Positive-negative epitope-tagging of beta amyloid precursor protein to identify inhibitors of A beta processing

Brain Res Mol Brain Res. 2000 Dec 8;84(1-2):115-26. doi: 10.1016/s0169-328x(00)00230-8.

Abstract

In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.

MeSH terms

  • Alzheimer Disease / drug therapy
  • Alzheimer Disease / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Amyloid Precursor Protein Secretases
  • Amyloid beta-Protein Precursor / chemistry
  • Amyloid beta-Protein Precursor / genetics
  • Amyloid beta-Protein Precursor / immunology
  • Amyloid beta-Protein Precursor / metabolism*
  • Antibodies, Monoclonal / immunology
  • Aspartic Acid Endopeptidases
  • Blotting, Western
  • Cell Line
  • Culture Media, Conditioned / chemistry
  • Culture Media, Conditioned / metabolism
  • Dipeptides / pharmacology
  • Endopeptidases / metabolism*
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / chemistry
  • Epitopes / genetics
  • Epitopes / immunology
  • Epitopes / metabolism*
  • Humans
  • Immunohistochemistry
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / metabolism
  • Protease Inhibitors / analysis
  • Protease Inhibitors / pharmacology*
  • Protease Inhibitors / therapeutic use
  • Protein Processing, Post-Translational / drug effects*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / metabolism*
  • Sensitivity and Specificity
  • Transfection

Substances

  • Amyloid beta-Protein Precursor
  • Antibodies, Monoclonal
  • Culture Media, Conditioned
  • Dipeptides
  • Epitopes
  • Isoenzymes
  • Protease Inhibitors
  • Recombinant Fusion Proteins
  • Amyloid Precursor Protein Secretases
  • Endopeptidases
  • Aspartic Acid Endopeptidases
  • BACE1 protein, human
  • calpain inhibitor III