β-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE

Science

Volumn 286, Issue 5440, 1999, Pages 735-741

  Vassar, Robert ^a   Bennett, Brian D ^a   Babu Khan, Safura ^a   Kahn, Steve ^a   Mendiaz, Elizabeth A ^a   Denis, Paul ^a   Teplow, David B ^b   Ross, Sandra ^a   Amarante, Patricia ^a   Loeloff, Richard ^a   Luo, Yi ^a   Fisher, Seth ^a   Fuller, Janis ^a   Edenson, Steven ^a   Lile, Jackson ^a   Jarosinski, Mark A ^a   Biere, Anja Leona ^a   Curran, Eileen ^a   Burgess, Teresa ^a   Louis, Jean Claude ^a   Collins, Frank ^a   Treanor, James ^a   Rogers, Gary ^a   Citron, Martin ^a   more..

^a AMGEN INC   (United States)

^b MASSACHUSETTS GENERAL HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMYLOID BETA PROTEIN; AMYLOID PRECURSOR PROTEIN; ANTISENSE OLIGONUCLEOTIDE; ASPARTIC PROTEINASE; BETA SECRETASE; MEMBRANE ENZYME; MESSENGER RNA; PROTEINASE; UNCLASSIFIED DRUG;

ALZHEIMER DISEASE; ARTICLE; CONTROLLED STUDY; ENZYME ACTIVITY; ENZYME INHIBITION; ENZYME LOCALIZATION; ENZYME SPECIFICITY; GENE OVEREXPRESSION; HUMAN; HUMAN CELL; HUMAN TISSUE; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN DEGRADATION; PROTEIN PROCESSING;

ALZHEIMER DISEASE; AMINO ACID MOTIFS; AMINO ACID SEQUENCE; AMYLOID BETA-PROTEIN; AMYLOID BETA-PROTEIN PRECURSOR; AMYLOID PRECURSOR PROTEIN SECRETASES; ANIMALS; ASPARTIC ENDOPEPTIDASES; BINDING SITES; BRAIN; CELL LINE; CLONING, MOLECULAR; ENDOPEPTIDASES; ENDOSOMES; GENE EXPRESSION; GENE LIBRARY; GOLGI APPARATUS; HUMANS; HYDROGEN-ION CONCENTRATION; MOLECULAR SEQUENCE DATA; OLIGONUCLEOTIDES, ANTISENSE; PEPTIDES; PROTEASE INHIBITORS; RECOMBINANT FUSION PROTEINS; RNA, MESSENGER; TRANSFECTION;

EID: 0033595706     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5440.735     Document Type: Article

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32. Immunocytochemistry of paraformalderiyde-fixed Triton X-100 permeabilized cells followed standard protocols (12). Primary antibodies and dilutions were the following: anti-HA monoclonal or polyclonal antibodies (Covance) 1:100; anti-58K (Sigma) 1:20; and anti-transferrin receptor (Dako) 1:20; anti-APPsβsw 1:500. Alexa 488 (green) and Alexa 594 (red) secondary antibodies (Molecular Probes) were used at 1:200 dilution.
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36. For the soluble BACE-IgG cell line, we generated a fusion construct encoding BACE amino acids 1-460 (ending at the start of the predicted transmembrane sequence) fused in frame with a three-amino acid linker (alanine-valine-threonine) and the Fc portion of human IgG1 (starting at amino acid 29; GenBank accession number X70421). BACE-IgG was purified from conditioned media of stably transfected 293T cells with Protein A columns. Substrate peptides were synthesized and labeled with dinitrophenol at P8′ to allow easy ultraviolet detection of cleavage products after reversed phase high performance liquid chromatography. The APPwt substrate sequence was TTRPGSGLTNIKTEEISEVKMDAEFRHDK(dnp)G; in the Swedish mutant variant, KM is substituted by NL, and in the MV mutant, M is substituted by V. All assays were performed with the same batch of BACE-IgG. Substrates were used at 30 μM in a 50-μl assay. All assays were buffered with 50 mM acetic acid (pH 4.5) unless otherwise indicated. Enzyme and substrate were incubated between 30 min and 18 hours at room temperature, then the reaction mixtures were quenched and analyzed by reversed-phase HPLC Both product and substrate were monitored by absorbance at 360 nm. Products were identified by retention time comparison with a reference peptide run under identical conditions.
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41. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X indicates any residue.
42. We thank E. H. Koo for monoclonal antibody 5A3, T. Woolf for technical advice on antisense experiments, G. Zajic for help with microscopy, and N. Davidson, T. Livelli, and J. Ngai for helpful discussions. We also thank D. Paulin, D. Olivares, and G. Zajic for preparation of figures.