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© The Rockefeller University Press, 0021-9525/1997//671 $5.00
The Journal of Cell Biology, Volume 138, Number 3, , 1997 671-680


Article

Novel β-Secretase Cleavage of β-Amyloid Precursor Protein in the Endoplasmic Reticulum/Intermediate Compartment of NT2N Cells



Abraham S.C. Chyung*, Barry D. Greenberg{ddagger}, David G. Cook*, Robert W. Doms*, and Virginia M.-Y. Lee*

* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and {ddagger} Cephalon, Incorporated, West Chester, Pennsylvania 19380

Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type β-amyloid precursor protein (APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by β-secretase cleavage (APPβ) is indeed produced from the endogenous full length APP (APPFL). Pulse–chase studies demonstrated a precursor–product relationship between APPFL and APPβ as well as intracellular and secreted APPβ fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APPβ recovered from the cell lysates. Furthermore, the production of intracellular APPβ from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPβ was not detected in several non-neuronal cell lines. Significantly, production of APPβ occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.


Abbreviations used in this paper: Aβ, amyloid β; AD, Alzheimer's disease; APP, amyloid precursor protein; BFA, brefeldin A; CNS, central nervous system; Endo H, endoglycosidase H; IC, intermediate compartment; Nglyc F, N-glycosidase F; SFV, Semliki Forest virus.

Please address all correspondence to Dr. Virginia M.-Y. Lee, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Third Floor Maloney, HUP, Philadelphia, PA 19104-4283. Tel.: (215) 662-6427; Fax: (215) 349-5909; E-mail: vmylee{at}mail.med.upenn.edu



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