A monomer-dimer equilibrium of a cellular prion protein (PrPC) not observed with recombinant PrP

被引:78
作者
Meyer, RK
Lustig, A
Oesch, B
Fatzer, R
Zurbriggen, A
Vandevelde, M
机构
[1] Univ Bern, Inst Anim Neurol, TSE Reference Ctr, CH-3012 Bern, Switzerland
[2] Univ Basel, Bioctr, CH-4056 Basel, Switzerland
[3] Univ Zurich, Prionics Ltd, CH-8057 Zurich, Switzerland
关键词
D O I
10.1074/jbc.M007114200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Both the purified normal (protease-sensitive) isoform of the prion protein (PrPC) (Pergami, P., Jaffe, Il., 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, Ri., Fletterick, R., Cohen, F. E., Vandlen, R., Henner, D., and Prusiner, S. B. (1996) Biochemistry 35, 5528-5537; and this paper), and therefore PrPC-PrPC 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 PrPC exists as a monomer-dimer equilibrium. A bovine PrPC-specific immunosandwich ELISA was developed and calibrated with recombinant PrP (Meyer, R. It., Oesch, B., Fatter, R., Zurbriggen, A., and Vandevelde, M. (1999) J. Virol. 73, 9386-9392). By this ELISA we identified a distinct PrPC fraction and partially purified this protein. When serial dilutions of brain homogenate or partially purified PrPC 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 PrPC 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 degreesC). 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 PrPC cross-linked by the homobifunctional cross-linker BS3. Finally, size exclusion chromatography of partially purified PrPC fractions revealed an additional shoulder not observed with recombinant PrP. The difference in respect of dimer formation between native PrPC and recombinant PrP could be explained by the lack of glycosylation of the latter.
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页码:38081 / 38087
页数:7
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