Abstract: Recombinant baculovirus was used to overexpress human Alzheimer β/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 immuno‐blotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti‐human 695APP645–694 or an antibody against the amino terminus of β/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 Sf 9 cells or Sf 9 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 β/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 β/A4‐amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited β/A4‐amyloid. The identification of potentially amyloidogenic fragments in recom‐binantly engineered Sf 9 cells may provide a useful experimental system for determination of alternative sites of APP proteolysis and investigation of the processing mechanisms involved.
|Original language||English (US)|
|Number of pages||4|
|Journal||Journal of Neurochemistry|
|State||Published - Jan 1992|
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience