Secretion of beta-amyloid precursor protein involves multiple cleavage sites.
A major histopathological feature of Alzheimer's disease is deposits of a approximately 4-kDa beta-amyloid peptide derived by proteolytic processing from a precursor, the beta-amyloid precursor protein (beta-APP). Proteolytic cleavage of beta-APP within the approximately 4-kDa beta-amyloid domain permits the secretion of the amino-terminal portion of beta-APP while concomitantly producing a membrane bound approximately 9-kDa carboxyl-terminal fragment. We have characterized the proteolytic cleavage site for beta-APP secretion by amino acid sequence analysis of the approximately 9-kDa beta-APP carboxyl-terminal cleavage product produced by recombinant and native expression systems. Recombinant beta-APP was generated by a vaccinia virus expression system in CV-1 monkey fibroblasts; endogenous beta-APP was obtained using a fibroblast 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 cleavage site is after Lys16 of the beta-amyloid domain as reported previously; however, an additional cleavage site is seen after Phe19 (40%). Radiosequence analysis of the carboxyl-terminal fragment purified from Down'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 occurred after the retained Lys16 but were after Glu11 and His13. These data suggest that secretion of beta-APP involves multiple proteinases and that the composition of these proteinases may vary within different cell backgrounds.
Zhong
Z
Z
Scios Nova Inc., Mountain View, California 94043.
Higaki
J
J
Murakami
K
K
Wang
Y
Y
Catalano
R
R
Quon
D
D
Cordell
B
B
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004314:Down Syndrome / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism; D000882:Haplorhini; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017421:Sequence Analysis / Q000379:methods; D014616:Vaccinia virus / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D009838:Oligodeoxyribonucleotides