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ELISA analysis of β‐secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways
Author(s) -
Steinhilb Michelle L.,
Turner R. Scott,
Gaut James R.
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.0022-3042.2002.00764.x
Subject(s) - endocytic cycle , amyloid precursor protein , amyloid precursor protein secretase , endoplasmic reticulum , endocytosis , golgi apparatus , cleavage (geology) , secretory pathway , alpha secretase , presenilin , secretion , amyloid beta , microbiology and biotechnology , biology , chemistry , biochemistry , alzheimer's disease , medicine , peptide , cell , paleontology , disease , fracture (geology)
Limiting beta amyloid (Aβ) production could become an important therapeutic target in Alzheimer's disease (AD). Aβ is derived by the sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Aβ40 and Aβ42 production. Aβ production and, therefore, β‐secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of β‐secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the β‐cleaved product, APPswβ. Using this ELISA, we provide new information regarding the relative amount of β‐secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the β‐secretase cleavage was still detected. Experiments utilizing endocytosis‐impaired mutants of APPsw revealed that little or no β‐secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswβ and Aβ secretion, suggesting that protein interactions with this region normally impede β‐secretase cleavage. These results suggest that APPsw is cleaved by β‐secretase late in the secretory pathway.