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

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Title:
Identification of BACE2 as an avid B-amyloid-degrading protease
Physical Description:
Mixed Material
Language:
English
Creator:
Abdul-Hay, Samer O
Sahara, Tomoko
McBride, Melinda
Kang, Dongcheul
Leissring, Malcolm A.
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BioMed Central
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Notes

Abstract:
Background: 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. Results: 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 endothelinconverting enzyme-1 (ECE1), while showing comparable effectiveness to IDE. Conclusions: 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. Keywords: Amyloid-ß-protein, Alzheimer disease, ß-site APP-cleaving enzyme-1, ß-site APP-cleaving enzyme-2, Functional screen, Gene therapy, Protease, Proteolytic degradation
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The Institutional Repository at the University of Florida community, with research, news, outreach, and educational materials.
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Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46; pgs. 1-12 http://www.molecularneurodegeneration.com/content/7/1/46
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doi:10.1186/1750-1326-7-46 Cite this article as: Abdul-Hay et al.: Identification of BACE2 as an avid ß-amyloid-degrading protease. Molecular Neurodegeneration 2012 7:46.

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Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
http://www.molecularneurodegeneration.com/content/7/1/46


MOLECULAR
NEURODEGENERATION


Identification of BACE2 as an avid

B-amyloid-degrading protease

Samer O Abdul-Hay, Tomoko Sahara, Melinda McBride, Dongcheul Kang and Malcolm A Leissring*


Abstract
Background: Proteases that degrade the amyloid B-protein (AB) 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 AB-degrading proteases (ARDPs), we conducted an unbiased, genome-scale, functional cDNA screen
designed to identify proteases capable of lowering net AR levels produced by cells, which were subsequently
characterized for AB-degrading activity using an array of downstream assays.
Results: The top hit emerging from the screen was B-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 AB.
BACE2 is known to be capable of lowering AR levels via non-amyloidogenic processing of APP. However, in vitro,
BACE2 was also found to be a particularly avid ABDP, with a catalytic efficiency exceeding all known ARDPs except
insulin-degrading enzyme (IDE). BACE1 was also found to degrade AR, albeit -150-fold less efficiently than BACE2.
AB 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 AR
levels to a greater extent than multiple, well-established ARDPs, including neprilysin (NEP) and endothelin
converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE.
Conclusions: This study identifies a new functional role for BACE2 as a potent ABDP. Based on its high catalytic
efficiency, its ability to degrade AB intracellularly, and other characteristics, BACE2 represents a particular strong
therapeutic candidate for the treatment or prevention of AD.
Keywords: Amyloid-B-protein, Alzheimer disease, B-site APP-cleaving enzyme-1, B-site APP-cleaving enzyme-2,
Functional screen, Gene therapy, Protease, Proteolytic degradation


Background
Alzheimer disease (AD) is a progressive and presently
incurable neurodegenerative disorder characterized by
abnormal accumulation of the amyloid P-protein (A3) in
brain regions important for mnemonic and cognitive
functions. AB is a heterogeneous mixture of peptides
ranging from 37 to 43 amino acids in length [1] pro-
duced via sequential cleavage of the amyloid precursor
protein (APP) by BACE1 and the presenilin/y-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 AB or by increasing the rela-
tive production of longer, more amyloidogenic forms,

SCorrespondence leissring@mayoedu
Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Birdsal
Bldg, Rm 117, Jacksonville, FL 32224, USA


such as A642 [5]. Nevertheless, the precise mechanisms
underlying sporadic AD, which makes up the vast major-
ity of cases, remain to be elucidated.
AfB-degrading proteases (ABDPs) are potent regulators
of cerebral AB levels and, as such, represent important
players in the etiology and potential treatment of AD
[6]. Amyloidogenesis and downstream cytopathology can
be attenuated and even completely prevented by enhan-
cing the activity of any of several AfBDPs, while, con-
versely, genetic deletion of one or more ABDPs leads to
significant elevations in cerebral AB [7]. Significantly,
patients with sporadic AD were recently shown to ex-
hibit defects in the clearance of AB (rather than
increases in its production) [8] and, in light of the large
body of evidence implicating ABDPs in the regulation of
cerebral AB levels [7], it is reasonable to infer that
defects in one or more ABDPs could contribute to


S 2012 Abdul-Hay et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the term of the
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distribution, and reproduction in any medium, provided the original work is properly cited.






Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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impaired AB clearance. While more than twenty pro-
teases are now known to degrade AS [7], these were not
,i...!.,..i through any systematic approach, but instead
emerged haphazardously from a disconnected set of
largely serendipitous discoveries. Nevertheless, essen-
tially all ABDPs now known to regulate AB in vivo were
originally identified through exclusively in vitro or cell-
based approaches [9].
To discover new ABDPs more systematically, we con-
ducted an unbiased, ii i I .i functional screen of 352
proteases in the human genome. The top AB-lowering
protease emerging from this screen was B-site APP-
cleaving enzyme-2 Ji-':.. El- [10]. Previous studies have
shown that BACE2 can lower AB levels via a-secretase-
like cleavage of APP within the A1 sequence [11-16], an
activity that has been dubbed "0-secretase" [17]. How-
ever, we found that BACE2 is also a remarkably avid
AIDP, with a catalytic efficiency exceeding all other
known AfDPs except insulin-degrading enzyme (IDE).


Results and discussion
Functional screen for novel ARDPs
To identify novel AiBDPs, we performed a ..i-:,-.-:
functional screen using a commercial library '. ; ;_
of 352 '., i ,,I. sequence-verified, human cDNAs en-
...!ii' 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 AB were
found to be confounded by the highly dominant effect of
IDE, which mediates the vast majority of extracellular
AS degradation in cultured cells [18-20]. Transient
transfection of cDNAs into cell lines stably expressing
APP was also tried, but this approach .w:: ,. I from in-
complete transfection efficiency, which attenuated the
effect on net extracellular AB levels. We therefore
elected to conduct the screen by co-transfecting prote-
i. .. ..: i,- cDNAs, together with positive and nega-
tive controls, into a rodent cell line (CHO cells) together
with a plasmid encoding 1-iype human APP fused to
,: ,il .. phosphatase (AP) (see F~.' ... 1A; Methods). Use
of the APP-AP construct ensured that human AP pro-
duction was limited to cells also expressing :,Il:. .
AIDPs, while also I ... '.i.._ an internal control for
transfection efficiency (via AP activity). Importantly, the
co-transfection .i. t also increased the likelihood of
detecting ABDPs that degrade AB intracellularly, prior
to its secretion, in addition to those that act exclusively
extracellularly. Cytotoxicity was also quantified via an
MTT conversion assay, but no .. :!!.t. i,!! cell death was
detected so these data were not incorporated into subse-
quent analyses. The screen was performed in i- :.. ,,.l;-
cate and, for each well, the ratio of Ai40 concentration


Page 2 of 12


to AP activity was calculated, then normalized to appro-
priate intra-plate controls (Figure 1B).
From among the 352 proteases examined, by far the
largest decrease in normalized AB levels (97 + 1.2%) was
induced by BACE2, which was in fact the only protease
to lower AB levels more than i" our pre-determined
cut-off for viable hits (Figure 1B).


BACE2 transfection lowers A3 levels
To confirm and extend the results obtained in the
cDNA screen, we compared the degree to which overex-
pression of BACE2 and its homolog BACE1 [21] affected
the net production of different AB species. Consistent
with the results of the primary screen, BACE2 transfec-
tion in CHO cells decreased the levels of both AP40 and
Ap42 (Figure 1C). Overexpression of BACE1 in this cell
type, by contrast, had no effect on net AB levels
(Figure 1C). We note that BACE1 overexpression would
not be expected to increase AB production in CHO cells,
since previous studies have established that y-secretase,
rather than fi-secretase, is the rate-limiting step in Af
production in this cell type [22].


BACE2 and BACE1 degrade AR in vitro
Expression of BACE2 in cells could lower AB levels ei-
ther i, i1;, via proteolytic degradation, or indirectly,
via alternative mechanisms such as hydrolysis of APP or
APP C-terminal fragments (CTFs) [11-16]. To distin-
. .; 1, these ip. ..iJ,,. we tested the ability of recom-
binant BACE2 to hydrolyze synthetic Af in vitro, using
a well-established fluorescence polarization-based AB
degradation assay [23]. Recombinant BACE2 was found
to ..i: degrade A8 in this .. ..I.. 1 confirming that
BACE2 is indeed a bona fide ASDP (Figure 2A). Recorn-
binant BACE1 also hydrolyzed AS, indicating that it too
is an ASDP (Figure 2B). However, BACE1 was much less
efficient than BACE2, requiring 24 h to degrade Ai to a
similar extent as was achieved following a 10-min incu-
bation with BACE2 (Figure 2B). Based on these results,
the efficiency of BACE1 would appear to be -150-fold
lower than that of BACE2.


BACE2-mediated AB degradation is pH-dependent
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 AS40 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 -t ..-. ..:-. that BACE2
would not be operative at the cell surface or within the
extracellular space.






Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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pAPP-AP
C IYhAP r PIV




cotransfection medium change O/N incubation
into CHO cells

arrayed cDNA library





.0 B 0 a
a 1100 .0 0
o 00- *.**f*. *-- .0 J.9



.. ...................- .......................... .


SBACE2
. --------------o.....-----Q Ae -- -* lvr b- oI ---1---

... ... ... ... ... ... .. ... ... ... ... ... ... ..


AR ELISA
TT assay --T/,- AP assay


conditioned media


0 100 200 300 Control BACE1 BACE2
cDNA number
Figure 1 Overview and outcome of functional screening for novel A3-lowering proteases. A, Cartoon the overall design of the
screen. Briefly, an arrayed collection of 352 i cDNAs was cotransfected into CHO cells together with an APP-AP fusion construct.
Following a medium change and overnight incubation, A40 levels, AP activity, and cytotoxicity (via MTT assay) were analyzed in the resulting
conditioned media. B, Results of the screen, expressed as [AB40]/AP ratios normalized to intraplate controls. Data are mean of 4 replicates. Note
that the largest decrease in AB levels by far was acheived by BACE2. C, Confirmation of the results of the screen. Following scale-up and
sequence verification, cDNAs encoding BACE2 and its homolog, BACE1, were cotransfected together with APP-AP into CHO cells. Consistent with
the outcome of the medium-throughput screen, BACE2, but not BACE1, expression resulted in decreases in the levels of both A40


and AB42. Data are mean + SEM of 4 replicates, and are normalized to


BACE2 does not degrade fibrillar AB
Individual ABDPs can be categorized in terms of their
ability or inability to degrade fibrillar forms of A3. Many
well-established ABDPs, such as IDE and NEP, avidly de-
grade monomeric AB but cannot degrade fibrillar forms
and are therefore categorized as pure peptidases. Others,
such as plasmin, degrade AB fibrils and thus can also be
categorized as fibrilases [7]. To determine to which cat-
egory BACE2 belongs, we incubated recombinant
BACE2 with pre-formed fibrils of A642 and quantified
the degree of aggregation by thioflavin T fluorescence.
No significant reduction in aggregation was observed,
even following incubation at 37C for up to 3 d
(Figure 2D). These results suggest that, as is true for the
majority of ABDPs [7], BACE2 does not degrade A1B
fibrils.

