Biochemical characterization of PrP^CGPIThy-1 mice.

Abstract

(A) Schematic representation of the PrPCGPIThy-1 fusion protein. PrPC comprises an N-terminal and a C-terminal (GPI-anchor) signal sequence, both of them not being present in the mature protein. Positions of an octameric repeat region (OR), a hydrophobic domain (HD), two N-glycans (N), a disulfide bridge, the 3F4 tag and epitope (present in mouse lines L27 and L16) and of the cleavage site for the ADAM10-mediated shedding (scissors) are indicated. The substitution of the GPI-SS of PrPC for the one of Thy-1 is shown in the dotted box. The substitution of the GPI-SS of PrPC for the one of Thy-1 is indicated. (B) Relative amount of PrPC mRNA from WTPrPC (n = 5) and PrPCGPIThy-1 (n = 5) measured by RT-qPCR. WTPrPC mRNA is set to one. Error bars are SEM. (C) Representative western blot showing PrPCGPIThy-1 protein expression in brain compared to WTPrPC. Bar chart shows the mean of PrP relative intensity related to actin intensity (used as a loading control). WTPrPC is set to 100%. Error bars are SEM. (D) Triton X-114 phase partitioning assay. Both, WTPrPC and PrPCGPIThy-1 were mainly found in the insoluble phase (ip), indicating that both carry a GPI-anchor as described [28] (dp: detergent phase; sp: soluble phase). Diagram shows the relative signal intensity of PrP from 3 independent experiments. WTPrPC ip is set to 100%. Error bars are SEM. (E) Representative western blot showing total homogenates (TH) from PrP KO (Prnp0/0, used as a negative control), WTPrPC and PrPCGPIThy-1 brains used for PNGase treatment (to eliminate N-glycans) followed by PrP immunoprecipitation (IP), shown in the adjacent western blot. PNGase+IP treated samples were then used to isolate the GPI-anchors, showed in (F). (F) Dot blot analysis of the GPI-anchors from deglycosylated, immunoprecipitated PrP and PK digested samples from mouse brain. Phosphatidylinositol (PI), mannose (man) and sialic acid (sial. acid) were detected as described in Methods. Note that the amounts of PI and mannose are similar between PrPCGPIThy-1 and WTPrPC whereas sialic acid is almost absent in PrPCGPIThy-1. Sialic acid background signal in PrPCGPIThy-1 could be explained by deglycosylation not being 100% achieved as shown in blot in (E).</p