title: Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment.
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 (PrP(C)) and the disease-associated (PrP(Sc)) 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 PrP(Sc)-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP(C) and PrP(Sc). 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 epitope mapping 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-terminally truncated form of PrP(Sc) 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.
authors.affiliation: Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia.
authors.forename: Miha
authors.initials: M
authors.lastname: Kosmač
authors.forename: Simon
authors.initials: S
authors.lastname: Koren
authors.forename: Gabriele
authors.initials: G
authors.lastname: Giachin
authors.forename: Tatiana
authors.initials: T
authors.lastname: Stoilova
authors.forename: Renato
authors.initials: R
authors.lastname: Gennaro
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Legname
authors.forename: Vladka Čurin
authors.initials: VČ
authors.lastname: Serbec
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D018620:PrPSc Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D010446:Peptide Fragments; D018620:PrPSc Proteins