Identification of BACE2 as an avid ß-amyloid-degrading protease
© Abdul-Hay et al.; licensee BioMed Central Ltd. 2012
Received: 16 March 2012
Accepted: 16 August 2012
Published: 17 September 2012
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© Abdul-Hay et al.; licensee BioMed Central Ltd. 2012
Received: 16 March 2012
Accepted: 16 August 2012
Published: 17 September 2012
Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer’s disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays.
The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds—Phe19-Phe20, Phe20-Ala21, and Leu34-Met35—with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE.
This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.
Alzheimer disease (AD) is a progressive and presently incurable neurodegenerative disorder characterized by abnormal accumulation of the amyloid β-protein (Aβ) in brain regions important for mnemonic and cognitive functions. Aß is a heterogeneous mixture of peptides ranging from 37 to 43 amino acids in length  produced via sequential cleavage of the amyloid precursor protein (APP) by BACE1 and the presenilin/γ-secretase complex [2–4]. Autosomal-dominant mutations in 3 genes—APP and presenilin-1 and −2—are known to cause rare, familial forms of AD either by increasing the production of all forms of Aß or by increasing the relative production of longer, more amyloidogenic forms, such as Aß42 . Nevertheless, the precise mechanisms underlying sporadic AD, which makes up the vast majority of cases, remain to be elucidated.
Aß-degrading proteases (AßDPs) are potent regulators of cerebral Aß levels and, as such, represent important players in the etiology and potential treatment of AD . Amyloidogenesis and downstream cytopathology can be attenuated and even completely prevented by enhancing the activity of any of several AßDPs, while, conversely, genetic deletion of one or more AßDPs leads to significant elevations in cerebral Aß . Significantly, patients with sporadic AD were recently shown to exhibit defects in the clearance of Aß (rather than increases in its production)  and, in light of the large body of evidence implicating AßDPs in the regulation of cerebral Aß levels , it is reasonable to infer that defects in one or more AßDPs could contribute to impaired Aß clearance. While more than twenty proteases are now known to degrade Aß , these were not identified through any systematic approach, but instead emerged haphazardously from a disconnected set of largely serendipitous discoveries. Nevertheless, essentially all AßDPs now known to regulate Aß in vivo were originally identified through exclusively in vitro or cell-based approaches .
To discover new AßDPs more systematically, we conducted an unbiased, cell-based, functional screen of 352 proteases in the human genome. The top Aß-lowering protease emerging from this screen was ß-site APP-cleaving enzyme-2 (BACE2) . Previous studies have shown that BACE2 can lower Aß levels via α-secretase-like cleavage of APP within the Aß sequence [11–16], an activity that has been dubbed “θ-secretase” . However, we found that BACE2 is also a remarkably avid AßDP, with a catalytic efficiency exceeding all other known AßDPs except insulin-degrading enzyme (IDE).
From among the 352 proteases examined, by far the largest decrease in normalized Aß levels (97 ± 1.2%) was induced by BACE2, which was in fact the only protease to lower Aß levels more than 75%, our pre-determined cut-off for viable hits (Figure 1B).
To confirm and extend the results obtained in the cDNA screen, we compared the degree to which overexpression of BACE2 and its homolog BACE1  affected the net production of different Aß species. Consistent with the results of the primary screen, BACE2 transfection in CHO cells decreased the levels of both Aβ40 and Aβ42 (Figure 1C). Overexpression of BACE1 in this cell type, by contrast, had no effect on net Aß levels (Figure 1C). We note that BACE1 overexpression would not be expected to increase Aß production in CHO cells, since previous studies have established that γ-secretase, rather than ß-secretase, is the rate-limiting step in Aß production in this cell type .
As an aspartyl protease, the catalytic efficiency of BACE2 is expected to be pH-dependent. To confirm this, we compared the rate of hydrolysis of Aß40 across a range a pH values. Consistent with expectations, BACE2 was found to be maximally effective at pH 3.5 (Figure 2C), and decreasingly effective at higher pH values. These findings strongly suggest that BACE2 would not be operative at the cell surface or within the extracellular space.
