title: Evaluation of cathepsins D and G and EC 3.4.24.15 as candidate beta-secretase proteases using peptide and amyloid precursor protein substrates.
abstract: No single protease has emerged that possesses all the expected properties for beta-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of beta-amyloid peptide. 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 beta-amyloid peptide. Its molecular size of 55-60 kDa and inhibitory profile indicate 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 related peptides that contain substitutions reported to modulate beta-amyloid peptide production. We have also tested the ability of these enzymes to form carboxyl-terminal 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 beta-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate beta-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM --> NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-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 beta-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.
authors.affiliation: CNS Disorders, Wyeth-Ayerst Research, Princeton, New Jersey 08543, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Brown
authors.forename: D M
authors.initials: DM
authors.lastname: Tummolo
authors.forename: M A
authors.initials: MA
authors.lastname: Spruyt
authors.forename: J S
authors.initials: JS
authors.lastname: Jacobsen
authors.forename: J
authors.initials: J
authors.lastname: Sonnenberg-Reines
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001923:Brain Chemistry; D002402:Cathepsin D / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D012697:Serine Endopeptidases; D013379:Substrate Specificity
chemical_list: D016564:Amyloid beta-Protein Precursor; D010455:Peptides; D001224:Aspartic Acid; D008715:Methionine; D053829:Amyloid Precursor Protein Secretases; D002403:Cathepsins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D056649:Cathepsin G; D002402:Cathepsin D; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase