Abstract
Both the purified normal (protease-sensitive) isoform of the prion protein (PrP(C)) (Pergami, P., Jaffe, H., and Safar, J. (1996) Anal. Biochem. 236, 63-73) and recombinant prion protein (PrP) have been found to be in monomeric form (Mehlhorn, I., Groth, D., Stockel, J., Moffat, B., Reilly, D., Yansura, D., Willet, W. S., Baldwin, M., Fletterick, R., Cohen, F. E., Vandlen, R., Henner, D., and Prusiner, S. B. (1996) Biochemistry 35, 5528-5537; and this paper), and therefore PrP(C)-PrP(C) interactions were previously unknown. In this report we confirm recombinant PrP to be a monomer by analytical ultracentrifugation. However, by three lines of evidence (enzyme-linked immunosorbent assay (ELISA), cross-linking experiments, and size exclusion chromatography) we could also demonstrate that, under native conditions, at least part of the native bovine PrP(C) exists as a monomer-dimer equilibrium. A bovine PrP(C)-specific immuno-sandwich ELISA was developed and calibrated with recombinant PrP (Meyer, R. K., Oesch, B., Fatzer, R., Zurbriggen, A., and Vandevelde, M. (1999) J. Virol. 73, 9386-9392). By this ELISA we identified a distinct PrP(C) fraction and partially purified this protein. When serial dilutions of brain homogenate or partially purified PrP(C) were measured, using the peptide antibody C15S, a nonlinear dose-response curve was obtained. This nonlinearity was shown not to be due to an artifact of the procedure but to a monomer-dimer equilibrium of PrP(C) with preferential binding of the antibody to the dimer. From the curvature we could deduce the association constant (3.9 x 10(8) M(-1) at 37 degrees C). Accordingly, DeltaG degrees of the reaction was calculated (-48.6 kJ M(-1)), and DeltaH degrees (9.5 kJ M(-1)) as well as DeltaS degrees (0.2 kJ K(-1) M(-1)) were extrapolated from the van't Hoff plot. When serial dilutions of monomeric recombinant PrP were tested, only a straight line was obtained, supporting our hypothesis. Additional evidence of dimer formation was revealed by Western blotting of partially purified PrP(C) cross-linked by the homobifunctional cross-linker BS(3). Finally, size exclusion chromatography of partially purified PrP(C) fractions revealed an additional shoulder not observed with recombinant PrP. The difference in respect of dimer formation between native PrP(C) and recombinant PrP could be explained by the lack of glycosylation of the latter.
Highlights
The prion protein (PrP)1 was detected in attempts to identify the infective agent of transmissible spongiform encephalopathies [4]
Such protein-protein interactions were absent in bovine recombinant PrP [34], and highly purified PrPC has not been shown by others to form dimers in vitro [1]
To learn more about the nature of PrPC detected by this assay, PrPC was partially purified from normal bovine brain thalamus using the ELISA as a purification guide (Table I)
Summary
The prion protein (PrP) was detected in attempts to identify the infective agent of transmissible spongiform encephalopathies [4]. PrPC is converted into PrPSc by an unknown process [20, 23] Some prion diseases, such as familial Creutzfeldt-Jakob disease [24], Gerstmann-Straussler-Scheinker disease [25], and fatal familial insomnia [26] of humans, are caused by germline mutations of the PrP gene, which facilitate conversion into the pathological isoform. By antibody studies to monitor protein expression in native bovine brain tissues, we obtained convincing evidence of a monomer-dimer equlibrium of at least a fraction of PrPC This evidence was further confirmed by cross-linking and by size exclusion chromatography of partially purified PrPC. Such protein-protein interactions were absent in recombinant protein, showing for the first time a biochemical difference in respect to the native, glycosylated form
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