SECRETION OF BETA-AMYLOID PRECURSOR PROTEIN INVOLVES MULTIPLE CLEAVAGE SITES

A major histopathological feature of Alzheimer's disease is deposits o f a approximately 4-kDa beta-amyloid peptide derived by proteolytic pr ocessing from a precursor, the beta-amyloid precursor protein (beta-AP P). Proteolytic cleavage of beta-APP within the approximately 4-kDa be ta-amyloid domain permits the secretion of the amino-terminal portion of beta-APP while concomitantly producing a membrane bound approximate ly 9-kDa carboxyl-terminal fragment. We have characterized the proteol ytic cleavage site for beta-APP secretion by amino acid sequence analy sis of the approximately 9-kDa beta-APP carboxyl-terminal cleavage pro duct produced by recombinant and native expression systems. Recombinan t beta-APP was generated by a vaccinia virus expression system in CV-1 monkey fibroblasts; endogenous beta-APP was obtained using a fibrobla st line derived from an individual with Down's syndrome. The sequences of both unlabeled and metabolically radiolabeled approximately 9-kDa fragment from CV-1 cells reveal that the major (60%) secretory cleavag e site is after Lys16 of the beta-amyloid domain as reported previousl y; however, an additional cleavage site is seen after Phe19 (40%). Rad iosequence analysis of the carboxyl-terminal fragment purified from Do wn's syndrome fibroblasts indicates cleavage sites after Phe19, Glu22, and Gly25 and not after Lys16. CV-1 cells expressing beta-APP mutants lacking 4 and 6 amino acids adjacent to Lys16 yielded approximately 9 -kDa fragments with two identical cleavage sites, neither of which occ urred after the retained Lys16 but were after Glu11 and His13. These d ata suggest that secretion of 6-APP involves multiple proteinases and that the composition of these proteinases may vary within different ce ll backgrounds.