Submitted to: Prion
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 16, 2011
Publication Date: June 1, 2012
Citation: Silva, C.J. 2012. Using small molecule reagents to selectively modify epitopes based on their conformation. Prion. 6:(2)165-175. Interpretive Summary: Infectious prions and the non-infectious normal prion protein are both made of the same atoms, and they differ only in the three dimensional arrangement of their atoms. Some of these atoms are on the surface of the normal form, but are buried in the infectious form. Only those atoms on the surface are available to form new chemical bonds. Antibodies can recognize prion proteins by the arrangement of small groups of atoms at the surface. If the arrangement is disturbed by chemicals, then an antibody can no longer recognize the protein. We made chemicals that formed chemical bonds with the normal prion protein, but not with the infectious prion. Then we used antibodies to detect the prions in the presence of (non-binding) chemically modified normal prion protein. This method allows us to detect prions without using enzymes – an advantage that improves sensitivity of detection. In addition our method identifies the atoms on the surface of prions, to help scientists understand the structural difference between the normal and infectious forms.
Technical Abstract: PrPSc is an infectious protein. The only experimentally verified difference between PrPSc and their normal cellular isoform (PrPC) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrPSc isoform. The N-hydroxysuccinimide esters of acetic acid and 4-trimethylammoniumbutyric acid were synthesized and reacted with brain homogenates from Me7-infeced mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by Western blots probed with the antibodies 3F4, AH6, GE8 or MAB5424. The 3F4, AH6, and GE8 antibodies recognize an epitope that is encripted in the PrPSc, but not the PrPC isoform. These reagents permit the detection of prion infected brain homogenates without the need for proteinase K digestion. In addition they can be used, with an appropriate antibody to determine which amino acids of PrPSc are exposed on the surface and which are encrypted, thus providing useful structural information.