Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells.
Deposition of beta-amyloid occurs in the brains of all sufferers of Alzheimer's disease. beta-amyloid is proteolytically derived from the beta-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and beta-secretase cleavage site of the Alzheimer's amyloid precursor protein. The beta-secretase cleavage event occurs at the extreme N-terminus of the beta-amyloid peptide. Our antiserum to the N-terminus of the beta-amyloid peptide (NT beta 4) specifically recognises beta-secretase cleaved species as opposed to intact beta APP. NT beta 4 specifically immunoprecipitates a 13 kDa fragment of beta APP (p13) which is potentially amyloidogenic. We have used these antisera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic beta APP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of beta APP fragments possessing the Asp-1 N-terminus of beta-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network.
Stephens
D J
DJ
Department of Surgery, St. George's Hospital Medical School, London, United Kingdom.
Austen
B M
BM
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D020126:Brefeldin A; D002460:Cell Line; D002846:Chromatography, Affinity; D003517:Cyclopentanes / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019085:Fluorescent Antibody Technique, Direct; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human