Identification of BACE2 as an avid ß-amyloid-degrading protease

To identify novel AßDPs, we performed a cell-based functional screen using a commercial library consisting of 352 full-length, sequence-verified, human cDNAs encoding diverse members of all protease classes. We experimented with several approaches before settling on a final configuration for the primary screen. Assays designed to monitor degradation of exogenous Aß were found to be confounded by the highly dominant effect of IDE, which mediates the vast majority of extracellular Aß degradation in cultured cells [18‐20]. Transient transfection of cDNAs into cell lines stably expressing APP was also tried, but this approach suffered from incomplete transfection efficiency, which attenuated the effect on net extracellular Aß levels. We therefore elected to conduct the screen by co-transfecting protease-encoding cDNAs, together with positive and negative controls, into a rodent cell line (CHO cells) together with a plasmid encoding wild-type human APP fused to alkaline phosphatase (AP) (see Figure 1A; Methods). Use of the APP-AP construct ensured that human Aβ production was limited to cells also expressing candidate AßDPs, while also providing an internal control for transfection efficiency (via AP activity). Importantly, the co-transfection strategy also increased the likelihood of detecting AßDPs that degrade Aß intracellularly, prior to its secretion, in addition to those that act exclusively extracellularly. Cytotoxicity was also quantified via an MTT conversion assay, but no significant cell death was detected so these data were not incorporated into subsequent analyses. The screen was performed in quadruplicate and, for each well, the ratio of Aß40 concentration to AP activity was calculated, then normalized to appropriate intra-plate controls (Figure 1B).

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 [21] 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 [22].

Expression of BACE2 in cells could lower Aß levels either directly, via proteolytic degradation, or indirectly, via alternative mechanisms such as hydrolysis of APP or APP C-terminal fragments (CTFs) [11‐16]. To distinguish these possibilities, we tested the ability of recombinant BACE2 to hydrolyze synthetic Aß in vitro, using a well-established fluorescence polarization-based Aß degradation assay [23]. Recombinant BACE2 was found to avidly degrade Aß in this paradigm, confirming that BACE2 is indeed a bona fide AßDP (Figure 2A). Recombinant BACE1 also hydrolyzed Aß, indicating that it too is an AßDP (Figure 2B). However, BACE1 was much less efficient than BACE2, requiring 24 h to degrade Aß to a similar extent as was achieved following a 10-min incubation with BACE2 (Figure 2B). Based on these results, the efficiency of BACE1 would appear to be ~150-fold lower than that of BACE2.

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 [7]. 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 [7], BACE2 does not degrade Aß fibrils.

We next investigated which peptide bond(s) within Aβ are hydrolyzed by BACE2 and BACE1. To that end, we co-incubated N-terminally biotinylated Aβ40 or Aβ42 (300nM) with BACE2 (5 nM) and analyzed the products by immunoprecipitation/mass spectrometry (IP/MS) (see Methods). Within 1 h, BACE2 almost completely hydrolyzed both Aß species, generating the shorter fragment, Aβ34, in both cases (Figure 3A-D). To test whether any additional cleavages can occur, we incubated N-terminally biotinylated Aβ40 (300 nM) with a larger amount of BACE2 (25 nM) for 1 and 24 h. At these higher concentrations and longer incubation times, Aβ19 and Aβ20 were the principal N-terminal fragments remaining at the end of the reaction (Figure 3E-F). Collectively, these in vitro results suggest that BACE2 cleaves Aβ at three different positions: Phe19-Phe20, Phe20-Ala21, and Leu34-Met35, with the latter cleavage site being the initial and principal one, as is consistent with previous observations [13, 14, 24].

To confirm whether BACE2 cleaves Aβ at the same sites in a more physiological setting, we analyzed Aß species in the conditioned media of cells expressing APP-AP either alone or together with BACE2 by IP/MS (see Methods). As expected for cells expressing APP-AP alone, the medium from these cells contained Aβ42, Aβ40, Aβ39, Aβ38, and Aβ37 (Figure 4A). BACE2 expression suppressed the signal of all of these species, and new peaks corresponding to Aβ19, Aβ20, and Aβ34 emerged (Figure 4B), confirming that the cleavage sites mediated by BACE2 in vitro are also hydrolyzed in intact cells. The appearance of Aß34 is particularly notable, because cleavage at position 34 can only occur after production of full-length Aß, as this peptide bond is positioned within the transmembrane domain of APP, as has been shown previously [24]. Although this result clearly indicates that BACE2 does indeed degrade Aß after it is produced, it is not possible to quantify the extent to which the Aß19 and Aß20 peaks are the result of θ-secretase activity or subsequent degradation of the Aß34 fragment (or full-length Aß). As a consequence, it is difficult to estimate the exact extent to which the Aß-lowering effect of BACE2 can be assigned to non-amyloidogenic processing versus Aß degradation per se in experimental paradigms of this type.

