|Silva, Christopher - Chris|
|BADIOLA, JUAN - University Of Zaragoza|
|REQUENA, JESUS - University Of Santiago De Compostela|
|BOLEA, ROSA - University Of Zaragoza|
Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2016
Publication Date: 12/11/2016
Citation: Silva, C.J., Erickson-Beltran, M.L., Hui, C., Badiola, J.J., Nicholson, E.M., Requena, J.R., Bolea, R. 2016. Quantitating PrP polymorphisms present in prions from heterozygous scrapie-infected sheep. Analytical Chemistry. 89(1):854-861. doi:10.1021/acs.analchem.6b03822.
Interpretive Summary: Scrapie is a prion disease of sheep. Prions are infectious proteins that are able to convert a normal cellular prion protein into a prion to propagate an infection. The course of scrapie is controlled by substitutions (polymorphisms) in the normal cellular prion protein. Each sheep receives a copy of the gene producing the normal cellular prion protein from its parents. Animals with two different genes produce two differently substituted forms of the normal cellular prion protein and are referred to as heterozygotes. A sensitive method was developed to detect and quantify the normal cellular prion protein that makes up prions. Samples from heterozygous sheep were analyzed using this method and found to contain both polymorphisms in their prions. These results show that heterozygous animals contain prions that are made up of significant amounts of both normal cellular prion protein polymorphisms.
Technical Abstract: Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M132LGSXMSRPL141 (X = A or V), Y153XENMY158 (X= H or R), and Y166RPVDXY172 (X = H, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPC or PrPSc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPSc isolated from sheep heterozygous for their PrPC proteins. The limit of detection for these peptides was less than 50 attomole. Brain tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrPSc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrPSc and samples containing only the PK-resistant PrPSc. These results show that heterozygous animals contain PrPSc that is composed of significant amounts of both PrP polymorphisms.