Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris: immunodetection of alpha-, beta-, and gamma-secretase products.
betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.
Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.
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Le Brocque
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Henry
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Cappai
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Li
J E
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Tanner
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Galatis
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Gray
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Holmes
J R
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Underwood
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Beyreuther
C L
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Masters
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Evin
D000544:Alzheimer Disease / Q000378:metabolism*; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009447:Neuroblastoma; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human