BACE2 cleaves AB at 3 sites
We next investigated which peptide bond(s) within AP3
are hydrolyzed by BACE2 and BACE1. To that end, we
co-incubated N-terminally biotinylated AP40 or AP42
(300nM) with BACE2 (5 nM) and analyzed the products


/ector-only controls.


by immunoprecipitation/mass spectrometry (IP/MS)
(see Methods). Within 1 h, BACE2 almost completely
hydrolyzed both AB species, generating the shorter frag-
ment, AP34, in both cases (Figure 3A-D). To test
whether any additional cleavages can occur, we incu-
bated N-terminally biotinylated AP40 (300 nM) with a
larger amount of BACE2 (25 nM) for 1 and 24 h. At
these higher concentrations and longer incubation times,
Ap19 and AP20 were the principal N-terminal fragments
remaining at the end of the reaction (Figure 3E-F). Col-
lectively, these in vitro results suggest that BACE2
cleaves AP at three different positions: Phel9-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 AP at the same
sites in a more physiological setting, we analyzed AB
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 AP42,
AP40, AP39, AP38, and AP37 (Figure 4A). BACE2


Page 3 of 12


B



C
o 100

I


0







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reaction time


A B
100- 5nM BACE2 5nM BACE1
m1 lnM BACE2 ? 7 5nM BACE2
'i 75- : 76-



O. 0 !26 I2


2 10 30 60
reaction time (min)


C D
50-
S100
40- C
8 75-
530.
\ 0 50-
> 20- \1
10 I- 25-
00

3 4 5 A fib. -
pH BACE2 -


Figure 2 BACE2 degrades AB in vitro. A, Percent AB remaining follow
various lengths of time. Data are mean + SEM of 4 replicates, normalized
ability of recombinant BACE2 vs. BACE1. Note that 24 h incubation with
degradation as effected by BACE2 in 10 min. Data are mean + SEM of 3
dependent. Percent AB degradation catalyzed by equivalent amounts of
D, BACE2 does not degrade fibrillar AB. Lack of effect of BACE2 (10 nM) c
determined by thioflavin T fluorescence. Data are mean + SEM of 3 repli


expression suppressed the signal of all of these species,
and new peaks corresponding to A319, AP20, and AP34
emerged (Figure 4B), confirming that the cleavage sites
mediated by BACE2 in vitro are also hydrolyzed in intact
cells. The appearance of A634 is particularly notable, be-
cause cleavage at position 34 can only occur after pro-
duction of full-length AB, 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 A1B
after it is produced, it is not possible to quantify the ex-
tent to which the A619 and A620 peaks are the result of
6-secretase activity or subsequent degradation of the
A634 fragment (or full-length AB). As a consequence, it
is difficult to estimate the exact extent to which the A6B-
lowering effect of BACE2 can be assigned to non-
amyloidogenic processing versus AB degradation per se
in experimental paradigms of this type.

BACE2 degrades AR more efficiently than well-established
ABDPs
Having established BACE2 as an A6DP, we next investi-
gated how BACE2 compares to other known ABDPs in
terms the ability to degrade AB in vitro and to lower net


ng incubation with different concentrations of recombinant BACE2 for
to protease-free controls. B, Comparison the relative AB-degrading
3ACE1 was required to achieve approximately the same extent of
replicates, normalized to protease-free controls. C, BACE2 activity is pH
BACE2 at different pH values. Data are mean + SEM of 4 replicates.
pn preformed A42 fibrils following incubation at 37'C for 5 d, as
:ates.


AB levels in cells. To compare the relative efficiency of
BACE2 in vitro, we monitored the degradation of a fixed
amount of AB (200 nM) by recombinant BACE2 (5 nM)
as compared to equal quantities of several well-
established ABDPs, including IDE, NEP and plasmin.
Under these conditions, BACE2 hydrolyzed AB more ef-
ficiently than all other ABDPs except IDE (Figure 5A).
We note that the concentration of AB used in this ex-
periment was considerably lower than the I(M for each
of the proteases tested (see [23] and below), making the
initial velocity of this reaction a good index of the rela-
tive catalytic efficiency.

Kinetics of AR degradation by BACE2
To investigate the catalytic efficiency of BACE2 more
quantititatively, we determined the kinetics of degrad-
ation of both A640 and A642 by BACE2 (see Methods).
For this analysis, we were careful to use freshly prepared
batches of monomeric human AP40 and AP42 peptides,
which we routinely prepare by size-exclusion chroma-
tography and which have been extensively characterized
[25,26]. BACE2 cleaved both AB species with similar
kinetics, exhibiting apparent I(M values in the low micro-
molar range and albeit with apparent kcat values slightly


*6 +


Page 4 of 12







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DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGW(IA)


AP42 4
5nM BACE2
t=0






4t

2000 3000 4000 5000
miz



Ap42
5nM BACE2
t=lh






34' *34
4*

2000 3000 4000 5000
mtz


Ap40
4o 5nM BACE2
t=0
30 -


10


2000 3000 4000 5000
miz


D
50-

40-


Ap40
5nM BACE2
t=lh


20 -


C 30.

20-

10-

0-


40-

30-



10


343

2000 3000 4000 5000
miz


Ap40
25nM BACE2
t=lh


._ I .[
2000 3000 4000 5000
m/z



20 Ap40
25nM BACE2
t = 24h

34



19


2000 3000 4000 5000
miz


Figure 3 Determination of peptide bonds within Ai hydrolyzed by BACE2. Top, Summary of cleavage sites determined from data in A-F,
showing the major site (block arrow) and two minor sites (arrow heads). At t-0 (A, C), intact AB42 (A) and AB40 (C) represent the only species
present. Following incubation of AB42 and AB40 with 5nM BACE2 for 1 h (B,D, respectively), the full-length A species are essentially completely
absent and replaced by AB34. E,F, Additional A cleavage products are produced following incubation with larger amounts of BACE2 (25 nM) for
longer lengths of time. By 1 h (E), a new peak corresponding to AB20 is produced. By 24 h (F), AB20 becomes the major species present, and
AB 9 is also produced. Double-charged fragments are denoted by "+", and "" represents the modification of a fragment by AEBSF, which leads
to a 183-Da increase in MW, as previously reported [46].


higher for A440 relative to A642 (0.135 + 0.016 min 1
and 0.025 0.005 min respectively; Table 1). In terms
of catalytic efficiency (kcat/KM), BACE2 degrades AP40
approximately 4-fold more efficiently than AP42
(Table 1). These parameters exceed the published values
for most other well-characterized ABDPs, including NEP
[23], ECE1 [27], and plasmin [23], while being compar-
able to those of IDE [23,28]. Consequently, these values
are in good agreement with the side-by-side comparison
of AB degradation in vitro discussed above (Figure 5A).
To investigate the relative ability of BACE2 to lower
AB levels under more physiological conditions, we co-
transfected CHO cells with APP together with BACE2
or several other ABDPs, then quantified net A640 and
A642 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 AB-lowering effect can also be
mediated to an undetermined degree by BACE2-mediated
0-secretase activity. Nevertheless, the results were in good
agreement with the in vitro findings: BACE2 lowered net
A640 and A642 levels to a comparable extent as IDE,
with both of the latter being significantly more effective
than NEP or plasmin (Figure 5B, C).

BACE2 colocalizes with AP intracellularly
Having determined that BACE2 is functionally among
the most efficient ABDPs yet discovered, we subse-
quently investigated the subcellular localization of
BACE2, focusing in particular on the extent to which it
colocalizes with AP in acidic compartments, where
BACE2 is expected to be operative. In agreement with
other published findings [29], application of fluores-
cently tagged AB to live cells resulted in its


Page 5 of 12


30 -

._,
C 20 -


10 -


0 -







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accumulation at intracellular sites largely overlapping
with lysosomes (Figure 6A). To test whether BACE2 is
also localized to lysosomes and/or other compartments
containing AB, we analyzed CHO cells expressing
BACE2 tagged at its N-terminus with green fluorescent
protein (BACE2-GFP). As determined by confocal mi-
croscopy, BACE2-GFP was found to be present in lyso-
somes (Figure 6B) and also to overlap significantly with
fluorescently labeled AB (Figure 6C).

BACE2 degrades AR at intracellular sites
To directly assess whether BACE2 degrades A1f at intra-
cellular sites, we tested the ability of BACE2-expressing
cells to degrade exogenously applied AB by multiple
methods. Cells overexpressing BACE2-GFP and loaded
with fluorescently tagged A640 showed significantly
reduced intracellular AB 1 h after washing, but this was
not the case for cells overexpressing GFP alone
(Figure 7A). Consistent with this, levels of intracellular
AB, 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 intra-
cellular AB were observed both 5 min and 2 h after
washing in multiple paradigms. Collectively, these results
strongly suggest that BACE2 is a bona fide A6DP that
avidly degrades AB within acidic compartments.

Conclusions
One of the most fruitful outcomes of the genomic revo-
lution is the emergence of genome-scale collections of
full-length, sequence verified cDNAs. Combined with


appropriate functional assays, cDNA libraries have cat-
alyzed significant advances in our understanding of
AD pathogenesis, including the seminal discovery that
B-secretase activity, the first step in the production of
AB, is mediated by BACE1 [21]. Here, we utilized a similar
approach to discover new candidate ABDPs, using a func-
tional assay sensitive to both extracellular and intracellular
AB degradation (as well as other potential ABf-lowering
mechanisms). Rather unexpectedly, the top hit emerging
from a screen of 352 proteases was BACE2, a close homo-
log of BACE1. Subsequent characterization confirmed
that, in addition to BACE2's established ability to lower
AB production via 0-secretase-mediated processing of
APP [11-16], BACE2 also avidly degrades AB with a cata-
lytic efficiency exceeding almost all well-established
ABDPs.
The finding that BACE2 is an avid A6DP 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 AB, via P-secretase
cleavage of APP, instead [15,16]. However, most evi-
dence now suggests that BACE2 does not contribute
appreciably to AB production in vivo [3]. For instance,
cultured neurons from BACE2 knockout mice did not
show reductions in AB following transfection with APP
[30] and conversely, overexpression of BACE2 in APP
transgenic mice failed to increase cerebral AB levels, as
would be expected if BACE2 possessed B-secretase-like
activity.
In addition to its potent ability to degrade AB, BACE2
also possesses a second ABf-lowering function for


Page 6 of 12


A B
30 25

25 40 0 20
20-


10-1
\ 15 45-


0- 0-




mlz mlz
Figure 4 Overexpression of BACE2 in cells yields AB fragments identical to those produced in vitro. A,B, Spectra ofAB fragments
determined by IP/MS analysis of the conditioned media ofCHO cells transfected with APP and empty vector (A) or APP and BACE2 (B) (see
Methods). A, APP expression alone produces peaks corresponding to AB42, AB40, AB39, AB38 and AB37. 3, Co-expression of APP and BACE2
results in decreases in the relative abundance of the aforementioned AB species and the appearance of three new fragments: AB34, AB20 and
AB19. Double-charged fragments are denoted by "+", and "" represents the modification of a fragment by AEBSF, which leads to a 183-Da
increase in MW, as previously reported [46].







Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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BACE2, one that is quite independent of AB degradation.
Specifically, BACE2 has been shown to cleave APP and
the B-secretase-derived APP-CTF within the AB se-
quence, in a manner analogous to a-secretase [11-16].
This activity, dubbed 0-secretase [17], occurs at positions
19 and 20 within the AB sequence, precisely the same
cleavage sites identified in the present study [13,14]. As is
true for a-secretase, 0-secretase activity lowers AB levels
by shuttling APP away from the amyloidogenic proces-
sing pathway [11-16].

Table 1 Kinetic parameters of AB40 and AB42
degradation by BACE2
AB40 Ai42


KM (pM)
Vmax (IM min-1)
kcat (min'1)
kcatlKM (M-1 min-1)


2.8 00.7
0.68 .083
0.135 0.016
4.82 x 107


2.3 0.6
0.12 0.025
0.025 0.005
1.07 x 10'


As confirmed by previous work [24], we found that
BACE2 also cleaves AB 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 AB by 6- and
y-secretase, because this peptide bond in APP or in APP
CTFs is normally embedded within the cell membrane
[24]. This fact, together with the finding that A634 is pro-
duced in cells overexpressing of BACE2 and APP, provides
clear evidence that the AB-degrading activity of BACE2
contributes significantly to the overall AB-lowering effect
of BACE2 overexpression, even in the context of concur-
rent 0-secretase activity.
Given that BACE2 can lower AB 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 crit-
ically on precisely where and to what extent BACE2 is
expressed in vivo. Although BACE2 protein is readily


Page 7 of 12


A
A M BACE2
100ooIDE
I NEP
I Plasmin
75.
e-3
E
1 50-
I I |







0 5 10 25 40 100
t (min)

B C
125- M A40 125- C Ap42

100- 1 5100-


so. 50

S25] H 25




Figure 5 Comparison of the efficacy of BACE2 relative to other well-established ABDPs in vitro and in cultured cells. A, Degradation of
AB in vitro by equivalent nominal concentrations (5 nM) of recombinant BACE2, IDE, NEP and plasmin. Note that BACE2 degrades AB at a faster
rate than NEP and plasmin, but nt IDE. B,C, Effects on AB40 (A) and AB42 (C) levels following cotransfection of CHO cells with APP together with
equivalent quantities of cDNAs BACE2, ECE b and IDE. In good agreement with the results in vitro (A), BACE2 lowers the levels of both
AB species to an extent exceeding NEP and ECE b, but comparable to IDE. Data are mean SEM of 4 replications, normalized to controls
cotransfected with emntv vector (V\-






Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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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 [30].
To the extent that these findings apply to humans,
0-secretase cleavage of APP by BACE2 would be un-
likely 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 [31]. 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 0-secretase activity of BACE2 may, to some extent,
contribute to the overall economy of brain AB. The


pathophysiological relevance of BACE2's function as
an A6DP 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 AB [37], but the contribution of
intra-astrocytic AB degradation relative to intraneuro-
nal or extracellular degradation in vivo remains to be
established. As was true for other ABDPs first identi-
fied in cells [9], the answer to these questions will re-
quire 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 can-
didate, particularly for gene therapy-based approaches.
BACE2 can lower AB catalytically via two independent
mechanisms, and its ABf-degrading ability alone exceeds
that of most other ABDPs, some of which are being


Page 8 of 12


B










C









Figure 6 BACE2 is localized to intracellular compartments relevant to AB degradation. A, Exogenous administration of fluorescently
labeled A40 (green) to CHO cells results in accumulation at intracellular sites overlapping with lysosomes, as labelled by Lysotracker Red (ed)
and visualized by confocal microscopy. BACE2 is expressed in multiple intracellular compartments, including lysosomes. Distribution of GFP-
tagged BACE2 (green) in cells labeled with Lysotracker Red (ed) shows I localization within lysosomes (yellow). C, BACE2 colocalizes with
exogenously administered AB. Confocal images showing i I overlap (yellow) between BACE2 (green) and fluorescently labeled A (red). For
these experiments, cells were imaged within 5 minutes of washing in cold PBS to remove medium containing excess fluorescently labeled AB.
Note that the the majority of BACE2-GFP-expressing cells contianed very low levels of fluorescent AS (see Figure 7), and the particular cell shown
exhibited relatively high levels of internalized AB, allowing us to highlight the overlap with BACE2.







Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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Page 9 of 12


A


A640
*
*


Vector: + + Vector: + +
BACE2: + + BACE2: + +
Chase time(min): 5 120 5 120 Chase time(min): 5 120 5 120
Figure 7 BACE2 degrades Ai at intracellular sites. A, CHO cells expressing GFP-BACE2 (green, left), but not those expressing GFP alone (green,
right), exhibit marked reductions in intracellular AB (red). For these experiments, cells were loaded for 6 h with 400 nM fluorescently labeled AB40,
washed, then incubated at 37'C for 1 h prior to imaging by conventional fluorescence microscopy. B,C, BACE2 overexpression I lowers
intracellular AB. B, Quantification of intracellular pools of fluorescently labeled A40 in CHO cells 0 and 2 hours after loading. C, Relative levels of
intracellular (unmodified) AB40 in CHO cells 0 and 2 hours after loading, as quantified by ELISA. Data are mean SEM of 3 replicates, normalized
to vector-only controls. *P <0.05 by Tukey's multiple comparisons test.


125

100.

o 75

50
IL 2
d 25


TAMRA-AS40


considered for gene therapy clinical trials [38]. More-
over, as an aspartyl protease, BACE2 possesses distinct
advantages relative to other ABDPs. First, it is operative
with subcellular compartments most relevant to AB pro-
duction-i.e., those containing active 6- and y-secretase,
which are both aspartyl proteases-thus allowing it to
impact AB levels prior to secretion. In this connection,
there is growing evidence that intracellular AB may rep-
resent an especially pathogenic role in AD [39], so
modulation of this pool may be particularly appropriate
therapeutically. Second, because BACE2 is operative ex-
clusively at intracellular sites, its expression could be
readily restricted to the site of administration. This is in


contrast to many other ABDPs 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 A6DP. This newly identified func-
tion of BACE2, together with its established ability to
also lower AB production via 0-secretase activity, sug-
gests 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 pres-
ently incurable disease.


Brgh fel rihtfil






Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
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Methods
cDNA screening
A library of 352 full-length, sequence verified, human
cDNAs encoding diverse members of all protease classes
was purchase from a commercial source (OriGene Tech-
nologies, Inc.) in 96-well format (100 :,'. .11' For nega-
tive and positive controls, a subset of blank wells on
each plate were supplemented with empty vector or a
construct expressing a well-established A6DP, human
ECElb [27], respectively (100 ng/well). As a source of
human A13 and also as a transfection control, each well
was cotransfected with a hybrid construct, APP-AP
(60 .-- '..- II.!. comprised of a vector expressing wild-type
human APP fused at its N-terminus with ii1 .i::.. phos-
phatase (AP) [42]. ,..i1.' ...i blank wells were left un-
treated for cell-free background controls. CHO cells
(4.8 x 1, ..I!) suspended in DMEM/Opti-MEM sup-
plemented 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 i..... I to
grow overnight under standard cell culture conditions
(5% CO2; 37C; 95% humidity) then the medium was
exchanged. 24 h later, the ..:I. v-!r,.-:1 media were col-
lected for downstream analysis (see below). All experi-
ments were conducted in compliance with and with
approval by the Mayo Clinic Institutional Review Board.


AP activity
Eii .... ij heat treatment to inactivate endogenous phos-
phatases (65C for 15 min) present in the media, condi-
tioned media (30 pL/well) was added to 9 ...-!I plates
containing AP substrate, 4-nitrophenylphosphate (170 [iL/
well, 2 mg/mL), dissolved in AP buffer (1 M diethanola-
mine, 0.5 mM MgC12, 10 mM L-homoarginine, pH 9.8).
Plates were incubated for 30 min and AP activity was
determined from absorbance (ODios) using a Spectra-
Max" M5e multilabel plate reader (Molecular Devices).


AP ELISA
A3 levels were q' ...ii, using a sandwich ELISA sys-
tem based on intl.. I, pairs 33.1.1/13.1.1 for .. In and
2.1.3/4 G8 for AP42 as described previously [43]. Condi-
tioned media were ,i: 'i.1 i .1 with CompleteTM Pro-
tease Inhibitor Cocktail (Roche) just after collection and
analyzed ..... i I, For experiments quantifying
intracellular A9, cells were plated in in 96-well plates
(2 x 104 cells per well) and transfected with BACE2-
.... I-c cDNA or empty vector, washed, then incubated
with 400 nM synthetic A8 for 6 h. After washing with
PBS, intracellular AB was extracted with 5 M guanidi-
nium isothiocyanate and quantified using a commer-
cially .. ui.ii,. ELISA (Wako Chemicals USA, Inc.)


Page 10 of 12


after 10-fold dilution in the manufacturer-provided di-
lution buffer.

Mass spectrometry
The cleavage sites within AP40 and AP42 hydrolyzed by
BACE2 and BACE1 were determined essentially as
,i .... i..1 [44] with minor modifications. Briefly, AP
peptides or biotinylated AP peptides were incubated for
various lengths of time with recombinant BACE2 en-
zyme in Assay Buffer (25 mM acetate buffer, pH 4.0,
supplemented with 0.1% BSA). The reaction was stopped
by addition of protease inhibitor <., I ; ..i and pH adjust-
ment. AP fragments were immediately precipitated by
magnetic beads coated with streptavidin (for biotinylated
AP) or magnetic beads coated with Ab9 .!!l...i; [45]
(for unmodified AP). 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% trifluor-
oacetic acid in 50% acetonitrile and water. D;.-. i..1 pro-
ducts were iul,. .1 onto a gold chip, dried, and analyzed
using a Ciphergen ProteinChip SELDI time-of-flight sys-
tem T..; -T:,.'." Mass spectra were acquired automatically
in a linear positive mode at 1350 shots per spectrum.
P. d;.1, containing a183-Da increase in MW were iden-
1.1,. I as being :...l.t,. I by AEBSF, as previously
reported [46]. Same procedure was :.pph!.:l to detect the
endogenous AP fragments produced by CHO cells trans-
fected with APP and BACE2 (using Ab9 as a capture
antibody).

