Altered processing characteristics of beta-amyloid-containing peptides in cytosol and in media of familial Alzheimer's disease cells.
By pulse-chase analysis of cytosol and extracellular proteins, lymphoblastoid cells derived from patients with early- and late-onset familial Alzheimer's disease exhibit delayed and deficient processing characteristics of beta-amyloid precursor protein and its fragments. During 120 min chase incubation, 70-80% of the 16-kDa fragment with beta/A4-amyloid and cytoplasmic domains is removed in both cytosol and serum-free media of normal cells, whereas it remained unprocessed or even accumulated in familial Alzheimer's disease cells. Two-dimensional gel analysis further clarified that the 16-kDa peptide in familial Alzheimer's disease cells is highly phosphorylated compared with normals, and almost all of the accumulated 16-kDa preamyloid after 60 min chase incubation is phosphorylated in these cells. Tris-tricine gel analysis revealed a difference of processing characteristics between cytosol and extracellular beta/A4-containing peptides. Cytosol APP as the 16-kDa band in Tris-glycine gel was further separated into five peptides with molecular mass of 11 kDa-16.5 kDa, and some early-onset familial Alzheimer's disease cells contain 3-6 kDa peptides with beta/A4 domain, that are generated by proteolysis around transmembrane domain. Extracellular APP secreted during 24 h serum-free culture, however, exhibits only beta/A4-cytoplasmic domain-containing peptides with 12 kDa band as major component, but no 3-6 kDa peptides both in normal and familial Alzheimer's disease cells. These findings suggest that familial Alzheimer's disease lymphoblastoid cells harbour biochemical abnormality in the APP processing step to generate beta/A4-cytoplasmic domain peptides, and intracellular processing is crucial for eventual accumulation of beta/A4-amyloid in lymphoblastoid cells.
Matsumoto
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Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000378:metabolism
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010455:Peptides; D012697:Serine Endopeptidases