Fig. 4
From: Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
Membrane attachment and topology of PrPC impact on its shedding. a Schematic representation of the expected subdomain localization of transmembrane PrP (PrP-TM; outside of lipid rafts) and PrP with an altered GPI-anchor signal sequence (PrPGPI-Thy1; in the core of lipid rafts) compared to wild-type PrP (PrP-WT; in the periphery of lipid rafts) at the plasma membrane. b Confocal microscopy showing that all PrP constructs are expressed and present at the cell surface upon transfection into PrP-KO N2a cells. c,d,e Representative western blots showing comparable expression of the constructs in lysates (c), revealing presence of both fl-PrP and C1 upon deglycosylation (d), and indicating correct processing and sorting as there is no altered glycopattern upon digestion of samples with Endo H (e). f Biochemical analysis upon incubation of cells with phospholipase C (PI-PLC) to cleave GPI-anchor structures. For PrP-WT, a reduction in cell-associated fl-PrP is accompanied by appearance of PrP in supernatants. Lack of this release in the case of PrP-TM confirms absence of a GPI-anchor and supports membrane attachment via a transmembrane domain. g Representative western blot analysis using the sPrPG228 Ab reveals lack of shed PrP for PrP-TM and reduced shedding in cells expressing PrPGPI-Thy1 compared to PrP-WT. Quantification of the ratio of shed PrP (g) referred to fl-PrP (c) is shown in H (PrP-WT was set to one; p = 0.00005 for PrP-TM to PrP-WT; p = 0.001 for PrPGPI-Thy1 to PrP-WT; n = 3)