Fig. 2
From: Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
PrP glycosylation mutants are differentially shed. Representative western blot characterization of PrPC depleted N2a cells (PrP-KO) transfected with wild-type PrP (PrP-WT) or the PrP glycomutants N180Q mutant (PrP-G1), N196Q (PrP-G2) and N180Q/N196Q (PrP-G3) showing the glycopattern in untreated lysates (a) as well as upon deglycosylation with PNGase F (b). c Digestion of lysates with Endo H does not reveal obvious alterations compared to the glycopattern in undigested samples (a), proving correct sorting and processing of the glycomutants. The differentially glycosylated C1 fragments resulting from α-cleavage of PrPC are also detected in A,B and C. Actin was detected as loading control. d Analysis in precipitated media supernatant using the sPrPG228 Ab reveals that not only PrP but likewise its C1 fragments are shed in PrP-WT and all glycomutants. e Quantification of PrP shedding. For normalization, intensity of sPrP signals in media was referred to total amounts of PrP in cell lysates. A trend of reduced shedding is observed for all glycomutants when compared to PrP-WT and a significant decrease is found for unglycosylated PrP-G3 (n = 3; p = 0.018). f Confocal microscopy of PrP surface staining confirms presence of all glycomutants at the cell surface (scale bar = 10 μm). g PNGase F digestion of precipitated cell culture supernatant. h For the unglycosylated G3 mutant we found a significant shift in the C1 to PrP ratio between cell lysates (membrane-bound forms; see asterisk in a) and supernatants (shed forms; see asterisk in b). Intensity for PrP-C1 in supernatants or lysates was referred to the respective fl-PrP signal. Shed C1 is significantly increased compared to membrane-bound C1 (n = 3; p = 0.007). i Schematic representation of PrP shedding summarizing the reduced shedding and the relative preference for C1 in mutants with impaired glycosylation