In vitro analyses of Ap degradation by BACE2
The kinetics of AP40 and AP342 degradation by
BACE2 were determined using freshly prepared,
monomeric AP peptides separated from aggregated
species by size-exclusion chromatography and charac-
terized as described -',.] AP peptides were diluted
in neutral Dilution Buffer (20 mM Tris, pH 8.0 .1,..I.
mented with 0.1% BSA) and reactions were initiated by
transfer into 20 times more volume of Assay Buffer sup-
plemented with pt...'i .1 recombinant BACE2 (R&D Sys-
tems, nominal concentration 1 or 5 nM) or, as a control
for non-specific loss of AP, the latter buffer lacking
BACE2. Where required, reactions were terminated
by supplementation with protease inhibitor cocktail
and !.i, .!! i..i to neutral pH. For ELISA-based
experiments, AP42 and A340 were quantified using
the sandwich ELISAs -..I.1 above. For determin-
ation of kinetic parameters, ELISAs were used to quan-
tify the initial velocities of degradation of a range of
.:iir....,r concentrations of A640 (0.2 to 16 pM) or
Af42 (0.6 to 16 pM) by a fixed amount of recombinant
BACE2 (5 nM) in Assay Buffer, and KM and v,, values







Abdul-Hay et al. Molecular Neurodegeneration 2012, 7:46
http://www.molecularneurodegeneration.com/content/7/1/46




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 AP
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 AP was
determined using a well-characterized fluorescence
polarization-based activity Ai degradation assay, as
d. i... ii [23]. For comparison of the rate of degrad-
ation of AP by different proteases, we incubated 200 nM
of AP fluorescent substrate (FA3B) with 5nM of differ-
ent 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 reac-
tions were stopped by addition of protease inhibitor
cocktail, 500nM streptavidin, and adjustment to neutral
pH. The degree of AP hydrolysis was immediately deter-
mined using a polarization-based AG 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 per-
formed at 37C.

Fluorescence microscopy
CHO cells (106 ....l!- .. r.2) were plated onto 8-well poly-
D-lysine-coated, glass-bottom chambers (MatTek Corp.)
in culture medium :( 1 i iOpti-MEM supplemented
with 5%FBS). For BACE2 transfections, cell were trans-
fected with a construct encoding BACE2 tagged at its
C-terminus with GFP (OriGene Technologies, Inc. Cat.
No. RG04860) using Fugene 6.0 transfection reagent
I. ,.... to manufacturer's recommendations (Pro-
mega Corp.). For AP colocalization experiments, cells
were washed twice in fresh culture medium, then incu-
bated in the latter medium supplemented with either
A640 (500 nM) labeled at the N-terminus with HiLight
FluorTM 488 or HiLight FluorTn 555 (AnaSpec, Inc.). For
lysosomal staining, cells were incubated with Lysotracker
Red according to manufacturer's recommendations (Invi-
trogen Corp.), then washed 2 times with fresh culture
medium prior to imaging. For confocal microscopy, cells
were washed with fresh medium then imaged immedi-
ately 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 iini. 1. ,lli i AS, cells were washed with
fresh medium, then incubated at 37C for 1 h prior to
imaging using a Nikon Labophot 2 fluorescent micro-
scope ,r J. .... Inc.).


Page 11 of 12


Abbreviations
AB' Amyloid B-protein; ABDP
AP Alkaline phosphatase; AP
cleaving enzyme-1; BACE2 B
converting enzyme-1; IDE In

Competing interests
The authors declare they hav

Authors' contributions
SA- contriouted to the desi
fol ow up experiments, analy
assisted with the execution c


SAB-degrading protease; AD Alzheimer disease;
P Amyloid precursor protein; BACE1 site APP
-site APP-cleaving enzyme-2; ECE1 Endothelin
sulin-degrading enzyme; NEP Neprilysin


ve no competing interests


gn of experiments, executed the screen and al
zed data, and drafted the manuscript TS
)f the primary screen MM and DK assisted with


the maintenance of cell cultures ML conceived of the experiment
approach, designed eperment anayed data and wrote the manuscript
All a ore ad and approved the final manuscript

Acknowledgements
We thank Dr Terrone Newberry and Wllaam Tay for providing monomeric
and fibriar AB peptides and Drs Iodd Goide and Kevin Felsenstein for
contibuting lhe APP AP construct Supported by a gran r fiom ine Coins ro
Alzheimet's Trust Iund to ML

Received: 16 March 2012 Accepted: 16 August 2012
Published: 17 September 2012

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doi:1 0.1186/1750-1326-7-46
Cite this article as: Abdul-Hay et al Identification of BACE2 as an avid
B-amyloid-degrading protease. Molecular Neurodegeneration 2012 746


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RESEARCHARTICLEOpenAccessIdentificationofBACE2asanavid -amyloid-degradingproteaseSamerOAbdul-Hay,TomokoSahara,MelindaMcBride,DongcheulKangandMalcolmALeissring*AbstractBackground: Proteasesthatdegradetheamyloid-protein(A)haveemergedaskeyplayersintheetiologyand potentialtreatmentofAlzheimer ’ sdisease(AD),butitisunlikelythatallsuchproteaseshavebeenidentified.To discovernewA-degradingproteases(ADPs),weconductedanunbiased,genome-scale,functionalcDNAscreen designedtoidentifyproteasescapableofloweringnetAlevelsproducedbycells,whichweresubsequently characterizedforA-degradingactivityusinganarrayofdownstreamassays. Results: Thetophitemergingfromthescreenwas-siteamyloidprecursorprotein-cleavingenzyme2(BACE2),a ratherunexpectedfindinggiventhewell-establishedroleofitsclosehomolog,BACE1,intheproductionofA. BACE2isknowntobecapableofloweringAlevelsvianon-amyloidogenicprocessingofAPP.However,invitro, BACE2wasalsofoundtobeaparticularlyavidADP,withacatalyticefficiencyexceedingallknownADPsexcept insulin-degradingenzyme(IDE).BACE1wasalsofoundtodegradeA,albeit~150-foldlessefficientlythanBACE2. AiscleavedbyBACE2atthreepeptidebonds — Phe19-Phe20,Phe20-Ala21,andLeu34-Met35 — withthelatter cleavagesitebeingtheinitialandprincipalone.BACE2overexpressioninculturedcellswasfoundtolowernetA levelstoagreaterextentthanmultiple,well-establishedADPs,includingneprilysin(NEP)andendothelinconvertingenzyme-1(ECE1),whileshowingcomparableeffectivenesstoIDE. Conclusions: ThisstudyidentifiesanewfunctionalroleforBACE2asapotentADP.Basedonitshighcatalytic efficiency,itsabilitytodegradeAintracellularly,andothercharacteristics,BACE2representsaparticularystrong therapeuticcandidateforthetreatmentorpreventionofAD. Keywords: Amyloid--protein,Alzheimerdisease,-siteAPP-cleavingenzyme-1,-siteAPP-cleavingenzyme-2, Functionalscreen,Genetherapy,Protease,ProteolyticdegradationBackgroundAlzheimerdisease(AD)isaprogressiveandpresently incurableneurodegenerativedisordercharacterizedby abnormalaccumulationoftheamyloid -protein(A )in brainregionsimportantformnemonicandcognitive functions.Aisaheterogeneousmixtureofpeptides rangingfrom37to43aminoacidsinlength[1]producedviasequentialcleavageoftheamyloidprecursor protein(APP)byBACE1andthepresenilin/ -secretase complex[2-4].Autosomal-dominantmutationsin3 genes — APPandpresenilin-1and 2 — areknownto causerare,familialformsofADeitherbyincreasingthe productionofallformsofAorbyincreasingtherelativeproductionoflonger,moreamyloidogenicforms, suchasA42[5].Nevertheless,theprecisemechanisms underlyingsporadicAD,whichmakesupthevastmajorityofcases,remaintobeelucidated. A-degradingproteases(ADPs)arepotentregulators ofcerebralAlevelsand,assuch,representimportant playersintheetiologyandpotentialtreatmentofAD [6].Amyloidogenesisanddownstreamcytopathologycan beattenuatedandevencompletelypreventedbyenhancingtheactivityofanyofseveralADPs,while,conversely,geneticdeletionofoneormoreADPsleadsto significantelevationsincerebralA[7].Significantly, patientswithsporadicADwererecentlyshowntoexhibitdefectsintheclearanceofA(ratherthan increasesinitsproduction)[8]and,inlightofthelarge bodyofevidenceimplicatingADPsintheregulationof cerebralAlevels[7],itisreasonabletoinferthat defectsinoneormoreADPscouldcontributeto *Correspondence: leissring@mayo.edu DepartmentofNeuroscience,MayoClinic,4500SanPabloRoad,Birdsall Bldg.,Rm.117,Jacksonville,FL32224,USA 2012Abdul-Hayetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46 http://www.molecularneurodegeneration.com/content/7/1/46

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impairedAclearance.WhilemorethantwentyproteasesarenowknowntodegradeA[7],thesewerenot identifiedthroughanysystematicapproach,butinstead emergedhaphazardouslyfromadisconnectedsetof largelyserendipitousdiscoveries.Nevertheless,essentiallyallADPsnowknowntoregulateAinvivowere originallyidentifiedthroughexclusivelyinvitroorcellbasedapproaches[9]. TodiscovernewADPsmoresystematically,weconductedanunbiased,cell-based,functionalscreenof352 proteasesinthehumangenome.ThetopA-lowering proteaseemergingfromthisscreenwas-siteAPPcleavingenzyme-2(BACE2)[10].Previousstudieshave shownthatBACE2canlowerAlevelsvia -secretaselikecleavageofAPPwithintheAsequence[11-16],an activitythathasbeendubbed “ -secretase ” [17].However,wefoundthatBACE2isalsoaremarkablyavid ADP,withacatalyticefficiencyexceedingallother knownADPsexceptinsulin-degradingenzyme(IDE).ResultsanddiscussionFunctionalscreenfornovelADPsToidentifynovelADPs,weperformedacell-based functionalscreenusingacommerciallibraryconsisting of352full-length,sequence-verified,humancDNAsencodingdiversemembersofallproteaseclasses.We experimentedwithseveralapproachesbeforesettlingon afinalconfigurationfortheprimaryscreen.Assays designedtomonitordegradationofexogenousAwere foundtobeconfoundedbythehighlydominanteffectof IDE,whichmediatesthevastmajorityofextracellular Adegradationinculturedcells[18-20].Transient transfectionofcDNAsintocelllinesstablyexpressing APPwasalsotried,butthisapproachsufferedfromincompletetransfectionefficiency,whichattenuatedthe effectonnetextracellularAlevels.Wetherefore electedtoconductthescreenbyco-transfectingprotease-encodingcDNAs,togetherwithpositiveandnegativecontrols,intoarodentcellline(CHOcells)together withaplasmidencodingwild-typehumanAPPfusedto alkalinephosphatase(AP)(seeFigure1A; Methods ).Use oftheAPP-APconstructensuredthathumanA productionwaslimitedtocellsalsoexpressingcandidate ADPs,whilealsoprovidinganinternalcontrolfor transfectionefficiency(viaAPactivity).Importantly,the co-transfectionstrategyalsoincreasedthelikelihoodof detectingADPsthatdegradeAintracellularly,prior toitssecretion,inadditiontothosethatactexclusively extracellularly.Cytotoxicitywasalsoquantifiedviaan MTTconversionassay,butnosignificantcelldeathwas detectedsothesedatawerenotincorporatedintosubsequentanalyses.Thescreenwasperformedinquadruplicateand,foreachwell,theratioofA40concentration toAPactivitywascalculated,thennormalizedtoappropriateintra-platecontrols(Figure1B). Fromamongthe352proteasesexamined,byfarthe largestdecreaseinnormalizedAlevels(971.2%)was inducedbyBACE2,whichwasinfacttheonlyprotease tolowerAlevelsmorethan75%,ourpre-determined cut-offforviablehits(Figure1B).BACE2transfectionlowersA levelsToconfirmandextendtheresultsobtainedinthe cDNAscreen,wecomparedthedegreetowhichoverexpressionofBACE2anditshomologBACE1[21]affected thenetproductionofdifferentAspecies.Consistent withtheresultsoftheprimaryscreen,BACE2transfectioninCHOcellsdecreasedthelevelsofbothA 40and A 42(Figure1C).OverexpressionofBACE1inthiscell type,bycontrast,hadnoeffectonnetAlevels (Figure1C).WenotethatBACE1overexpressionwould notbeexpectedtoincreaseAproductioninCHOcells, sincepreviousstudieshaveestablishedthat -secretase, ratherthan-secretase,istherate-limitingstepinA productioninthiscelltype[22].BACE2andBACE1degradeAinvitroExpressionofBACE2incellscouldlowerAlevelseitherdirectly,viaproteolyticdegradation,orindirectly, viaalternativemechanismssuchashydrolysisofAPPor APPC terminalfragments(CTFs)[11-16].Todistinguishthesepossibilities,wetestedtheabilityofrecombinantBACE2tohydrolyzesyntheticAinvitro,using awell-establishedfluorescencepolarization-basedA degradationassay[23].RecombinantBACE2wasfound toavidlydegradeAinthisparadigm,confirmingthat BACE2isindeedabonafideADP(Figure2A).RecombinantBACE1alsohydrolyzedA,indicatingthatittoo isanADP(Figure2B).However,BACE1wasmuchless efficientthanBACE2,requiring24htodegradeAtoa similarextentaswasachievedfollowinga10-minincubationwithBACE2(Figure2B).Basedontheseresults, theefficiencyofBACE1wouldappeartobe~150-fold lowerthanthatofBACE2.BACE2-mediatedAdegradationispH-dependentAsanaspartylprotease,thecatalyticefficiencyof BACE2isexpectedtobepH-dependent.Toconfirm this,wecomparedtherateofhydrolysisofA40across arangeapHvalues.Consistentwithexpectations, BACE2wasfoundtobemaximallyeffectiveatpH3.5 (Figure2C),anddecreasinglyeffectiveathigherpH values.ThesefindingsstronglysuggestthatBACE2 wouldnotbeoperativeatthecellsurfaceorwithinthe extracellularspace.Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page2of12 http://www.molecularneurodegeneration.com/content/7/1/46

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BACE2doesnotdegradefibrillarA IndividualADPscanbecategorizedintermsoftheir abilityorinabilitytodegradefibrillarformsofA.Many well-establishedADPs,suchasIDEandNEP,avidlydegrademonomericAbutcannotdegradefibrillarforms andarethereforecategorizedaspurepeptidases.Others, suchasplasmin,degradeAfibrilsandthuscanalsobe categorizedasfibrilases[7].TodeterminetowhichcategoryBACE2belongs,weincubatedrecombinant BACE2withpre-formedfibrilsofA42andquantified thedegreeofaggregationbythioflavinTfluorescence. Nosignificantreductioninaggregationwasobserved, evenfollowingincubationat37Cforupto3d (Figure2D).Theseresultssuggestthat,asistrueforthe majorityofADPs[7],BACE2doesnotdegradeA fibrils. BACE2cleavesAat3sites Wenextinvestigatedwhichpeptidebond(s)withinA arehydrolyzedbyBACE2andBACE1.Tothatend,we co-incubatedN-terminallybiotinylatedA 40orA 42 (300nM)withBACE2(5nM)andanalyzedtheproducts byimmunoprecipitation/massspectrometry(IP/MS) (see Methods ).Within1h,BACE2almostcompletely hydrolyzedbothAspecies,generatingtheshorterfragment,A 34,inbothcases(Figure3A-D).Totest whetheranyadditionalcleavagescanoccur,weincubatedN-terminallybiotinylatedA 40(300nM)witha largeramountofBACE2(25nM)for1and24h.At thesehigherconcentrationsandlongerincubationtimes, A 19andA 20weretheprincipalN-terminalfragments remainingattheendofthereaction(Figure3E-F).Collectively,theseinvitroresultssuggestthatBACE2 cleavesA atthreedifferentpositions:Phe19-Phe20, Phe20-Ala21,andLeu34-Met35,withthelattercleavage sitebeingtheinitialandprincipalone,asisconsistent withpreviousobservations[13,14,24]. ToconfirmwhetherBACE2cleavesA atthesame sitesinamorephysiologicalsetting,weanalyzedA speciesintheconditionedmediaofcellsexpressing APP-APeitheraloneortogetherwithBACE2byIP/MS (see Methods ).AsexpectedforcellsexpressingAPP-AP alone,themediumfromthesecellscontainedA 42, A 40,A 39,A 38,andA 37(Figure4A).BACE2 Figure1 OverviewandoutcomeoffunctionalscreeningfornovelA-loweringproteases. A ,Cartoonillustratingtheoveralldesignofthe screen.Briefly,anarrayedcollectionof352protease-encodingcDNAswascotransfectedintoCHOcellstogetherwithanAPP-APfusionconstruct. Followingamediumchangeandovernightincubation,A40levels,APactivity,andcytotoxicity(viaMTTassay)wereanalyizedintheresulting conditionedmedia. B ,Resultsofthescreen,expressedas[A40]/APratiosnormalizedtointraplatecontrols.Dataaremeanof4replicates.Note thatthelargestdecreaseinAlevelsbyfarwasacheivedbyBACE2. C ,Confirmationoftheresultsofthescreen.Followingscale-upand sequenceverification,cDNAsencodingBACE2anditshomolog,BACE1,werecotransfectedtogetherwithAPP-APintoCHOcells.Consistentwith theoutcomeofthemedium-throughputscreen,BACE2,butnotBACE1,expressionresultedinsignificantdecreasesinthelevelsofbothA40 andA42.DataaremeanSEMof4replicates,andarenormalizedtovector-onlycontrols. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page3of12 http://www.molecularneurodegeneration.com/content/7/1/46

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expressionsuppressedthesignalofallofthesespecies, andnewpeakscorrespondingtoA 19,A 20,andA 34 emerged(Figure4B),confirmingthatthecleavagesites mediatedbyBACE2invitroarealsohydrolyzedinintact cells.TheappearanceofA34isparticularlynotable,becausecleavageatposition34canonlyoccurafterproductionoffull-lengthA,asthispeptidebondis positionedwithinthetransmembranedomainofAPP,as hasbeenshownpreviously[24].Althoughthisresult clearlyindicatesthatBACE2doesindeeddegradeA afteritisproduced,itisnotpossibletoquantifytheextenttowhichtheA19andA20peaksaretheresultof -secretaseactivityorsubsequentdegradationofthe A34fragment(orfull-lengthA).Asaconsequence,it isdifficulttoestimatetheexactextenttowhichtheAloweringeffectofBACE2canbeassignedtononamyloidogenicprocessingversusAdegradationperse inexperimentalparadigmsofthistype. BACE2degradesAmoreefficientlythanwell-established ADPs HavingestablishedBACE2asanADP,wenextinvestigatedhowBACE2comparestootherknownADPsin termstheabilitytodegradeAinvitroandtolowernet Alevelsincells.Tocomparetherelativeefficiencyof BACE2invitro,wemonitoredthedegradationofafixed amountofA(200nM)byrecombinantBACE2(5nM) ascomparedtoequalquantitiesofseveralwellestablishedADPs,includingIDE,NEPandplasmin. Undertheseconditions,BACE2hydrolyzedAmoreefficientlythanallotherADPsexceptIDE(Figure5A). WenotethattheconcentrationofAusedinthisexperimentwasconsiderablylowerthanthe K M foreach oftheproteasestested(see[23]andbelow),makingthe initialvelocityofthisreactionagoodindexoftherelativecatalyticefficiency. KineticsofAdegradationbyBACE2 ToinvestigatethecatalyticefficiencyofBACE2more quantititatively,wedeterminedthekineticsofdegradationofbothA40andA42byBACE2(see Methods ). Forthisanalysis,wewerecarefultousefreshlyprepared batchesofmonomerichumanA 40andA 42peptides, whichweroutinelypreparebysize-exclusionchromatographyandwhichhavebeenextensivelycharacterized [25,26].BACE2cleavedbothAspecieswithsimilar kinetics,exhibitingapparent K M valuesinthelowmicromolarrangeandalbeitwithapparent k cat valuesslightly Figure2 BACE2degradesAinvitro. A ,PercentAremainingfollowingincubationwithdifferentconcentrationsofrecombinantBACE2for variouslengthsoftime.DataaremeanSEMof4replicates,normalizedtoprotease-freecontrols. B ,ComparisontherelativeA-degrading abilityofrecombinantBACE2vs.BACE1.Notethat24hincubationwithBACE1wasrequiredtoachieveapproximatelythesameextentof degradationaseffectedbyBACE2in10min.DataaremeanSEMof3replicates,normalizedtoprotease-freecontrols. C ,BACE2activityispH dependent.PercentAdegradationcatalyzedbyequivalentamountsofBACE2atdifferentpHvalues.DataaremeanSEMof4replicates. D ,BACE2doesnotdegradefibrillarA.LackofeffectofBACE2(10nM)onpreformedA42fibrilsfollowingincubationat37Cfor5d,as determinedbythioflavinTfluorescence.DataaremeanSEMof3replicates. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page4of12 http://www.molecularneurodegeneration.com/content/7/1/46

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higherforA40relativetoA42(0.1350.016min -1 and0.0250.005min -1 ,respectively;Table1).Interms ofcatalyticefficiency( k cat / K M ),BACE2degradesA 40 approximately4-foldmoreefficientlythanA 42 (Table1).Theseparametersexceedthepublishedvalues formostotherwell-characterizedADPs,includingNEP [23],ECE1[27],andplasmin[23],whilebeingcomparabletothoseofIDE[23,28].Consequently,thesevalues areingoodagreementwiththeside-by-sidecomparison ofAdegradationinvitrodiscussedabove(Figure5A). ToinvestigatetherelativeabilityofBACE2tolower Alevelsundermorephysiologicalconditions,wecotransfectedCHOcellswithAPPtogetherwithBACE2 orseveralotherADPs,thenquantifiednetA40and A42levelsintheconditionedmediumbyELISA.We emphasizethatthisapproachcannotcontrolforintrinsic differencesintranscriptionortranslationefficiency,and, inthecaseofBACE2,theA-loweringeffectcanalsobe mediatedtoanundetermineddegreebyBACE2-mediated -secretaseactivity.Nevertheless,theresultswereingood agreementwiththeinvitrofindings:BACE2lowerednet A40andA42levelstoacomparableextentasIDE, withbothofthelatterbeingsignificantlymoreeffective thanNEPorplasmin(Figure5B,C). BACE2colocalizeswithA intracellularly HavingdeterminedthatBACE2isfunctionallyamong themostefficientADPsyetdiscovered,wesubsequentlyinvestigatedthesubcellularlocalizationof BACE2,focusinginparticularontheextenttowhichit colocalizeswithA inacidiccompartments,where BACE2isexpectedtobeoperative.Inagreementwith otherpublishedfindings[29],applicationoffluorescentlytaggedAtolivecellsresultedinits Figure3 DeterminationofpeptidebondswithinAhydrolyzedbyBACE2. Top ,Summaryofcleavagesitesdeterminedfromdatain A F showingthemajorsite( blockarrow )andtwominorsites( arrowheads ).Att=0( A C ),intactA42( A )andA40( C )representtheonlyspecies present.FollowingincubationofA42andA40with5nMBACE2for1h( B D ,respectively),thefull-lengthAspeciesareessentiallycompletely absentandreplacedbyA34. E F ,AdditionalAcleavageproductsareproducedfollowingincubationwithlargeramountsofBACE2(25nM)for longerlengthsoftime.By1h( E ),anewpeakcorrespondingtoA20isproduced.By24h( F ),A20becomesthemajorspeciespresent,and A19isalsoproduced.Double-chargedfragmentsaredenotedby “ + ” ,and “ ” representsthemodificationofafragmentbyAEBSF,whichleads toa183-DaincreaseinMW,aspreviouslyreported[46]. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page5of12 http://www.molecularneurodegeneration.com/content/7/1/46

