Figure 7
From: The G127V variant of the prion protein interferes with dimer formation in vitro but not in cellulo
The V127PrP variant is complex glycosylated, located at the plasma membrane and forms homodimers similarly to G127PrP. (a) PrP forms disulfide bond-stabilized homodimers under non-reducing conditions in Western blot analysis. HeLa cells were transiently transfected with WTPrP (G127) or V127PrP containing a serine (S) or cysteine (C) at aa 132. The cysteine (C132) enables the formation of an intermolecular disulfide bond and allows separation of PrP dimers and PrP monomers on non-reducing SDS-PAGE. Cell lysates were prepared and denatured by boiling in Laemmli sample buffer with (+ ME) or without reducing agent (− ME). A white arrowhead represents monomeric PrP, the black arrowhead PrP homodimers. Right panel: quantifications of the dimerization efficiency measured densitometrically, ns not significant. Data represent mean ± SD of 4 independent experiments. (b) HeLa cells were transiently transfected with the V127PrP containing a serine (S132) or cysteine (C132) at aa 132 and were analyzed by indirect immunofluorescence. Fixed cells were either permeabilized or non-permeabilized and were detected with the 3F4 antibody. (c) Western blot analysis shows that dimerization of PrP does not interfere with complex glycosylation. HeLa cells were transiently transfected with the indicated constructs. To determine the glycosylation status lysates were treated either with peptide: N-Glycosidase F (PNGase F +, left panel) that cleaves high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins or endoglycosidase H, which cleaves only mannose rich oligosaccharides (Endo H +, right panel). Please note that the reaction buffer for PNGase F and Endo H contains a reducing agent, therefore only PrP monomers are seen in the PNGase F- and Endo H-treated samples. White arrowhead: monomer, black arrowhead: dimer.