Individual AßDPs can be categorized in terms of their ability or inability to degrade fibrillar forms of Aß. Many well-established AßDPs, such as IDE and NEP, avidly degrade monomeric Aß but cannot degrade fibrillar forms and are therefore categorized as pure peptidases. Others, such as plasmin, degrade Aß fibrils and thus can also be categorized as fibrilases . To determine to which category BACE2 belongs, we incubated recombinant BACE2 with pre-formed fibrils of Aß42 and quantified the degree of aggregation by thioflavin T fluorescence. No significant reduction in aggregation was observed, even following incubation at 37°C for up to 3 d (Figure 2D). These results suggest that, as is true for the majority of AßDPs , BACE2 does not degrade Aß fibrils.
Kinetic parameters of Aß40 and Aß42 degradation by BACE2
K M (μM)
2.8 ± 0.7
2.3 ± 0.6
V max (μM min -1 )
0.68 ± .083
0.12 ± 0.025
k cat (min -1 )
0.135 ± 0.016
0.025 ± 0.005
k cat / K M (M -1 min -1 )
4.82 x 107
1.07 x 107
To investigate the relative ability of BACE2 to lower Aß levels under more physiological conditions, we co-transfected CHO cells with APP together with BACE2 or several other AßDPs, then quantified net Aß40 and Aß42 levels in the conditioned medium by ELISA. We emphasize that this approach cannot control for intrinsic differences in transcription or translation efficiency, and, in the case of BACE 2, the Aß-lowering effect can also be mediated to an undetermined degree by BACE2-mediated θ-secretase activity. Nevertheless, the results were in good agreement with the in vitro findings: BACE2 lowered net Aß40 and Aß42 levels to a comparable extent as IDE, with both of the latter being significantly more effective than NEP or plasmin (Figure 5B, C).
One of the most fruitful outcomes of the genomic revolution is the emergence of genome-scale collections of full-length, sequence verified cDNAs. Combined with appropriate functional assays, cDNA libraries have catalyzed significant advances in our understanding of AD pathogenesis, including the seminal discovery that ß-secretase activity, the first step in the production of Aß, is mediated by BACE1 . Here, we utilized a similar approach to discover new candidate AßDPs, using a functional assay sensitive to both extracellular and intracellular Aß degradation (as well as other potential Aß-lowering mechanisms). Rather unexpectedly, the top hit emerging from a screen of 352 proteases was BACE2, a close homolog of BACE1. Subsequent characterization confirmed that, in addition to BACE2’s established ability to lower Aß production via θ-secretase-mediated processing of APP [11–16], BACE2 also avidly degrades Aß with a catalytic efficiency exceeding almost all well-established AßDPs.
The finding that BACE2 is an avid AßDP suggests a novel and unexpected role for this protease in the pathogenesis of AD. Indeed, given its close homology with BACE1, it was initially hypothesized that BACE2 might mediate the production of Aß, via β-secretase cleavage of APP, instead [15, 16]. However, most evidence now suggests that BACE2 does not contribute appreciably to Aß production in vivo . For instance, cultured neurons from BACE2 knockout mice did not show reductions in Aß following transfection with APP  and conversely, overexpression of BACE2 in APP transgenic mice failed to increase cerebral Aß levels, as would be expected if BACE2 possessed ß-secretase-like activity.
In addition to its potent ability to degrade Aß, BACE2 also possesses a second Aß-lowering function for BACE2, one that is quite independent of Aß degradation. Specifically, BACE2 has been shown to cleave APP and the ß-secretase-derived APP-CTF within the Aß sequence, in a manner analogous to α-secretase [11–16]. This activity, dubbed θ-secretase , occurs at positions 19 and 20 within the Aß sequence, precisely the same cleavage sites identified in the present study [13, 14]. As is true for α-secretase, θ-secretase activity lowers Aß levels by shuttling APP away from the amyloidogenic processing pathway [11–16].