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accumulationatintracellularsiteslargelyoverlapping withlysosomes(Figure6A).TotestwhetherBACE2is alsolocalizedtolysosomesand/orothercompartments containingA,weanalyzedCHOcellsexpressing BACE2taggedatitsN-terminuswithgreenfluorescent protein(BACE2-GFP).Asdeterminedbyconfocalmicroscopy,BACE2-GFPwasfoundtobepresentinlysosomes(Figure6B)andalsotooverlapsignificantlywith fluorescentlylabeledA(Figure6C). BACE2degradesAatintracellularsites TodirectlyassesswhetherBACE2degradesAatintracellularsites,wetestedtheabilityofBACE2-expressing cellstodegradeexogenouslyappliedAbymultiple methods.CellsoverexpressingBACE2-GFPandloaded withfluorescentlytaggedA40showedsignificantly reducedintracellularA1hafterwashing,butthiswas notthecaseforcellsoverexpressingGFPalone (Figure7A).Consistentwiththis,levelsofintracellular A,bothfluorescentlytaggedandunmodified,were foundtobeconsistentlylowerincellsoverexpressing (untagged)BACE2relativetovector-trasfectedcontrols (Figure7B,C).Notably,significantlylowerlevelsofintracellularAwereobservedboth5minand2hafter washinginmultipleparadigms.Collectively,theseresults stronglysuggestthatBACE2isabonafideADPthat avidlydegradesAwithinacidiccompartments. Conclusions Oneofthemostfruitfuloutcomesofthegenomicrevolutionistheemergenceofgenome-scalecollectionsof full-length,sequenceverifiedcDNAs.Combinedwith appropriatefunctionalassays,cDNAlibrarieshavecatalyzedsignificantadvancesinourunderstandingof ADpathogenesis,includingtheseminaldiscoverythat -secretaseactivity,thefirststepintheproductionof A,ismediatedbyBACE1[21].Here,weutilizedasimilar approachtodiscovernewcandidateADPs,usingafunctionalassaysensitivetobothextracellularandintracellular Adegradation(aswellasotherpotentialA-lowering mechanisms).Ratherunexpectedly,thetophitemerging fromascreenof352proteaseswasBACE2,aclosehomologofBACE1.Subsequentcharacterizationconfirmed that,inadditiontoBACE2 ’ sestablishedabilitytolower Aproductionvia -secretase-mediatedprocessingof APP[11-16],BACE2alsoavidlydegradesAwithacatalyticefficiencyexceedingalmostallwell-established ADPs. ThefindingthatBACE2isanavidADPsuggestsa novelandunexpectedroleforthisproteaseinthe pathogenesisofAD.Indeed,givenitsclosehomology withBACE1,itwasinitiallyhypothesizedthatBACE2 mightmediatethe production ofA,via -secretase cleavageofAPP,instead[15,16].However,mostevidencenowsuggeststhatBACE2doesnotcontribute appreciablytoAproductioninvivo[3].Forinstance, culturedneuronsfromBACE2knockoutmicedidnot showreductionsinAfollowingtransfectionwithAPP [30]andconversely,overexpressionofBACE2inAPP transgenicmicefailedtoincreasecerebralAlevels,as wouldbeexpectedifBACE2possessed-secretase-like activity. InadditiontoitspotentabilitytodegradeA,BACE2 alsopossessesasecondA-loweringfunctionfor Figure4 OverexpressionofBACE2incellsyieldsAfragmentsidenticaltothoseproducedinvitro.A B ,SpectraofAfragments determinedbyIP/MSanalysisoftheconditionedmediaofCHOcellstransfectedwithAPPandemptyvector( A )orAPPandBACE2( B )(see Methods ). A ,APPexpressionaloneproducespeakscorrespondingtoA42,A40,A39,A38andA37. B ,Co-expressionofAPPandBACE2 resultsindecreasesintherelativeabundanceoftheaforementionedAspeciesandtheappearanceofthreenewfragments:A34,A20and A19.Double-chargedfragmentsaredenotedby “ + ” ,and “ ” representsthemodificationofafragmentbyAEBSF,whichleadstoa183-Da increaseinMW,aspreviouslyreported[46]. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page6of12 http://www.molecularneurodegeneration.com/content/7/1/46

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BACE2,onethatisquiteindependentofAdegradation. Specifically,BACE2hasbeenshowntocleaveAPPand the-secretase-derivedAPP-CTFwithintheAsequence,inamanneranalogousto -secretase[11-16]. Thisactivity,dubbed -secretase[17],occursatpositions 19and20withintheAsequence,preciselythesame cleavagesitesidentifiedinthepresentstudy[13,14].Asis truefor -secretase, -secretaseactivitylowersAlevels byshuttlingAPPawayfromtheamyloidogenicprocessingpathway[11-16]. Asconfirmedbypreviouswork[24],wefoundthat BACE2alsocleavesAattheLeu34-Met35peptide bond,whichwasinfacttheinitialandprincipalsiteof cleavage.Notably,cleavageatthispositioncanonly occurafterproductionoffulllengthAby-and -secretase,becausethispeptidebondinAPPorinAPP CTFsisnormallyembeddedwithinthecellmembrane [24].Thisfact,togetherwiththefindingthatA34isproducedincellsoverexpressingofBACE2andAPP,provides clearevidencethattheA-degradingactivityofBACE2 contributessignificantlytotheoverallA-loweringeffect ofBACE2overexpression,eveninthecontextofconcurrent -secretaseactivity. GiventhatBACE2canlowerAbothbydecreasing itsproductionandbymediatingitsdegradation,which ofthesemechanismsarerelevanttothepathogenesisor thepotentialtreatmentofAD?TheanswerdependscriticallyonpreciselywhereandtowhatextentBACE2is expressedinvivo.AlthoughBACE2proteinisreadily Figure5 ComparisonoftheefficacyofBACE2relativetootherwell-establishedADPsinvitroandinculturedcells. A ,Degradationof Ainvitrobyequivalentnominalconcentrations(5nM)ofrecombinantBACE2,IDE,NEPandplasmin.NotethatBACE2degradesAatafaster ratethanNEPandplasmin,butnotIDE. B C ,EffectsonA40( A )andA42( C )levelsfollowingcotransfectionofCHOcellswithAPPtogetherwith equivalentquantitiesofcDNAsencodingBACE2,ECE1bandIDE.Ingoodagreementwiththeresultsinvitro( A ),BACE2lowersthelevelsofboth AspeciestoanextentexceedingNEPandECE1b,butcomparabletoIDE.DataaremeanSEMof4replications,normalizedtocontrols cotransfectedwithemptyvector(V o ). Table1KineticparametersofA40andA42 degradationbyBACE2 A40A42 K M ( M) 2.80.72.30.6 V max ( Mmin -1 ) 0.68.0830.120.025 k cat (min -1 ) 0.1350.0160.0250.005 k cat / K M (M -1 min -1 ) 4.82x10 7 1.07x10 7 Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page7of12 http://www.molecularneurodegeneration.com/content/7/1/46

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detectedinbrainextracts[15,30-36],anditsactivityhas evenbeenshowntobecomparabletothatofBACE1in post-mortembrain[31,33],thereisconflictingevidence aboutwhichcelltypesexpressBACE2.Studiesinmice, ontheonehand,suggestthattheproteaseisexpressed abundantlyingliabutonlyminimallyinneurons[30]. Totheextentthatthesefindingsapplytohumans, -secretasecleavageofAPPbyBACE2wouldbeunlikelytoplayanysignificantpathophysiologicalrolein AD,giventhatAPPitselfisexpressedpredominantlyin neurons,withonlymodestexpressionlevelsinnonneuronalbraincells[31].Ontheotherhand,multiple studiesinpost-mortemhumanbraintissuehave reporteddetectableBACE2expressionnotonlyin astrocytes,butalsoinneurons[15,33],suggestingthat the -secretaseactivityofBACE2may,tosomeextent, contributetotheoveralleconomyofbrainA.The pathophysiologicalrelevanceofBACE2 ’ sfunctionas anADPissimilarlydifficulttopredictandlikewise dependentontheextenttowhichtheproteaseis expressedinneurons.Ast rocytesareknownmediate theclearanceofA[37],butthecontributionof intra-astrocyticAdegradationrelativetointraneuronalorextracellulardegradationinvivoremainstobe established.AswastrueforotherADPsfirstidentifiedincells[9],theanswertothesequestionswillrequirefurtherstudyinrelevantanimalmodels. NotwithstandinguncertaintyaboutitsroleinAD pathogenesis,anumberofconsiderationssuggestthat BACE2representsanespeciallystrongtherapeuticcandidate,particularlyforgenetherapy-basedapproaches. BACE2canlowerAcatalyticallyviatwoindependent mechanisms,anditsA-degradingabilityaloneexceeds thatofmostotherADPs,someofwhicharebeing Figure6 BACE2islocalizedtointracellularcompartmentsrelevanttoAdegradation. A ,Exogenousadministrationoffluorescently labeledA40( green )toCHOcellsresultsinaccumulationatintracellularsitesoverlappingwithlysosomes,aslabelledbyLysotrackerRed( red ) andvisualizedbyconfocalmicroscopy. B ,BACE2isexpressedinmultipleintracellularcompartments,includinglysosomes.DistributionofGFPtaggedBACE2( green )incellslabeledwithLysotrackerRed( red )showssignificantlocalizationwithinlysosomes( yellow ). C ,BACE2colocalizeswith exogenouslyadministeredA.Confocalimagesshowingsignificantoverlap( yellow )betweenBACE2( green )andfluorescentlylabeledA( red ).For theseexperiments,cellswereimagedwithin5minutesofwashingincoldPBStoremovemediumcontainingexcessfluorescentlylabeledA. NotethatthethemajorityofBACE2-GFP-expressingcellscontianedverylowlevelsoffluorescentA(seeFigure7),andtheparticularcellshown exhibitedrelativelyhighlevelsofinternalizedA,allowingustohighlighttheoverlapwithBACE2. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page8of12 http://www.molecularneurodegeneration.com/content/7/1/46

