Marty Lehto

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of β-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

Current and future molecular diagnostics for prion diseases.

It is now widely held that the infectious agents underlying the transmissible spongiform encephalopathies are prions, which are primarily composed of a misfolded, protease-resistant isoform of the host prion protein. Untreatable prion disorders include some human diseases, such as Creutzfeldt–Jakob disease, and diseases of economically important animals, such as bovine spongiform encephalopathy (cattle) and chronic wasting disease (deer and elk). Detection and diagnosis of prion disease (and presymptomatic incubation) is contingent upon developing novel assays, which exploit properties uniquely possessed by this misfolded protein complex, rather than targeting an agent-specific nucleic acid. This review highlights some of the conventional and disruptive technologies developed to respond to this challenge.

Reply to “Properties of a disease-specific prion probe”

To the editor: In a recently published article1, Paramithiotis et al. describe antibodies specific for the prion Tyr-Tyr-Arg (YYR) repeat motif. These antibodies interact with the pathological isoform of the prion protein (PrPSc), but not with the normal cellular isoform (PrPC). Because of this restricted specificity, they suggest that YYR-specific antibodies could be useful for the diagnosis and treatment of prion diseases (Fig. 1). The monoclonal antibodies, all of the IgM isotype, were produced by immunizing mice with a synthetic peptide (CYYRRYYRYY). When coupled to magnetic beads, these YYR-specific antibodies immunoprecipitate PrPSc much more efficiently than PrPC. Notably, the Paramithiotis study did not rely on antibodies to YYR for specific detection of PrP. Their immunoblots were not ultimately probed with PrPSc-specific antibodies, but rather with ‘regular’ antibodies. The latter can detect PrP (but do not distinguish between PrPSc and PrPC) in a precipitate that could include any protein containing solvent-accessible tyrosine and arginine residues. This report is notably similar to that of Korth et al.2, who described a PrpSc-specific IgM (designated 15B3) after immunizing with full-length recombinant bovine PrP. The 15B3 epitope consists of three separate, linear segments of PrP (15B3-1, 15B3-2 and 15B3-3). The YYR epitope (bold) identified by Paramithiotis et al. is included in or located near two of the 15B3 segments (underlined): GSDYEDRYYR (15B3-1) and YYRPVDQYS (15B3-2). Thus, these two independent studies relying on the same method of immunoprecipitation have identified similar IgM antibodies interacting with the same region on PrP, and possibly with the same YYR motifs. The new reagents described could put an end to the quest for a PrPSc-specific antibody. Because the authors envision therapeutic use of the described antibodies, however, it seems appropriate to emphasize that YYR-specific antibodies could interact with any protein with tyrosine and arginine residues on its surface. In regard to PrPSc detection, it should be noted that no diagnostic application of 15B3 has been reported since the report of Korth et al. was published in in 1997. The availability of new diagnostic tests sensitive enough to ensure the protection of public health is an important issue3. For the design of such tests, there remains the choice between high-affinity antibodies that recognize both PrPSc and PrPC but require prior elimination of PrPC, and lower-affinity but PrPSc-specific antibodies.