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

• Published in: Prion 2012-04, Vol.6 (2), p.163-173
• Main Author: Silva, Christopher J.
• Format: Article
• Language: English
• Subjects: 263K; Amino Acid Sequence; Animals; antibody; Binding; Biology; Bioscience; Blotting, Western; Brain Chemistry; Calcium; Cancer; Cell; chemical modification; conformation; Cricetinae; Cycle; epitope; Epitopes - chemistry; Esters - chemistry; Landes; mass spectrometry; Me7; Mesocricetus; Mice; Molecular Sequence Data; Organogenesis; prion; Protein Conformation; Proteins; PrPSc Proteins - analysis; PrPSc Proteins - chemistry; Quaternary Ammonium Compounds - chemistry; Research Paper; Succinimides - chemistry
• ISSN: 1933-6896; 1933-690X
• DOI: 10.4161/pri.18795

PrP Sc is an infectious protein. The only experimentally verified difference between PrP Sc 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 PrP Sc 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 PrP Sc isoform, but exposed in the PrP C 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 PrP Sc 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 hamster-adapted scrapie without the use of proteinase K.