Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein

Abstract

beta-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPPbeta and its C-terminal fragment CTFbeta. CTFbeta is subsequently cleaved by gamma-secretase to produce the neurotoxic/synaptotoxic amyloid-beta peptide (Abeta) that accumulates in Alzheimer's disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPIBACE substantially up-regulates the secretion of both sAPPbeta and amyloid-beta peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-beta peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695 isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770 was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased beta-amyloid production.

Fig. 1.

Schematic diagram of MycHis-tagged human BACE (WT-BACE) and the GPI-anchored construct (GPI-BACE). The amino acid sequence of WT-BACE at the start of the transmembrane domain (bold) is shown. In GPI-BACE, the transmembrane, cytosolic, and MycHis domains were replaced with the 24-aa GPI anchor signal sequence (bold, underlined) of human carboxypeptidase M. The GPI attachment site is marked (*).

Fig. 2.

Expression of WT-BACE and GPI-BACE in SH-SY5Y cells. SH-SY5Y cells were stably transfected with either WT-BACE or GPI-BACE as described in Experimental Procedures. Cell lysates were prepared from transfected (WT or GPI) and untransfected (Un) cells, and immunoblotted by using the monoclonal 9B21 antibody to BACE or a polyclonal β-actin antibody.

Fig. 3.

Release of GPI-BACE from cells by exogenous PI-PLC treatment. Confluent SH-SY5Y cells stably expressing WT-BACE or GPI-BACE were incubated in OptiMEM ± PI-PLC for 7 h, after which the medium was collected, centrifuged at 150,000 × g for 30 min, and concentrated. Equal amounts of protein from media samples were analyzed by SDS/PAGE and immunoblotted with the monoclonal 9B21 antibody to BACE.

Fig. 4.

Isolation of lipid rafts from SH-SY5Y cells stably expressing WT-BACE or GPI-BACE. Lipid rafts were prepared as described in Experimental Procedures from cells stably expressing WT-BACE (A) or GPI-BACE (B). Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed by SDS/PAGE and immunoblotted with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody.

Fig. 5.

Comparison of APP cleavage by WT-BACE and GPI-BACE. Confluent untransfected SH-SY5Y cells or cells stably expressing WT-BACE or GPI-BACE were incubated in OptiMEM for 16 h, after which the medium was collected and concentrated and cell lysates were prepared. Equal amounts of protein from media and lysates were analyzed by SDS/PAGE and immunoblotting. (A) Detection of full-length APP and β-actin in cell lysates with the polyclonal Ab54 antibody to APP and a polyclonal β-actin antibody. (B) Detection of sAPPα in media by using the monoclonal 6E10 antibody. (C) Detection of sAPPβ in media by using antibody G26, which recognizes the epitope of APP produced by β-site cleavage. Densitometric analysis of immunoblots was carried out, and the mean results ± SEM (n = 3) are shown graphically.

Fig. 6.

Analysis of sAPP isoforms secreted by WT-BACE- and GPI-BACE-expressing cells. Media were collected from untransfected SH-SY5Y cells or cells stably expressing WT-BACE or GPI-BACE as described in Fig. 5. (A) Detection of KPI-containing APP isoforms by using an anti-KPI antibody. (B) Media samples were treated with neuraminidase and O-glycosidase as described in Experimental Procedures. Treated and untreated samples were analyzed by SDS/PAGE and immunoblotting with the G26 antibody.

Fig. 7.

The effect of lovastatin and MβCD treatment on APP cleavage by GPI-BACE. Confluent SH-SY5Y cells stably expressing WT-BACE or GPI-BACE were treated with lovastatin and methyl-β-cyclodextrin as described in Experimental Procedures, or incubated in OptiMEM for equal lengths of time. The medium was collected, and lipid rafts were prepared as described. (A) Sucrose gradients were harvested in 0.5-ml fractions (fraction 1, top of gradient; fraction 9, bottom of gradient), and each fraction was analyzed for BACE and flotillin by SDS/PAGE and immunoblotting with the monoclonal 9B21 BACE antibody or a monoclonal flotillin antibody. (B) Media collected from WTBACE- or GPI-BACE-expressing cells incubated in the absence or presence of lovastatin was concentrated, and equal amounts of protein were analyzed by SDS/PAGE and immunoblotting with the G26 antibody to sAPPβ. Densitometric analysis was carried out, and the mean results ± SEM (n = 3) are shown graphically (black bars, untreated cells; gray bars, treated cells).