Using small molecule reagents to selectively modify epitopes based on their conformation

Abstract

PrPSc is an infectious protein. The only experimentally verified difference between PrPSc and its normal cellular isoform (PrP C) 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 detergent-solubilized brain extracts from Me7-infected mice, uninfected mice, 263K-infected hamsters or uninfected hamsters. These reaction mixtures were analyzed by western blots probed with the antibodies 3F4, 6D11, 7D9, AG4, AH6, GE8 or MAB5424. The 3F4, 6D11, AH6 and GE8 antibodies recognize an epitope that is encrypted in the PrPSc isoform, but exposed in the PrPC isoform. These reagents permit the detection of prion infected brain extracts 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. This approach was used to distinguish between the 263K and drowsy strains of hamsteradapted scrapie without the use of proteinase K.