Maturation and Endosomal Targeting of β-Site Amyloid Precursor Protein-cleaving Enzyme

Abstract

The amyloidogenic A␤ peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, ␤-secretase and ␥-secretase. Recently, a type I membrane protein termed BACE (␤-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the ␤-secretase. We undertook a series of biochemical and morphological investigations designed to characterize the basic properties of this protein. Initial studies indicated that BACE undergoes Nlinked glycosylation at three of four potential sites. Metabolic pulse-chase experiments revealed that after core glycosylation, BACE is rapidly and efficiently transported to the Golgi apparatus and distal secretory pathway. BACE was also found to be quite stable, being turned over with a t1 ⁄2 of ϳ16 h. Retention of BACE in the endoplasmic reticulum by introduction of a C-terminal dilysine motif prevented complex carbohydrate processing and demonstrated that propeptide cleavage occurs after exit from this organelle. BACE exhibited intramolecular disulfide bonding but did not form oligomeric structures by standard SDS-polyacrylamide gel electrophoresis analysis and sedimented as a monomer in sucrose velocity gradients. Immunofluorescence studies showed a largely vesicular staining pattern for BACE that colocalized well with endosomal, but not lysosomal, markers. Measurable levels of BACE were also detected on the plasma membrane by both immunostaining and cell surface biotinylation, and cycling of the protein between the cell membrane and the endosomes was documented. A cytoplasmic dileucine motif was found to be necessary for normal targeting of BACE to the endosomal system and accumulation of the protein in this intracellular site.

Trafficking and Proteolytic Processing of APP

“…During maturation, BACE1 undergoes a number of post/co-translational modifications, including N-glycosylation, disulfide bridge formation, and palmitoylation (Bennett et al 2000b;Capell et al 2000;Huse et al 2000;Benjannet et al 2001). The propeptide of immature BACE1 is removed by Furin and related proteases during maturation (Capell et al 2000;Creemers et al 2001).…”

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

“…Cell surface biotinylation assay was performed as reported [12], and all procedures were carried on ice unless otherwise stated. Briefly, HEK293 cells were labeled with EZ-Link TM Sulfo-NHS-SS-Biotin (Pierce) for 30 min followed by washing with 100 mM glycine to remove the free biotin.…”

“…Because BACE is known to be internalized, and sorted to endosomes and the Golgi apparatus to exert its cleavage activity [3,12], we thus studied whether RTK activation affects the intracellular distribution of BACE. As shown in Figure 5, BACE was localized in endosomes (EEA-1 as the marker) and Golgi apparatus (Golgi 58K protein as the marker), with little in lysosomes (LAMP-1 as the marker), consistent with other reports [3,15].…”

“…Recent studies revealed that BACE is synthesized as an immature and inactive form in the endoplasmic reticulum [11], and undergoes maturation during its transport to cell surface along the secretory pathway [12][13][14][15][16]. The mature BACE is then internalized from cell surface to endosomes [3,12], followed by sorting to the trans golgi network (TGN) for recycling or to lysosomes for degradation [15,16].…”

“…The mature BACE is then internalized from cell surface to endosomes [3,12], followed by sorting to the trans golgi network (TGN) for recycling or to lysosomes for degradation [15,16]. Interestingly, endosomal accumulation of Aβ was also found in AD patients at the early disease stage [17].…”