Ca(2+)-dependent 68-kilodalton protease in familial Alzheimer's disease cells cleaves the N-terminus of beta-amyloid.
Lymphoblastoid cells derived from patients with early- and late-onset familial Alzheimer's disease express a Ca(2+)-dependent, 68-kDa protease which forms an SDS-stable and heat-labile complex with the beta-amyloid precursor protein. Utilizing this property, we prepared the protein by heat-dissociation of its immunoprecipitate with an antibody raised against the extracellular part of the beta-amyloid precursor protein. Disuccinimidyl suberate cross-linking analysis showed that in the presence of Ca2+ this protein binds to a synthetic oligopeptide corresponding to the first 12 amino acids of beta/A4-amyloid and its N-terminal flanks. Thin-layer chromatography of a reaction mixture of the 68-kDa protein and the oligopeptide demonstrated its proteolytic activity in the presence of Ca2+. Subsequent N-terminal amino acid sequencing of the cleaved fragment showed the cleavage site of the oligopeptide to be the Lys-2-Met-1-Asp beta A4-1 bonds. This protease also cleaves a natural substrate of 110-kDa beta-amyloid precursor protein, thereby generating the 16-kDa preamyloid peptide that accumulates abnormally in familial Alzheimer's disease lymphoblastoid cells. It does not, however, cleave the Gln beta A4-15-Lys16-Leu17 bond that is regarded to be the normal proteolytic site for the secretion of the beta-amyloid precursor protein. Analysis of the effects of protease inhibitors suggests that this 68-kDa protease is a Ca(2+)-dependent serine protease.
Matsumoto
A
A
Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
Fujiwara
Y
Y
D000544:Alzheimer Disease / Q000201:enzymology* / Q000235:genetics; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D003432:Cross-Linking Reagents; D012697:Serine Endopeptidases; D002118:Calcium