Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1996 International Society for Neurochemistry Evaluation of Cathepsins D and G and EC 3.4.24.15 as Candidate ,@-Secretase Proteases Using Peptide and Amyloid Precursor Protein Substrates Abraham M. Brown, Donna M. Tummolo, Michael A. Spruyt, J. Steven Jacobsen, and June Sonnenberg-Reines CNS Disorders, Wyeth-Avers! Research, Princeton, New Jersey, U.S.A. Abstract: No single protease has emerged that pos- sesses all the expected properties for /3-secretase, in- cluding brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of /3-amyloid pep- tide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of /3-amyloid peptide. Its molecular size of 55—60 kDa and inhibitory profile indi- cate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or re- lated peptides that contain substitudons reported to modulate /3-amyloid peptide production. We have also tested the ability of these enzymes to form carboxy-termi- nal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM —~ NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of ~3-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate /~-secretase. Although cathepsin G cleaves the model peptide cor- rectly, it displays poor ability to cleave the Swedish KM —~ NL peptide and does not generate carboxy-termi- nal fragments that are immunoreactive with amino-termi- nal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish “NL peptide” and “NL precursor” substrates appropriately. Our results suggest that cathepsin D could act as /3- secretase in the Swedish type of familial Alzheimer’s dis- ease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases. Key Words: /3-Secretase—Cathepsin D — Cathepsin G—EC 3.4.24.15—Amyloid precursor pro- tein — Proteolytic processing—Swedish-type familial Alz- heimer’s disease. J. Neurochem. 66, 2436—2445 (1996). Alzheimer’s disease (AD), clinically diagnosed by specific memory and behavioral changes, is typically confirmed at autopsy by the observation of senile plaques in the brain that contain deposits of insoluble /3-amyloid peptide (/3AP). At present, the identity of the endogenous enzymes involved in generating /3AP is unknown. Soluble ~AP is formed by two sequential proteolytic events: one that releases its amino-terminus (N-terminus) and the other its carboxy-terminus (C- terminus) from amyloid precursor protein (APP) (Si- sodia eta!., 1990; Lai eta!., 1995). The amino-termi- nal cleaving activity, which has been referred to as /3- secretase, cleaves APP at the peptide bond between residues methionine-596 and aspartic acid-597 (Met! Asp) in APP 695 [numbering according to Kang et al. (1987)], corresponding to aspartic acid-I (Asp1) in the ~AP sequence. This is implied from experiments demonstrating that cerebrovascular /3AP from AD or Down’s syndrome brain (Glenner and Wong, 1984) or from the cerebral cortex of AD brains (Mon et al., 1992) and soluble /3AP isolated from CSF (Vigo- Received October 19, 1995; revised manuscript received January 5, 1996; accepted January 15, 1996. Address correspondence and reprint requests to Dr. J. Sonnenberg- Reines at CNS Disorders, Wyeth-Ayerst Research, Princeton, NJ 08543, U.S.A. The present address of Dr. A. M. Brown is Department of Denien- tin Research, Burke Medical Research Institute. 785 Mamaroneck Avenue, White Plains, NY 10605, U.S.A. The present address of M. A. Spruyt is Cadus Pharm. Inc., 777 Old Saw Mill Road, Tarrytown, NY 10591, U.S.A. Abbreviations used: AD, Alzheimer’s disease; NAP, /~-amyloid peptide; APP, amyloid precursor protein; APP-REP, truncated amy- bid precursor protein7si; APP-REPNI, truncated amyloid precursor protein:si containing the NL Swedish mutation; CHO, Chinese ham- ster ovary; Cpp-A-A-F-pAB, N-Fl -(R,S)-carhoxy-3-phenylpropyl 1- Ala-Ala-Phe-p-aminobenzoate; C-99, a 99-amino-acid carboxy-ter- minal fragment of amyloid precursor protein; Cd 02. a 102-amino- acid carboxy-terminal fragment of amyloid precursor protein that has three additional amino acids at its amino-terminus; C-terminus, carboxy-terminus; CTF, carboxy-terminal fragmcnl; DTT, dithio- threitol; 1PB, immunoprecipitation buffer; MES, 2- ( N-rnorpholino )- ethanesulfonic acid; N-terminus, amino-terminus; SPA, scintillation proximity assay; T-PEN, N,N,N’,N’-tetrakis- ( 2-pyridylmethyl )eth- ylenediamine. 2436