Fig. 4

Cellular interaction between meprin β and APP occurs prior to endocytosis. a APP695myc and meprin β were overexpressed in HEK-293 T cells. Total lysates were immunoprecipitated with 9E10 (for APP) or anti-meprin β antibody. Co-purified proteins were detected using antibodies as indicated (WB: 9E10 for APP and reciprocal WB: meprin β). 20 μg of each lysate was used as transfection control (lysate input). As control, lysates of cells separately expressing either APP (“L1”) or meprin β (“L2”), were mixed post-lysis (lanes “Mix L1 + L2”). Co-purified proteins were immunoblotted as indicated. APP and meprin β specifically interacted only in co-transfected cells, but not post-lysis. b MEF cells were transiently co-transfected with APP695ΔNPxY and meprin β. Surface staining was performed, using IC16 antibody for detection of APP and an anti-meprin β antibody. Secondary antibodies Alexa-Fluor546 and Alexa-Fluor488 were used, respectively. The confocal image shows colocalization of APP (red) and meprin β (green). Single channels are depicted aside. The right image shows the cell morphology as bright-field picture (Scale bar: 10 nm). (C,D,E) GFP fluorescence shows colocalizing APP and meprin β (depicted in green) (Scale bar: 10 nm). c Only little colocalization of APP and meprin β can be found in the endoplasmic reticulum (PDI; depicted in red). d Colocalization was mostly found within the cis-golgi compartment (GM130; depicted in red). e Colocalization in early endosomes was hardly detectable (EEA1; depicted in red). f, g HEK-293 T cells were transiently co-transfected with APP695wt or APPΔNPxY, and meprin β or empty vector. Detection of proteins was carried out using specific antibodies. f 24 h post transfection, surface proteins were labeled using sulfo-NHS-LC-LC biotin and precipitated with NeutrAvidin agarose beads. g Quantification of biotinylated surface APP (graph shows mean ± S.E. (n = 3); statistical significance: * < 0.05, ***p < 0.001; t-test). h Aβ variants of conditioned medium was immunoprecipitated using IC16-conjugated dynabeads and separated on 8 M urea SDS-Page. Alternatively, cells were treated overnight with 100 nM tripartite BACE-1 inhibitor [26]. Samples were run on one gel, but rearranged for better presentation. Shown is one representative of six independent experiments. Generation of Aβ variants by meprin β was preserved in endocytosis impaired APPΔNPxY, indicating that cleavage occurred on the way to or at the cell surface