Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris:: Immunodetection of α-, β-, and γ-secretase products

beta A4 (A beta) 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 beta A4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP(695) 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 beta A4 and corresponds to sAPP alpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPP beta (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 beta A4 domain yielded a 4 kDa product with the same electrophoretic mobility as beta A4 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 beta A4 amyloid.