Carsten Korth

Prion (PrPSc)-specific epitope defined by a monoclonal antibody

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPCand PrPScare evident from increased β-sheet content and protease resistance in PrPSc(refs 1,2,3). Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models forthe PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

Recombinant full‐length murine prion protein, mPrP(23–231): purification and spectroscopic characterization

The cellular prion protein of the mouse, mPrPC, consists of 208 amino acids (residues 23–231). It contains a carboxy‐terminal domain, mPrP(121–231), which represents an autonomous folding unit with three α‐helices and a two‐stranded antiparallel β‐sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23–231), in the cytoplasm of Escherichia coli. mPrP(23–231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques. Comparison of near‐UV circular dichroism, fluorescence and one‐dimensional 1H‐NMR spectra of mPrP(23–231) and mPrP(121–231) shows that the amino‐terminal segment 23–120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three‐dimensional structure as detected by these techniques, indicating that the residues 121–231 might be the only polypeptide segment of PrPC with a defined three‐dimensional structure.

Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein.

Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. When considering presenting an antigen to a mouse with the goal of obtaining specific monoclonal antibodies against a misfolded or aggregated form of a host protein, it is desirable to increase the definition of a subtle conformational difference. This can be achieved by immunizing an antigen knockout mouse that has not developed self-tolerance against the respective antigen. Furthermore, if conformational isoforms and/or oligomeric forms of a protein sequence are understood to exist in an equilibrium, high and pure amounts of recombinant protein may increase the likelihood that a particular population of protein conformation passes an antigenic threshold necessary to start an immunogenic response. Pulling out the monoclonal antibodies by correct screening is essential. Screening against the pure misfolded or aggregated protein is often complicated by its poor solubility and hence the ability to immobilize. In the present case, immobilization of disease-associated PrP on nitrocellulose had been established as a conformation-sensitive screening method, allowing to freeze PrP in its distinguishable, disease-associated conformation. We are cautious to generalize conclusions of how to assess the generation of monoclonal antibodies against these particular protein isoforms to other diseases of protein misfolding and/or aggregation, but ultimately the present approach may inspire respective experiments.