As confirmed by previous work , we found that BACE2 also cleaves Aß at the Leu34-Met35 peptide bond, which was in fact the initial and principal site of cleavage. Notably, cleavage at this position can only occur after production of full length Aß by ß- and γ-secretase, because this peptide bond in APP or in APP CTFs is normally embedded within the cell membrane . This fact, together with the finding that Aß34 is produced in cells overexpressing of BACE2 and APP, provides clear evidence that the Aß-degrading activity of BACE2 contributes significantly to the overall Aß-lowering effect of BACE2 overexpression, even in the context of concurrent θ-secretase activity.
Given that BACE2 can lower Aß both by decreasing its production and by mediating its degradation, which of these mechanisms are relevant to the pathogenesis or the potential treatment of AD? The answer depends critically on precisely where and to what extent BACE2 is expressed in vivo. Although BACE2 protein is readily detected in brain extracts [15, 30–36], and its activity has even been shown to be comparable to that of BACE1 in post-mortem brain [31, 33], there is conflicting evidence about which cell types express BACE2. Studies in mice, on the one hand, suggest that the protease is expressed abundantly in glia but only minimally in neurons . To the extent that these findings apply to humans, θ-secretase cleavage of APP by BACE2 would be unlikely to play any significant pathophysiological role in AD, given that APP itself is expressed predominantly in neurons, with only modest expression levels in non-neuronal brain cells . On the other hand, multiple studies in post-mortem human brain tissue have reported detectable BACE2 expression not only in astrocytes, but also in neurons [15, 33], suggesting that the θ-secretase activity of BACE2 may, to some extent, contribute to the overall economy of brain Aß. The pathophysiological relevance of BACE2’s function as an AßDP is similarly difficult to predict and likewise dependent on the extent to which the protease is expressed in neurons. Astrocytes are known mediate the clearance of Aß , but the contribution of intra-astrocytic Aß degradation relative to intraneuronal or extracellular degradation in vivo remains to be established. As was true for other AßDPs first identified in cells , the answer to these questions will require further study in relevant animal models.
Notwithstanding uncertainty about its role in AD pathogenesis, a number of considerations suggest that BACE2 represents an especially strong therapeutic candidate, particularly for gene therapy-based approaches. BACE2 can lower Aß catalytically via two independent mechanisms, and its Aß-degrading ability alone exceeds that of most other AßDPs, some of which are being considered for gene therapy clinical trials . Moreover, as an aspartyl protease, BACE2 possesses distinct advantages relative to other AßDPs. First, it is operative with subcellular compartments most relevant to Aß production—i.e., those containing active ß- and γ-secretase, which are both aspartyl proteases—thus allowing it to impact Aß levels prior to secretion. In this connection, there is growing evidence that intracellular Aß may represent an especially pathogenic role in AD , so modulation of this pool may be particularly appropriate therapeutically. Second, because BACE2 is operative exclusively at intracellular sites, its expression could be readily restricted to the site of administration. This is in contrast to many other AßDPs which are secreted and/or active extracellularly [19, 40, 41] and thus less capable of being confined to specific regions.
In conclusion, this study identifies BACE2 as a novel and highly efficient AßDP. This newly identified function of BACE2, together with its established ability to also lower Aß production via θ-secretase activity, suggests that BACE2 may play a significant role in AD pathogenesis. Moreover, even if BACE2 plays no role in the etiology of AD, BACE2 nevertheless represents a particularly attractive candidate for gene therapeutic approaches to the treatment of prevention of this presently incurable disease.
