Fig. 1
From: Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
A new antibody directed against shed PrPC reveals important aspects of the ADAM10-mediated cleavage. a Shedding of murine PrPC by ADAM10 at the plasma membrane (PM). The sPrPG228 Ab is directed against the C-terminus Gly228 exposed after release of shed PrP into the extracellular space (ex). b Representative western blot of forebrain homogenates from Prnp0/0, wild-type control, ADAM10 cKO and tga20 mice, first probed with sPrPG228 Ab and re-probed with POM2 against fl-PrP. An asterisk indicates position of signal from the initial detection of sPrP (in controls and tga20 resulting in overexposure and blurred appearance when re-probed with POM2) and demonstrates a small shift in molecular weight between sPrP and fl-PrP. c Western blot of tga20 brain shows that all PrPC glycoforms can be shed, though a clear preference exists for the diglycosylated form. d Glycopattern analysis in three C57BL/6 mouse forebrains and quantification of glycoform proportions using sPrPG228 Ab and POM1 reveals a shift towards the diglycosylated form in sPrP compared to fl-PrP. e Western blot showing inhibitory effects of dominant-negative ADAM10 in forebrains of transgenic mice (A10 d.n.) compared to controls on PrPC shedding. Premature/mature ADAM10 and C-terminal fragments (CTF) confirm genotypes. Quantification shows relative levels of sPrP referred to fl-PrP signal (controls set to one; n = 4; p = 0.014). f, g Representative analysis of precipitated supernatants and cell lysates of N2a cells treated with resveratrol (Resv), tamibarotene (Am80) or GI254023X (GI), GM6001 and Batimastat. Resveratrol and Am80 increased shedding (f), whereas the ADAM10-specific inhibitor GI (f) and metalloprotease inhibitors GM6001 and Batimastat (g) completely abolished it. f Re-probing the supernatant blot with POM2 shows an increase in fl-PrP released by other mechanisms. g No obvious effect on fl-PrP (here deglycosylated (PNGase F)) or N1/C1 levels was detected upon treatment. h Glycopattern comparison between sPrP (supernatants) and fl-PrP (lysates; n = 3) reveals predominance of shed diglycosylated PrP in N2a cells similar to findings in mice (d). β-actin = loading control; “d, m, u” = di-, mono-, unglycosylated PrP