Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

Nobuto Kakuda,Satoru Funamoto,Sousuke Yagishita,Mako Takami,Satoko Osawa,Naoshi Dohmae,Yasuo Ihara

doi:10.1074/jbc.m513453200

Abstract

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.

Highlights

Amyloid ␤-protein (A␤)4 is the major component of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (AD)
We found that PS1/2 and amyloid precursor protein (APP) carrying familial AD (FAD) mutations increase the proportion of APP intracellular domains (AICD)-(49 –99) [14]
As FAD mutations cause an increase in the A␤42/A␤40 ratio [1], we assumed that relationships exist between ␥- and ␧-cleavage sites

Summary

Introduction

Amyloid ␤-protein (A␤)4 is the major component of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (AD). A␤s longer than A␤43 have slower mobilities (data not shown and see Ref. 22) and can be separated from the major A␤ band at about 4 kDa. The longer A␤s appear to be present in very small amounts, as they constituted around 10% of the total amount estimated from densitometric scanning of the Western blot (gel I; Fig. 1A and Table 1), assuming that the retention efficiency for each A␤ species is equal. Such high activity of ␥-secretase in our system made it possible to use conventional Western blotting to accurately quantify the amounts of A␤ and AICD produced.

Results

Conclusion