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consideredforgenetherapyclinicaltrials[38].Moreover,asanaspartylprotease,BACE2possessesdistinct advantagesrelativetootherADPs.First,itisoperative withsubcellularcompartmentsmostrelevanttoAproduction — i.e.,thosecontainingactive-and -secretase, whicharebothaspartylproteases — thusallowingitto impactAlevelspriortosecretion.Inthisconnection, thereisgrowingevidencethatintracellularAmayrepresentanespeciallypathogenicroleinAD[39],so modulationofthispoolmaybeparticularlyappropriate therapeutically.Second,becauseBACE2isoperativeexclusivelyatintracellularsites,itsexpressioncouldbe readilyrestrictedtothesiteofadministration.Thisisin contrasttomanyotherADPswhicharesecretedand/ oractiveextracellularly[19,40,41]andthuslesscapable ofbeingconfinedtospecificregions. Inconclusion,thisstudyidentifiesBACE2asanovel andhighlyefficientADP.ThisnewlyidentifiedfunctionofBACE2,togetherwithitsestablishedabilityto alsolowerAproductionvia -secretaseactivity,suggeststhatBACE2mayplayasignificantroleinAD pathogenesis.Moreover,evenifBACE2playsnorolein theetiologyofAD,BACE2neverthelessrepresentsa particularlyattractivecandidateforgenetherapeutic approachestothetreatmentofpreventionofthispresentlyincurabledisease. Figure7 BACE2degradesAatintracellularsites. A ,CHOcellsexpressingGFP-BACE2( green,left ),butnotthoseexpressingGFPalone( green, right ),exhibitmarkedreductionsinintracellularA( red ).Fortheseexperiments,cellswereloadedfor6hwith400nMfluorescentlylabeledA40, washed,thenincubatedat37Cfor1hpriortoimagingbyconventionalfluorescencemicroscopy. B C ,BACE2overexpressionsignificantlylowers intracellularA. B ,QuantificationofintracellularpoolsoffluorescentlylabeledA40inCHOcells0and2hoursafterloading. C ,Relativelevelsof intracellular(unmodified)A40inCHOcells0and2hoursafterloading,asquantifiedbyELISA.DataaremeanSEMof3replicates,normalized tovector-onlycontrols.* P <0.05byTukey ’ smultiplecomparisonstest. Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page9of12 http://www.molecularneurodegeneration.com/content/7/1/46

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MethodscDNAscreeningAlibraryof352full-length,sequenceverified,human cDNAsencodingdiversemembersofallproteaseclasses waspurchasefromacommercialsource(OriGeneTechnologies,Inc.)in96-wellformat(100ng/well).Fornegativeandpositivecontrols,asubsetofblankwellson eachplateweresupplementedwithemptyvectorora constructexpressingawell-establishedADP,human ECE1b[27],respectively(100ng/well).Asasourceof humanAandalsoasatransfectioncontrol,eachwell wascotransfectedwithahybridconstruct,APP-AP (60ng/well),comprisedofavectorexpressingwild-type humanAPPfusedatitsN-terminuswithalkalinephosphatase(AP)[42].Additionalblankwellswereleftuntreatedforcell-freebackgroundcontrols.CHOcells (4.8x104/well)suspendedinDMEM/Opti-MEMsupplementedwith5%FBSwerethenco-transfectedwith APP-APandprotease-encodingcDNAsusingFugene 6.0,accordingtomanufacturer ’ srecommendations (PromegaCorp.).Transfectedcellswereallowedto growovernightunderstandardcellcultureconditions (5%CO2;37C;95%humidity)thenthemediumwas exchanged.24hlater,theconditionedmediawerecollectedfordownstreamanalysis(seebelow).Allexperimentswereconductedincompliancewithandwith approvalbytheMayoClinicInstitutionalReviewBoard.APactivityFollowingheattreatmenttoinactivateendogenousphosphatases(65Cfor15min)presentinthemedia,conditionedmedia(30 L/well)wasaddedto96-wellplates containingAPsubstrate,4-nitrophenylphosphate(170 L/ well,2mg/mL),dissolvedinAPbuffer(1Mdiethanolamine,0.5mMMgCl2,10mML-homoarginine,pH9.8). Plateswereincubatedfor30minandAPactivitywas determinedfromabsorbance(OD405)usingaSpectraMaxWM5emultilabelplatereader(MolecularDevices).A ELISAA levelswerequantifiedusingasandwichELISAsystembasedonantibodypairs33.1.1/13.1.1forA 40and 2.1.3/4G8forA 42asdescribedpreviously[43].ConditionedmediaweresupplementedwithCompleteTMProteaseInhibitorCocktail(Roche)justaftercollectionand analyzedimmediately.Forexperimentsquantifying intracellularA,cellswereplatedinin96-wellplates (2x104cellsperwell)andtransfectedwithBACE2encodingcDNAoremptyvector,washed,thenincubated with400nMsyntheticAfor6h.Afterwashingwith PBS,intracellularAwasextractedwith5MguanidiniumisothiocyanateandquantifiedusingacommerciallyavailableELISA(WakoChemicalsUSA,Inc.) after10-folddilutioninthemanufacturer-provideddilutionbuffer.MassspectrometryThecleavagesiteswithinA 40andA 42hydrolyzedby BACE2andBACE1weredeterminedessentiallyas described[44]withminormodifications.Briefly,A peptidesorbiotinylatedA peptideswereincubatedfor variouslengthsoftimewithrecombinantBACE2enzymeinAssayBuffer(25mMacetatebuffer,pH4.0, supplementedwith0.1%BSA).Thereactionwasstopped byadditionofproteaseinhibitorcocktailandpHadjustment.A fragmentswereimmediatelyprecipitatedby magneticbeadscoatedwithstreptavidin(forbiotinylated A )ormagneticbeadscoatedwithAb9antibody[45] (forunmodifiedA ).Beadswerewashedwith10mM NH4CO3,pH8.0,andpeptidefragmentswereeluted using0.5%trifluoroaceticacidin75%acetonitrilein water,followedbytheadditionofanequalvolumeofa saturatedsinapicacidsolutiondissolvedin0.5%trifluoroaceticacidin50%acetonitrileandwater.Digestedproductswerespottedontoagoldchip,dried,andanalyzed usingaCiphergenProteinChipSELDItime-of-flightsystem(Bio-Rad).Massspectrawereacquiredautomatically inalinearpositivemodeat1350shotsperspectrum. Peptidescontaininga183-DaincreaseinMWwereidentifiedasbeingmodifiedbyAEBSF,aspreviously reported[46].Sameprocedurewasappliedtodetectthe endogenousA fragmentsproducedbyCHOcellstransfectedwithAPPandBACE2(usingAb9asacapture antibody).InvitroanalysesofA degradationbyBACE2ThekineticsofA 40andA 42degradationby BACE2weredeterminedusingfreshlyprepared, monomericA peptidesseparatedfromaggregated speciesbysize-exclusionchromatographyandcharacterizedasdescribed[25,26].A peptideswerediluted inneutralDilutionBuffer(20mMTris,pH8.0supplementedwith0.1%BSA)andreactionswereinitiatedby transferinto20timesmorevolumeofAssayBuffersupplementedwithpurifiedrecombinantBACE2(R&DSystems,nominalconcentration1or5nM)or,asacontrol fornon-specificlossofA ,thelatterbufferlacking BACE2.Whererequired,reactionswereterminated bysupplementationwithproteaseinhibitorcocktail andadjustmenttoneutralpH.ForELISA-based experiments,A 42andA 40werequantifiedusing thesandwichELISAsdescribedabove.Fordeterminationofkineticparameters,ELISAswereusedtoquantifytheinitialvelocitiesofdegradationofarangeof differentconcentrationsofA40(0.2to16 M)or A42(0.6to16 M)byafixedamountofrecombinant BACE2(5nM)inAssayBuffer,and KMand vmaxvaluesAbdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page10of12 http://www.molecularneurodegeneration.com/content/7/1/46

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weredeterminedintriplicatebyfittingahyperbolic curvetothesedatainPrism5.0(GraphPadSoftware, Inc.).FordeterminationofthepHdependenceofA degradation,experimentswerecarriedsameas describedabove,usingAssayBufferatdifferentpH values(3.0,3.5,4.0,4.5,5.0,5.5).Thereactionswere stoppedat10minandtheremaining200nMofA was determinedusingawell-characterizedfluorescence polarization-basedactivityAdegradationassay,as described[23].ForcomparisonoftherateofdegradationofA bydifferentproteases,weincubated200nM ofA fluorescentsubstrate(FA B)with5nMofdifferentproteaseintheircorrespondingbuffers:BACE1and BACE2usingAssayBufferandIDE,NEP,andplasmin inPBS,pH7.4supplementedwith0.1%BSA.Thereactionswerestoppedbyadditionofproteaseinhibitor cocktail,500nMstreptavidin,andadjustmenttoneutral pH.ThedegreeofA hydrolysiswasimmediatelydeterminedusingapolarization-basedAdegradationassay [23].RecombinantBACE2(R&DSystems)andplasmin (EMDBiosciences)werepurchasedfromcommercial sources,whilerecombinantIDEandsecretedNEP(i.e., lackingthetransmembranedomain)weregenerated andpurifiedasdescribed[23].Allreactionswereperformedat37C.FluorescencemicroscopyCHOcells(106cells/cm2)wereplatedonto8-wellpolyD-lysine-coated,glass-bottomchambers(MatTekCorp.) inculturemedium(DMEM/Opti-MEMsupplemented with5%FBS).ForBACE2transfections,cellweretransfectedwithaconstructencodingBACE2taggedatits C-terminuswithGFP(OriGeneTechnologies,Inc.Cat. No.RG04860)usingFugene6.0transfectionreagent accordingtomanufacturer ’ srecommendations(PromegaCorp.).ForA colocalizationexperiments,cells werewashedtwiceinfreshculturemedium,thenincubatedinthelattermediumsupplementedwitheither A40(500nM)labeledattheN-terminuswithHiLight FluorTM488orHiLightFluorTM555(AnaSpec,Inc.).For lysosomalstaining,cellswereincubatedwithLysotracker Redaccordingtomanufacturer ’ srecommendations(InvitrogenCorp.),thenwashed2timeswithfreshculture mediumpriortoimaging.Forconfocalmicroscopy,cells werewashedwithfreshmediumthenimagedimmediatelyusingthe488-nmand543-nmlaserlinesonaZeiss LCM510METAconfocalmicroscope(CarlZeiss,Inc.). ImageswereprocessedandanalyzedusingMetaMorph softwareaccordingtomanufacturer ’ srecommendations (MolecularDevices,Inc.).Forconventionalfluorescence microscopyofintracellularA,cellswerewashedwith freshmedium,thenincubatedat37Cfor1hpriorto imagingusingaNikonLabophot2fluorescentmicroscope(NikonInc.).Abbreviations A:Amyloid-protein;ADP:A-degradingprotease;AD:Alzheimerdisease; AP:Alkalinephosphatase;APP:Amyloidprecursorprotein;BACE1:-siteAPPcleavingenzyme-1;BACE2:-siteAPP-cleavingenzyme-2;ECE1:Endothelinconvertingenzyme-1;IDE:Insulin-degradingenzyme;NEP:Neprilysin. Competinginterests Theauthorsdeclaretheyhavenocompetinginterests. Authors'contributions SA-Hcontributedtothedesignofexperiments,executedthescreenandall followupexperiments,analyzeddata,anddraftedthemanuscript.TS assistedwiththeexecutionoftheprimaryscreen.MMandDKassistedwith themaintenanceofcellcultures.MLconceivedoftheexperimental approach,designedexperiments,analyzeddataandwrotethemanuscript. Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements WethankDr.TerroneNewberryandWilliamTayforprovidingmonomeric andfibrillarApeptidesandDrs.ToddGoldeandKevinFelsensteinfor contributingtheAPP-APconstruct.SupportedbyagrantfromtheCoinsFor Alzheimer ’ sTrustFundtoML. Received:16March2012Accepted:16August2012 Published:17September2012 References1.SelkoeDJ: Translatingcellbiologyintotherapeuticadvancesin Alzheimer'sdisease. Nature 1999, 399: A23 – A31. 2.VassarR: Beta-secretase(BACE)asadrugtargetforAlzheimer'sdisease. AdvDrugDelivRev 2002, 54: 1589 – 1602. 3.DeStrooperB,VassarR,GoldeT: Thesecretases:enzymeswith therapeuticpotentialinAlzheimerdisease. NatRevNeurol 2010, 6: 99 – 107. 4.WolfeMS: Thegamma-secretasecomplex:membrane-embedded proteolyticensemble. Biochemistry 2006, 45: 7931 – 7939. 5.MuckeL: Neuroscience:Alzheimer'sdisease. 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Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Abdul-Hay etal.MolecularNeurodegeneration 2012, 7 :46Page12of12 http://www.molecularneurodegeneration.com/content/7/1/46


