Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrPC) and the disease-associated (PrPSc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrPSc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrPC and PrPSc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitopemapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminallytruncated form of PrPSc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: Identification of a novel C-terminally truncated prion fragment.

GENNARO, RENATO;
2011-01-01

Abstract

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrPC) and the disease-associated (PrPSc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrPSc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrPC and PrPSc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitopemapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminallytruncated form of PrPSc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2563286
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