Alzheimer beta/A4-amyloid precursor protein: evidence for putative amyloidogenic fragment.
Recombinant baculovirus was used to overexpress human Alzheimer beta/A4-amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl-terminal fragments of APP by an immunoblotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti-human 695APP645-694) or an antibody against the amino terminus of beta/A4-amyloid (rabbit anti-human 695APP586-606). Anti-human 695APP645-694 identified APP holoprotein, a 25-kDa species, and a prominent group of carboxyl-terminal fragments of 17, 16, and 14 kDa, whereas anti-human 695APP586-606 identified APP holoprotein and a single prominent low-molecular-mass protein species comigrating with the 17-kDa carboxyl-terminal fragment identified by anti-human 695APP645-694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf9 cells or Sf9 cells infected with wild-type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a beta/A4-amyloid amino-terminal epitope into a 17-kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl-terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl-terminal fragments of APP containing an intact beta/A4-amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited beta/A4-amyloid.(ABSTRACT TRUNCATED AT 250 WORDS)
Gandy
S E
SE
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, NY 10021.
Bhasin
R
R
Ramabhadran
T V
TV
Koo
E H
EH
Price
D L
DL
Goldgaber
D
D
Greengard
P
P
D000544:Alzheimer Disease / Q000378:metabolism*; D000682:Amyloid / Q000096:biosynthesis*; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011995:Recombination, Genetic; D014777:Virus Diseases / Q000378:metabolism
D000682:Amyloid; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D011498:Protein Precursors