The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

Abstract

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

“…3 , reproduced here for convenience. The reported epitopes are based on peptide competition and differential binding to PrP C mutants.…”

“…Hence CC2 binders acted similarly to pharmacological "partial antagonists", triggering opposite effects depending on the presence or absence of additional ligands. Crucially, neither POM2 nor POM3 sterically hinder POM1 binding 3,4 and the non-toxic GD antibody POM5, which did not compete for the POM1 epitope 3 ( Supplementary Fig. 9d) did not rescue neurotoxicity ( Supplementary Fig.…”

“…The scFvPOM2 gene, constructed previously using the phage display method 3 The clarified soluble protein extract was loaded onto a Ni-NTA agarose (Qiagen) column equilibrated with 50 mM Tris and 100 mM NaCl, pH 7.0. The unbound protein and impurities were washed off with 50 mM Tris, 100 mM NaCl and 50 mM Imidazole pH 7.0 and the elution was performed with 50 mM Tris, 100 mM NaCl and 500 mM Imidazole pH 7.0.…”

Nanopore analysis

“…(One intriguing method to overcome this would be to co-crystallize an antibody-peptide complex in which the antibody prevents an aggregate from forming.) 55,56 Nanopore analysis is ideally suited for studying peptides which can adopt multiple conformations since each molecule is interrogated individually. It is also very sensitive since in theory a single molecule can be detected.…”

Characterization of Conformation-dependent Prion Protein Epitopes

“…Because this approach is laborious, ELISA-based peptide scanning has been the preferred method to map the approximate locations of epitopes. However, surprisingly few anti-PrP mAbs are amenable to this form of characterization, leading previous investigators to infer the existence of discontinuous PrP epitopes (12)(13)(14)(15)(16)(17). To date, the involvement of specific amino acid residues in such hypothesized conformational epitopes has not been described.…”