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

doi:10.1083/jcb.138.3.671

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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, 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 Cephalon, Incorporated, West Chester, Pennsylvania 19380

Previous studies have demonstrated that NT2N neurons derivedfrom a human embryonal carcinoma cell line (NT2) constitutivelyprocess the endogenous wild-type β-amyloid precursor protein(APP) to amyloid β peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generatedby intracellular processing must also be present in these cells.Here we show that the NH₂-terminal fragment of APP generatedby β-secretase cleavage (APPβ) is indeed producedfrom the endogenous full length APP (APP_FL). Pulse–chasestudies demonstrated a precursor–product relationshipbetween APP_FL 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 intracellularAPPβ from wild-type APP appears to be a unique characteristicof postmitotic neurons, since intracellular APPβ was notdetected in several non-neuronal cell lines. Significantly,production of APPβ occurred even when APP was retainedin the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15°C, or by expression of exogenous APPbearing 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, Universityof 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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