Characterization of Recombinant, Soluble -Secretase from an Insect Cell Expression System WILLIAM D. MALLENDER, 1 DEBRA YAGER, LUISA ONSTEAD, MICHAEL R. NICHOLS, CHRISTOPHER ECKMAN, KUMAR SAMBAMURTI, LISA M. KOPCHO, JOVITA MARCINKEVICIENE, ROBERT A. COPELAND, and TERRONE L. ROSENBERRY Department of Pharmacology, Mayo Foundation for Medical Education and Research, and the Department of Research, Mayo Clinic Jacksonville, Jacksonville, Florida (W.D.M., D.Y., L.O., M.R.N., C.E., K.S., T.L.R.); and Department of Chemical Enzymology, The DuPont Pharmaceuticals Company, Wilmington, Delaware (L.M.K., J.M., R.A.C.) Received September 21, 2000; accepted December 1, 2000 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT The -site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N termi- nus of the amyloid  peptide, a major component of the plaques found in the brains of Alzheimer’s disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl pro- teases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the produc- tion of the A peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing A for the treatment of Alzheimer’s disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was conve- nient to purify and was enzymatically active and specific for cleaving the -secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme- linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately up- stream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide- based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer’s disease treatments. Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by neuronal loss in the brain and the presence of amyloid plaques and neurofibrillary tangles (Selkoe, 1991). The major components of amyloid plaque cores have been identified as two small peptide fragments derived from the amyloid precursor protein (APP), A42 and A40 (Glenner and Wong, 1984; Robakis et al., 1987; Tanzi et al., 1987; Miller et al., 1993). APP itself is a type I integral membrane protein with the A segment, which begins at D672 in the longest isoform, spanning the boundary of the exocytoplasmic region (28 amino acids) and the transmem- brane domain (12–14 amino acids). A is generated from APP by the proteolytic activity of the enzymes - and -secretase, which produce the amino- and carboxyl-terminal ends of A, respectively (Fig. 1) (see Checler, 1995). -Secretase cleavage also generates a soluble N-terminal fragment from APP (sAPP) (Seubert et al., 1993). Another enzyme, -secretase, cleaves APP at a position within the A sequence to produce a soluble APP (sAPP) (Esch et al., 1990). During the course of AD, A produced by the enzymes - and -secretase accu- mulates extracellularly in vivo and forms large, insoluble amyloid fibrils that elicit both cytotoxic and inflammatory responses after deposition in the brain (Cummings et al., 1996; Yankner, 1996). Thus, understanding the enzymes re- sponsible for the production of this toxic peptide is crucial to finding a therapeutic intervention point in AD (Selkoe, 1997; De Strooper and Konig, 1999). Whereas several studies suggested that known proteases like cathepsin D or caspase had activity similar to -secre- tase, it remained unclear whether these proteases were di- rectly responsible for the overproduction of A found in AD 1 Current address: Millennium Pharmaceuticals, Inc., Cambridge, Massa- chusetts. ABBREVIATIONS: AD, Alzheimer’s disease; APP, amyloid precursor protein; A, amyloid  peptide (those mentioned herein consist of 40 or 42 amino acids); sAPP, soluble APP; BACE, -site amyloid precursor protein cleaving enzyme; ELISA, enzyme-linked immunosorbent assay; HPLC, high-pressure liquid chromatography; AChE, acetylcholinesterase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TBS-T, Tris-buffered saline/Tween-20; HRP, horseradish peroxidase; CHO, Chinese hamster ovary; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DNP, dinitrophenol. 0026-895X/01/5903-619 –626$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 597/887115 Mol Pharmacol 59:619–626, 2001 Printed in U.S.A. 619 at ASPET Journals on July 20, 2018 molpharm.aspetjournals.org Downloaded from