Abstract
The 4-kDa beta-amyloid peptide (A beta), a major constituent of parenchymal amyloid deposits in Alzheimer's disease, is derived from larger amyloid precursor proteins (APP). We have examined the metabolism of APP in Madin-Darby canine kidney cells stably transfected with cDNA encoding either wild-type human APP-695 or human APP-695 that harbors the Swedish double mutation associated with familial early-onset Alzheimer's disease. Although approximately 90% of total soluble APP secreted from wild-type cells is secreted basolaterally following cleavage at the alpha-secretase site, soluble derivatives cleaved near or at the amino terminus of A beta (presumably the "beta-secretase" site) are preferentially secreted into the apical compartment of SWE cells. Concomitantly, levels of a specific A beta-containing carboxyl-terminal fragment are elevated in SWE cell lysates. Using domain-specific biotinylation and release assays, we failed to detect a beta-secretase-generated soluble derivative (APPs beta) released from the surface of SWE cells. However, APPs beta can be detected in SWE cell lysates, consistent with "beta-secretase" cleavage occurring in an intracellular compartment. Finally, we demonstrate that A beta is secreted into the basolateral compartment of SWE cells and that the majority of these A beta-related species contains an amino-terminal aspartate residue (+1).
Highlights
Detergentfilter supports at confluent density and thenlabeled metaboli- soluble lysates of cells harvested at the end of each chase period cally from the basolateral compartmentfor 4 h with [35Slme- were subjected to immunoprecipitation with Myc-I, whereas thionine
An aliquot of total radiolabeled conditioned medium aliquots of radiolabeled medium from either the apical or bafrom either the apical or basolateral compartments was frac- solateral compartmentwere immunoprecipitated with monoclonal antibody (mAb) tionated by SDS-PAGE
Because hippocampanl eurons and epithelial cells appear to use similar mechanisms to sort viral proteins, we examined, in polarized MDCK cells, the trafficking and processing of human amyloid precursor proteins (APP)-695 and human APP-695 harboring an FAD mutation
Summary
The values indicated representtheaveragevaluesobtained from fourdifferent clones intwoindependent experiments. m, apical; El, basolatePraaln. el C , polariszeecdretion of
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have