Endoproteolysis of -Secretase (-Site Amyloid Precursor Protein-cleaving Enzyme) within Its Catalytic Domain A POTENTIAL MECHANISM FOR REGULATION* Received for publication, December 31, 2002 Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213303200 Jason T. Huse‡, Damani Byant§, Yaxiong Yang§, Donald S. Pijak‡, Ian D’Souza§, James J. Lah¶, Virginia M.-Y. Lee, Robert W. Doms‡, and David G. Cook§** From the ‡Department of Microbiology and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, §Veterans Affairs Puget Sound Health Care System, Geriatric Research Education and Clinical Center and Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98108, and ¶Department of Neurology, Emory University School of Medicine, Atlanta, Georgia 30322 Sequential proteolysis of the amyloid precursor pro- tein (APP) by - and -secretase activities yields the amyloid  peptide that is widely deposited in the brains of individuals with Alzheimer’s disease. The membrane- anchored aspartyl protease -site APP-cleaving enzyme (BACE) exhibits all of the characteristics of a -secre- tase and has been shown to cleave APP at its -site in vitro and in vivo. We found that BACE undergoes cleav- age on a surface-exposed -helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoprote- olysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was pre- dominant, BACE was found primarily as endoproteo- lyzed fragments in pancreas, liver, and muscle. In addi- tion, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable of generating amyloid  peptide, suggesting that endopro- teolyzed BACE retains measurable -secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum, is en- hanced in the trans-Golgi network, and is sensitive to inhibitors of vesicular acidification. The membrane- bound proteases tumor necrosis factor -converting en- zyme and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of BACE on a post-translational level. Alzheimer’s disease (AD) 1 is characterized pathologically by the florid accumulation of insoluble amyloid  peptide (A) in the central nervous system (1). A causative role for A in AD progression has been accepted by many for some time (2, 3), and recent evidence from mouse models has underscored the importance of this peptide in the process of cognitive decline (4 – 6). A is produced by the sequential processing of the amy- loid precursor protein (APP) by two proteolytic activities that have historically been referred to as - and -secretase. Alter- natively, cleavage of APP by so-called -secretase activity within the A region of the protein precludes the generation of the peptide (7). Numerous studies have identified the mem- brane-anchored aspartyl protease BACE as the -secretase (8 –12), whereas evidence continues to mount that -secretase activity is mediated at least in part by the presenilin proteins (PS1 and PS2) (13–17). A group of metalloprotease disinte- grins, including tumor necrosis factor- converting enzyme (TACE), appears to be responsible for at least a significant fraction of cellular -secretase activity (18, 19). The central role of A in AD pathogenesis requires careful study of the proteins involved in APP/A processing and me- tabolism. The -secretase activity of TACE, for instance, may depend on C-terminal phosphorylation of the enzyme by pro- tein kinase C (18, 19). The presenilins have also been demon- strated to undergo phosphorylation (20 –22), although the func- tional impact of these modifications remains unclear. More thoroughly characterized is the endoproteolysis of PS1 and PS2, an event that generates distinct presenilin N- and C- terminal fragments that are thought to represent the biologi- cally active forms of the proteins. At endogenous levels the cleavage fragments of PS1 and PS2 predominate, and under conditions of overexpression, the vast majority of the preseni- lins exist as holoproteins, presumably due to the saturation of the endoproteolytic pathway (23–27). Despite its fairly recent discovery, BACE has already been the subject of numerous investigations designed to elucidate its basic cell biology. During maturation in the secretory pathway, BACE undergoes glycosylation at 3– 4 N-linked sites and is separated from its propeptide domain (28 –30). This latter proc- * This work was supported by National Institutes of Health Grant PO1 AG11542 (to R. W. D.), a Howard Hughes Medical Institute pre- doctoral fellowship (to J. T. H.), National Institutes of Health Grant NIA-R01 AG11762 (to G. Schellenberg), a Veterans Affairs Merit Award (to D. G. C.), and National Institute on Aging Grant AG05136 Alzhei- mer’s Disease Research Center/Project (to D. G. C.). The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Veterans Affairs Puget Sound Health Care System, Geriatric Research Education and Clinical Center (182B), 1660 S. Columbian Way, Seattle, WA 98108. Tel.: 206-768-5437; Fax: 206-764-2569; E-mail: dgcook@u.washington.edu. 1 The abbreviations used are: AD, Alzheimer’s disease; A, amyloid ; APP, amyloid precursor protein; BACE, -site APP-cleaving enzyme; BACE-Ct, BACE C-terminal fragment; BACE-Nt, BACE N-terminal fragment; TACE, tumor necrosis factor a converting enzyme; PS1, pre- senilin 1; PS2, presenilin 2; ER, endoplasmic reticulum; TGN, trans- Golgi network; HA, influenza hemagglutinin; FBS, fetal bovine serum; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation as- say; BHK, baby hamster kidney; DMEM, Dulbecco’s modified Eagle’s medium; SFV, Semliki Forest virus; mAb, monoclonal antibody. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 19, Issue of May 9, pp. 17141–17149, 2003 Printed in U.S.A. This paper is available on line at http://www.jbc.org 17141 by guest on May 25, 2020 http://www.jbc.org/ Downloaded from