A library of 352 full-length, sequence verified, human cDNAs encoding diverse members of all protease classes was purchase from a commercial source (OriGene Technologies, Inc.) in 96-well format (100 ng/well). For negative and positive controls, a subset of blank wells on each plate were supplemented with empty vector or a construct expressing a well-established AßDP, human ECE1b , respectively (100 ng/well). As a source of human Aß and also as a transfection control, each well was cotransfected with a hybrid construct, APP-AP (60 ng/well), comprised of a vector expressing wild-type human APP fused at its N-terminus with alkaline phosphatase (AP) . Additional blank wells were left untreated for cell-free background controls. CHO cells (4.8 x 104/well) suspended in DMEM/Opti-MEM supplemented with 5%FBS were then co-transfected with APP-AP and protease-encoding cDNAs using Fugene 6.0, according to manufacturer’s recommendations (Promega Corp.). Transfected cells were allowed to grow overnight under standard cell culture conditions (5% CO2; 37°C; 95% humidity) then the medium was exchanged. 24 h later, the conditioned media were collected for downstream analysis (see below). All experiments were conducted in compliance with and with approval by the Mayo Clinic Institutional Review Board.
Following heat treatment to inactivate endogenous phosphatases (65°C for 15 min) present in the media, conditioned media (30 μL/well) was added to 96-well plates containing AP substrate, 4-nitrophenylphosphate (170 μL/well, 2 mg/mL), dissolved in AP buffer (1 M diethanolamine, 0.5 mM MgCl2, 10 mM L-homoarginine, pH 9.8). Plates were incubated for 30 min and AP activity was determined from absorbance (OD405) using a SpectraMax® M5e multilabel plate reader (Molecular Devices).
Aβ levels were quantified using a sandwich ELISA system based on antibody pairs 33.1.1/13.1.1 for Aβ40 and 2.1.3/4 G8 for Aβ42 as described previously . Conditioned media were supplemented with CompleteTM Protease Inhibitor Cocktail (Roche) just after collection and analyzed immediately. For experiments quantifying intracellular Aß, cells were plated in in 96-well plates (2 x 104 cells per well) and transfected with BACE2-encoding cDNA or empty vector, washed, then incubated with 400 nM synthetic Aß for 6 h. After washing with PBS, intracellular Aß was extracted with 5 M guanidinium isothiocyanate and quantified using a commercially available ELISA (Wako Chemicals USA, Inc.) after 10-fold dilution in the manufacturer-provided dilution buffer.
The cleavage sites within Aβ40 and Aβ42 hydrolyzed by BACE2 and BACE1 were determined essentially as described  with minor modifications. Briefly, Aβ peptides or biotinylated Aβ peptides were incubated for various lengths of time with recombinant BACE2 enzyme in Assay Buffer (25 mM acetate buffer, pH 4.0, supplemented with 0.1% BSA). The reaction was stopped by addition of protease inhibitor cocktail and pH adjustment. Aβ fragments were immediately precipitated by magnetic beads coated with streptavidin (for biotinylated Aβ) or magnetic beads coated with Ab9 antibody  (for unmodified Aβ). Beads were washed with 10 mM NH4CO3, pH 8.0, and peptide fragments were eluted using 0.5% trifluoroacetic acid in 75% acetonitrile in water, followed by the addition of an equal volume of a saturated sinapic acid solution dissolved in 0.5% trifluoroacetic acid in 50% acetonitrile and water. Digested products were spotted onto a gold chip, dried, and analyzed using a Ciphergen ProteinChip SELDI time-of-flight system (Bio-Rad). Mass spectra were acquired automatically in a linear positive mode at 1350 shots per spectrum. Peptides containing a183-Da increase in MW were identified as being modified by AEBSF, as previously reported . Same procedure was applied to detect the endogenous Aβ fragments produced by CHO cells transfected with APP and BACE2 (using Ab9 as a capture antibody).
