Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells.
Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000235:genetics; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009842:Oligopeptides / Q000276:immunology; D010449:Peptide Mapping; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D013373:Substance P / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D000939:Epitopes; D009842:Oligopeptides; D013373:Substance P; D004744:Enkephalin, Methionine; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human