Having established BACE2 as an AßDP, we next investigated how BACE2 compares to other known AßDPs in terms the ability to degrade Aß in vitro and to lower net Aß levels in cells. To compare the relative efficiency of BACE2 in vitro, we monitored the degradation of a fixed amount of Aß (200 nM) by recombinant BACE2 (5 nM) as compared to equal quantities of several well-established AßDPs, including IDE, NEP and plasmin. Under these conditions, BACE2 hydrolyzed Aß more efficiently than all other AßDPs except IDE (Figure 5A). We note that the concentration of Aß used in this experiment was considerably lower than the K_M for each of the proteases tested (see [23] and below), making the initial velocity of this reaction a good index of the relative catalytic efficiency.

To investigate the catalytic efficiency of BACE2 more quantititatively, we determined the kinetics of degradation of both Aß40 and Aß42 by BACE2 (see Methods). For this analysis, we were careful to use freshly prepared batches of monomeric human Aβ40 and Aβ42 peptides, which we routinely prepare by size-exclusion chromatography and which have been extensively characterized [25, 26]. BACE2 cleaved both Aß species with similar kinetics, exhibiting apparent K_M values in the low micromolar range and albeit with apparent k_cat values slightly higher for Aß40 relative to Aß42 (0.135 ± 0.016 min^-1 and 0.025 ± 0.005 min^-1, respectively; Table 1). In terms of catalytic efficiency (k_cat/K_M), BACE2 degrades Aβ40 approximately 4-fold more efficiently than Aβ42 (Table 1). These parameters exceed the published values for most other well-characterized AßDPs, including NEP [23], ECE1 [27], and plasmin [23], while being comparable to those of IDE [23, 28]. Consequently, these values are in good agreement with the side-by-side comparison of Aß degradation in vitro discussed above (Figure 5A).

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).

Having determined that BACE2 is functionally among the most efficient AßDPs yet discovered, we subsequently investigated the subcellular localization of BACE2, focusing in particular on the extent to which it colocalizes with Aβ in acidic compartments, where BACE2 is expected to be operative. In agreement with other published findings [29], application of fluorescently tagged Aß to live cells resulted in its accumulation at intracellular sites largely overlapping with lysosomes (Figure 6A). To test whether BACE2 is also localized to lysosomes and/or other compartments containing Aß, we analyzed CHO cells expressing BACE2 tagged at its N-terminus with green fluorescent protein (BACE2-GFP). As determined by confocal microscopy, BACE2-GFP was found to be present in lysosomes (Figure 6B) and also to overlap significantly with fluorescently labeled Aß (Figure 6C).

To directly assess whether BACE2 degrades Aß at intracellular sites, we tested the ability of BACE2-expressing cells to degrade exogenously applied Aß by multiple methods. Cells overexpressing BACE2-GFP and loaded with fluorescently tagged Aß40 showed significantly reduced intracellular Aß 1 h after washing, but this was not the case for cells overexpressing GFP alone (Figure 7A). Consistent with this, levels of intracellular Aß, both fluorescently tagged and unmodified, were found to be consistently lower in cells overexpressing (untagged) BACE2 relative to vector-trasfected controls (Figure 7B,C). Notably, significantly lower levels of intracellular Aß were observed both 5 min and 2 h after washing in multiple paradigms. Collectively, these results strongly suggest that BACE2 is a bona fide AßDP that avidly degrades Aß within acidic compartments.

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 [27], 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) [42]. 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® M5^e 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 [43]. Conditioned media were supplemented with Complete^TM 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 [44] 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 [45] (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 [46]. 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 [23]. 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 [23]. 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 [23]. 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 Fluor^TM 488 or HiLight Fluor^TM 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.).