Location: Location not imported yet.Title: Probing the structure of GPI-less PrPSc by limited proteolysis(Abstract)) Author
|Silva, Christopher - Chris|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/9/2012
Publication Date: 5/12/2012
Citation: Vazquez-Fernandez, E., Pastrana, M., Ramos, A., Alonso, J., Stitz, L., Vindal, E., Dynin, I.A., Silva, C.J. 2012. Probing the structure of GPI-less PrPSc by limited proteolysis(Abstract). Meeting Abstract. Prion 6(supplement),26-27. Interpretive Summary:
Technical Abstract: Limited proteolysis is a very useful tool to pinpoint flexible regions within scrapie prion protein (PrPSc), but due to carbohydrate and glysosylphosphatidylinositol (GPI) moieties, and limitations of the analytical techniques, until now it was impossible to characterize accurately these regions. To provide more knowledge about the structure of PrPSc, we have worked with scrapie infected transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. This model made possible the exact detection and location of flexible, proteinase K (PK) susceptible regions by using limited proteolysis followed by western blot and mass spectrometry analysis. Samples were treated with 25 ug/mL of PK, deglycosylated, subjected to Tricine-SDS-PAGE, and probed with antibody R1, which recognizes epitope Y226-S231. We have detected 7 bands with apparent MWs of approximately 17, 14.6, 13, 12, 10.2, 8 and 6.7 kDa. Samples were also analyzed by MALDI, and 13 different peaks were identified, with exact MWs of 17147, 16726, 16371, 13606, 13463, 12173, 12041, 11171, 9687, 9573, 8358, 7436 and 6274. They match quite well bands identified by western blot analysis and correspond to cleavages at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179, respectively. The first 9 cleavages, up to position 153, match very well PK cleavage sites previously identified in wild type PrPSc by our group. These results reinforce the hypothesis that the structure of PrPSc consists of a series of short highly PK-resistant beta-sheet strands connected by short flexible loops and turns exhibiting higher sensitivity to PK, and that a sizeable C-terminal stretch of PrPSc is highly resistant to PK and therefore perhaps constituted by beta-sheet secondary structure.