PROCESSING OF THE ALZHEIMERS-DISEASE AMYLOID PRECURSOR PROTEIN IN PICHIA-PASTORIS - IMMUNODETECTION OF ALPHA-SECRETASE, BETA-SECRETASE, ANDGAMMA-SECRETASE PRODUCTS

beta A4 (A beta) amyloid peptide, a major component of Alzheimer's dis ease (AD) plaques, is a proteolytic product of the amyloid precursor p rotein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secreti on involves cleavage within the beta A4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeas t Pichia pastoris was transfected with human APP(695) cDNA. In additio n 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 als o contained minor secreted forms detected by a monoclonal antibody spe cific for sAPP beta (the ectodomain released by beta-secretase cleavag e). Analysis of the cell lysates with specific antibodies also detecte d membrane-associated C-terminal fragments corresponding to the produc ts of alpha and beta cleavages. Moreover, immunoprecipitation of the c ulture medium with three antibodies directed at distinct epitopes of t he beta A4 domain yielded a 4 kDa product with the same electrophoreti c mobility as beta A4 synthetic peptide. These results suggest that th e alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to id entify the proteases involved in the generation of AD beta A4 amyloid.