽  
Wild Type ◽  
Structural Determinants ◽  
C Terminus

Abstract The point mutations in the gene coding of prion protein (PrP) originate human familial prion protein (HuPrP) diseases. Such diseases are caused by several amino acid mutations of HuPrP including V176G, I215V, and E196A located at the second, third native helix and in their loop, respectively. Determining the transition from cellular prion protein (PrPc) to pathogenic conformer (PrPSc) in the globular domain of HuPrP that results in pathogenic mutations is the key issue. The effects of mutation on monomeric PrP are detected in the absence of an unstructured N-terminal domain only. A MD simulation for each of these wild type mutants is performed to examine their structure in the aqueous media. The structural determinants are discerned to be different for wild-type HuPrP (125–228) variants compare to that of HuPrP mutations. These three mutations exhibiting diverse effects on the dynamical properties of PrP are attributed to the variations in the secondary structure, solvent accessible surface areas (SASAs), and salt bridges in the globular domain of HuPrP. High fluctuations that are evidenced around residues of the C-terminus of the helix 1 for V176G cause Gerstmann-Straussler-Scheinker (GSS) syndrome. Conversely, the occurrence of fluctuations around residues of helix 2, helix 3, and the loss of salt bridges in these regions for E196A and I215V mutants is responsible for Creutzfeldt-Jakob disease. Furthermore, small changes in the overall SASAs mutations strongly influence the intermolecular interactions during the aggregation process. The comparative results in this study demonstrate that the three mutants undergo different pathogenic transformations.

scholarly journals PrP glycoforms are associated in a strain-specific ratio in native PrPSc

2005 ◽  
Vol 86 (9) ◽  
pp. 2635-2644 ◽  
Author(s):  
Azadeh Khalili-Shirazi ◽  
Linda Summers ◽  
Jacqueline Linehan ◽  
Gary Mallinson ◽  
David Anstee ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Prion Disease ◽  
Prion Diseases ◽  
Cellular Prion Protein ◽  
Normal Brain ◽  
Western Blots ◽  
Prion Strains ◽  
Specific Manner ◽  
First Time

Prion diseases involve conversion of host-encoded cellular prion protein (PrPC) to a disease-related isoform (PrPSc). Using recombinant human β-PrP, a panel of monoclonal antibodies was produced that efficiently immunoprecipitated native PrPSc and recognized epitopes between residues 93–105, indicating for the first time that this region is exposed in both human vCJD and mouse RML prions. In contrast, monoclonal antibodies raised to human α-PrP were more efficient in immunoprecipitating PrPC than PrPSc, and some of them could also distinguish between different PrP glycoforms. Using these monoclonal antibodies, the physical association of PrP glycoforms was studied in normal brain and in the brains of humans and mice with prion disease. It was shown that while PrPC glycoforms can be selectively immunoprecipitated, the differentially glycosylated molecules of native PrPSc are closely associated and always immunoprecipitate together. Furthermore, the ratio of glycoforms comprising immunoprecipitated native PrPSc from diverse prion strains was similar to those observed on denaturing Western blots. These studies are consistent with the view that the proportion of each glycoform incorporated into PrPSc is probably controlled in a strain-specific manner and that each PrPSc particle contains a mixture of glycoforms.

scholarly journals Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation

2004 ◽  
Vol 85 (11) ◽  
pp. 3473-3482 ◽  
Author(s):  
Chan-Lan Kim ◽  
Ayako Karino ◽  
Naotaka Ishiguro ◽  
Morikazu Shinagawa ◽  
Motoyoshi Sato ◽  
...  
Keyword(s):  
Cell Surface ◽  
Prion Protein ◽  
Flow Cytometric Analysis ◽  
Degradation Pathway ◽  
Surface Flow ◽  
Cellular Prion Protein ◽  
Terminal Portion ◽  
Persistently Infected ◽  
Prion Propagation ◽  
In Cells

The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrPSc), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrPSc accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrPSc formation reduced PrPSc accumulation in cells persistently infected with prions. The 50 % effective dose was as low as ∼1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrPC) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb–PrPC complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrPSc formation by interfering with the regular PrPC degradation pathway.

scholarly journals Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106–126

1999 ◽  
Vol 342 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Mario SALMONA ◽  
Paolo MALESANI ◽  
Luca DE GIOIA ◽  
Stefano GORLA ◽  
Maurizio BRUSCHI ◽  
...  
Keyword(s):  
Prion Protein ◽  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Physicochemical Characteristics ◽  
Cellular Prion Protein ◽  
Random Coil ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
C Terminus ◽  
Molecular Determinants

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrPC), known as PrPres. The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 HD, PrP106-126 A and PrP106-126 K, with L-His → D-His, His → Ala and His → Lys substitutions respectively at position 111, PrP106-126 NH2 with amidation of the C-terminus, PrP106-126 V with an Ala → Val substition at position 117, and PrP106-126 VNH2 with an Ala → Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His111 is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly126 yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala → Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH2 on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

Production and Characterization of a Panel of Monoclonal Antibodies Against Native Human Cellular Prion Protein

Hybridoma ◽  
2009 ◽  
Vol 28 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Michael Jones ◽  
Victoria McLoughlin ◽  
John G. Connolly ◽  
Christine F. Farquhar ◽  
Ian R. MacGregor ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Cellular Prion Protein

Activated platelets of patients with paroxysmal nocturnal hemoglobinuria express cellular prion protein

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 341-343 ◽  
Author(s):  
Karel Holada ◽  
Jan Simak ◽  
Antonio M. Risitano ◽  
Jaroslaw Maciejewski ◽  
Neal S. Young ◽  
...  
Keyword(s):  
Prion Protein ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Surface Expression ◽  
Cellular Prion Protein ◽  
Terminal Part ◽  
Western Blots ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Membrane Spanning ◽  
Gpi Proteins

Abstract Cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that contains a putative membrane-spanning section. Patients with paroxysmal nocturnal hemoglobinuria (PNH) lack GPI proteins on the surface of somatically mutated hematopoietic stem cell and its progeny. Platelet expression of PrPc was studied in 8 PNH patients. Resting PNH (CD55−) platelets were devoid of surface PrPc, but activation of platelets resulted in the surface expression of PrPc. Expressed PrPc was detected by 2 monoclonal antibodies (mAbs) against the N-terminal part of the molecule but not by mAb 6H4, which binds at the C-terminus beyond the membrane-spanning section. However, 6H4 detected PrPc on Western blots of PNH platelets, demonstrating that the lack of 6H4 binding was not caused by PrPc truncation. Our results indicate that in the absence of GPI anchor, PrPc can be expressed intracellularly and up-regulated on the platelet membrane, likely in a transmembrane form with the C-terminal part of the molecule inserted into the cytoplasm.

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