The kinetics of Aβ40 and Aβ42 degradation by BACE2 were determined using freshly prepared, monomeric Aβ peptides separated from aggregated species by size-exclusion chromatography and characterized as described [25, 26]. Aβ peptides were diluted in neutral Dilution Buffer (20 mM Tris, pH 8.0 supplemented with 0.1% BSA) and reactions were initiated by transfer into 20 times more volume of Assay Buffer supplemented with purified recombinant BACE2 (R&D Systems, nominal concentration 1 or 5 nM) or, as a control for non-specific loss of Aβ, the latter buffer lacking BACE2. Where required, reactions were terminated by supplementation with protease inhibitor cocktail and adjustment to neutral pH. For ELISA-based experiments, Aβ42 and Aβ40 were quantified using the sandwich ELISAs described above. For determination of kinetic parameters, ELISAs were used to quantify the initial velocities of degradation of a range of different concentrations of Aß40 (0.2 to 16 μM) or Aß42 (0.6 to 16 μM) by a fixed amount of recombinant BACE2 (5 nM) in Assay Buffer, and K M and v max values were determined in triplicate by fitting a hyperbolic curve to these data in Prism 5.0 (GraphPad Software, Inc.). For determination of the pH dependence of Aβ degradation, experiments were carried same as described above, using Assay Buffer at different pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5). The reactions were stopped at 10 min and the remaining 200nM of Aβ was determined using a well-characterized fluorescence polarization-based activity Aß degradation assay, as described . For comparison of the rate of degradation of Aβ by different proteases, we incubated 200 nM of Aβ fluorescent substrate (FAβB) with 5nM of different protease in their corresponding buffers: BACE1 and BACE2 using Assay Buffer and IDE, NEP, and plasmin in PBS, pH 7.4 supplemented with 0.1% BSA. The reactions were stopped by addition of protease inhibitor cocktail, 500nM streptavidin, and adjustment to neutral pH. The degree of Aβ hydrolysis was immediately determined using a polarization-based Aß degradation assay . Recombinant BACE2 (R&D Systems) and plasmin (EMD Biosciences) were purchased from commercial sources, while recombinant IDE and secreted NEP (i.e., lacking the transmembrane domain) were generated and purified as described . All reactions were performed at 37°C.
CHO cells (106 cells/cm2) were plated onto 8-well poly-D-lysine-coated, glass-bottom chambers (MatTek Corp.) in culture medium (DMEM/Opti-MEM supplemented with 5%FBS). For BACE2 transfections, cell were transfected with a construct encoding BACE2 tagged at its C-terminus with GFP (OriGene Technologies, Inc. Cat. No. RG04860) using Fugene 6.0 transfection reagent according to manufacturer’s recommendations (Promega Corp.). For Aβ colocalization experiments, cells were washed twice in fresh culture medium, then incubated in the latter medium supplemented with either Aß40 (500 nM) labeled at the N-terminus with HiLight FluorTM 488 or HiLight FluorTM 555 (AnaSpec, Inc.). For lysosomal staining, cells were incubated with Lysotracker Red according to manufacturer’s recommendations (Invitrogen Corp.), then washed 2 times with fresh culture medium prior to imaging. For confocal microscopy, cells were washed with fresh medium then imaged immediately using the 488-nm and 543-nm laser lines on a Zeiss LCM 510 META confocal microscope (Carl Zeiss, Inc.). Images were processed and analyzed using MetaMorph software according to manufacturer’s recommendations (Molecular Devices, Inc.). For conventional fluorescence microscopy of intracellular Aß, cells were washed with fresh medium, then incubated at 37°C for 1 h prior to imaging using a Nikon Labophot 2 fluorescent microscope (Nikon Inc.).
Amyloid precursor protein
ß-site APP-cleaving enzyme-1
ß-site APP-cleaving enzyme-2
We thank Dr. Terrone Newberry and William Tay for providing monomeric and fibrillar Aß peptides and Drs. Todd Golde and Kevin Felsenstein for contributing the APP-AP construct. Supported by a grant from the Coins For Alzheimer’s Trust Fund to ML.
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