Processing of the Alzheimer's Disease Amyloid Precursor Protein inPichia pastoris: Immunodetection of α-, β-, and γ-Secretase Products

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Abstract
βA4 (Aβ) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed β- and γ-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the βA4 domain by α-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 βA4 and corresponds to sAPPα, the product of α-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPβ (the ectodomain released by β-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of α and β cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the βA4 domain yielded a 4 kDa product with the same electrophoretic mobility as βA4 synthetic peptide. These results suggest that the α-, β-, and γ-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 βA4 amyloid.