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ui 1750-1326-7-46
ji 1750-1326
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dochead Research article
bibl
title
p Identification of BACE2 as an avid ß-amyloid-degrading protease
aug
au id A1 snm Abdul-Haymi Ofnm Samerinsr iid I1 email abdulhay.samer@mayo.edu
A2 SaharaTomokotomoko.sahara@mbi.ufl.edu
A3 McBrideMelindaleissring.malcolm@mayo.edu
A4 KangDongcheulkang.dongcheul@mayo.edu
A5 ca yes LeissringAMalcolmleissring@mayo.edu
insg
ins Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Birdsall Bldg., Rm. 117, Jacksonville, FL, 32224, USA
source Molecular Neurodegeneration
issn 1750-1326
pubdate 2012
volume 7
issue 1
fpage 46
url http://www.molecularneurodegeneration.com/content/7/1/46
xrefbib pubidlist pubid idtype doi 10.1186/1750-1326-7-46pmpid 22986058
history rec date day 16month 3year 2012acc 1682012pub 1792012
cpyrt 2012collab Abdul-Hay et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Amyloid-ß-protein
Alzheimer disease
ß-site APP-cleaving enzyme-1
ß-site APP-cleaving enzyme-2
Functional screen
Gene therapy
Protease
Proteolytic degradation
abs
sec
st
Abstract
Background
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.
Results
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.
Conclusions
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.
bdy
Background
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 abbrgrp
abbr bid B1 1
produced via sequential cleavage of the amyloid precursor protein (APP) by BACE1 and the presenilin/γ-secretase complex
B2 2
B3 3
B4 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
B5 5
. 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
B6 6
. 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
B7 7
. Significantly, patients with sporadic AD were recently shown to exhibit defects in the clearance of Aß (rather than increases in its production)
B8 8
and, in light of the large body of evidence implicating AßDPs in the regulation of cerebral Aß levels
7
, 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
7
, 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
B9 9
.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)
B10 10
. Previous studies have shown that BACE2 can lower Aß levels via α-secretase-like cleavage of APP within the Aß sequence
B11 11
B12 12
B13 13
B14 14
B15 15
B16 16
, an activity that has been dubbed “θ-secretase”
B17 17
. 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).
Results and discussion
Functional screen for novel AßDPs
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
B18 18
B19 19
B20 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 figr fid F1 1A; it 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).
fig Figure 1caption Overview and outcome of functional screening for novel Aß-lowering proteasestext
b Overview and outcome of functional screening for novel Aß-lowering proteases. A, Cartoon illustrating the overall design of the screen. Briefly, an arrayed collection of 352 protease-encoding cDNAs was cotransfected into CHO cells together with an APP-AP fusion construct. Following a medium change and overnight incubation, Aß40 levels, AP activity, and cytotoxicity (via MTT assay) were analyized in the resulting conditioned media. B, Results of the screen, expressed as [Aß40]/AP ratios normalized to intraplate controls. Data are mean of 4 replicates. Note that the largest decrease in Aß levels by far was acheived by BACE2. C, Confirmation of the results of the screen. Following scale-up and sequence verification, cDNAs encoding BACE2 and its homolog, BACE1, were cotransfected together with APP-AP into CHO cells. Consistent with the outcome of the medium-throughput screen, BACE2, but not BACE1, expression resulted in significant decreases in the levels of both Aß40 and Aß42. Data are mean ± SEM of 4 replicates, and are normalized to vector-only controls.
graphic file 1750-1326-7-46-1 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).
BACE2 transfection lowers Aβ levels
To confirm and extend the results obtained in the cDNA screen, we compared the degree to which overexpression of BACE2 and its homolog BACE1
B21 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
B22 22
.
BACE2 and BACE1 degrade Aß in vitro
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
12
13
14
15
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
B23 23
. Recombinant BACE2 was found to avidly degrade Aß in this paradigm, confirming that BACE2 is indeed a bona fide AßDP (Figure F2 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.
Figure 2BACE2 degrades Aß in vitro
BACE2 degrades Aß in vitro. A, Percent Aß remaining following incubation with different concentrations of recombinant BACE2 for various lengths of time. Data are mean ± SEM of 4 replicates, normalized to protease-free controls. B, Comparison the relative Aß-degrading ability of recombinant BACE2 vs. BACE1. Note that 24 h incubation with BACE1 was required to achieve approximately the same extent of degradation as effected by BACE2 in 10 min. Data are mean ± SEM of 3 replicates, normalized to protease-free controls. C, BACE2 activity is pH dependent. Percent Aß degradation catalyzed by equivalent amounts of BACE2 at different pH values. Data are mean ± SEM of 4 replicates. D, BACE2 does not degrade fibrillar Aß. Lack of effect of BACE2 (10 nM) on preformed Aß42 fibrils following incubation at 37°C for 5 d, as determined by thioflavin T fluorescence. Data are mean ± SEM of 3 replicates.
1750-1326-7-46-2
BACE2-mediated Aß degradation is pH-dependent
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.
BACE2 does not degrade fibrillar Aß
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.
BACE2 cleaves Aß at 3 sites
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 F3 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
B24 24
.
Figure 3Determination of peptide bonds within Aß hydrolyzed by BACE2
Determination of peptide bonds within Aß hydrolyzed by BACE2. Top, Summary of cleavage sites determined from data in AF, showing the major site (block arrow) and two minor sites (arrow heads). At t = 0 (A, C), intact Aß42 (A) and Aß40 (C) represent the only species present. Following incubation of Aß42 and Aß40 with 5nM BACE2 for 1 h (BD, respectively), the full-length Aß species are essentially completely absent and replaced by Aß34. E,F, Additional Aß cleavage products are produced following incubation with larger amounts of BACE2 (25 nM) for longer lengths of time. By 1 h (E), a new peak corresponding to Aß20 is produced. By 24 h (F), Aß20 becomes the major species present, and Aß19 is also produced. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported B46 46.
1750-1326-7-46-3 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 F4 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.
Figure 4Overexpression of BACE2 in cells yields Aß fragments identical to those produced in vitro
Overexpression of BACE2 in cells yields Aß fragments identical to those produced in vitro. AB, Spectra of Aß fragments determined by IP/MS analysis of the conditioned media of CHO cells transfected with APP and empty vector (A) or APP and BACE2 (B) (see Methods).A, APP expression alone produces peaks corresponding to Aß42, Aß40, Aß39, Aß38 and Aß37. B, Co-expression of APP and BACE2 results in decreases in the relative abundance of the aforementioned Aß species and the appearance of three new fragments: Aß34, Aß20 and Aß19. Double-charged fragments are denoted by “+”, and “*” represents the modification of a fragment by AEBSF, which leads to a 183-Da increase in MW, as previously reported 46.
1750-1326-7-46-4
BACE2 degrades Aß more efficiently than well-established AßDPs
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 F5 5A). We note that the concentration of Aß used in this experiment was considerably lower than the K
sub 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.
Figure 5Comparison of the efficacy of BACE2 relative to other well-established AßDPs in vitro and in cultured cells
Comparison of the efficacy of BACE2 relative to other well-established AßDPs in vitro and in cultured cells. A, Degradation of Aß in vitro by equivalent nominal concentrations (5 nM) of recombinant BACE2, IDE, NEP and plasmin. Note that BACE2 degrades Aß at a faster rate than NEP and plasmin, but not IDE. B,C, Effects on Aß40 (A) and Aß42 (C) levels following cotransfection of CHO cells with APP together with equivalent quantities of cDNAs encoding BACE2, ECE1b and IDE. In good agreement with the results in vitro (A), BACE2 lowers the levels of both Aß species to an extent exceeding NEP and ECE1b, but comparable to IDE. Data are mean ± SEM of 4 replications, normalized to controls cotransfected with empty vector (Vo).
1750-1326-7-46-5
Kinetics of Aß degradation by BACE2
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
B25 25
B26 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 minsup -1 and 0.025 ± 0.005 min-1, respectively; Table tblr tid T1 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
B27 27
, and plasmin
23
, while being comparable to those of IDE
23
B28 28
. Consequently, these values are in good agreement with the side-by-side comparison of Aß degradation in vitro discussed above (Figure 5A).
table
Table 1
Kinetic parameters of Aß40 and Aß42 degradation by BACE2
tgroup align left cols 3
colspec colname c1 colnum 1 colwidth 1*
center c2 2
c3
thead valign top
row rowsep
entry
Aß40
Aß42
tbody
K
M
(μM)
char ±
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).
BACE2 colocalizes with Aβ intracellularly
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
B29 29
, application of fluorescently tagged Aß to live cells resulted in its accumulation at intracellular sites largely overlapping with lysosomes (Figure F6 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).
Figure 6BACE2 is localized to intracellular compartments relevant to Aß degradation
BACE2 is localized to intracellular compartments relevant to Aß degradation. A, Exogenous administration of fluorescently labeled Aß40 (green) to CHO cells results in accumulation at intracellular sites overlapping with lysosomes, as labelled by Lysotracker Red (red) and visualized by confocal microscopy. B, BACE2 is expressed in multiple intracellular compartments, including lysosomes. Distribution of GFP-tagged BACE2 (green) in cells labeled with Lysotracker Red (red) shows significant localization within lysosomes (yellow). C, BACE2 colocalizes with exogenously administered Aß. Confocal images showing significant overlap (yellow) between BACE2 (green) and fluorescently labeled Aß (red). For these experiments, cells were imaged within 5 minutes of washing in cold PBS to remove medium containing excess fluorescently labeled Aß. Note that the the majority of BACE2-GFP-expressing cells contianed very low levels of fluorescent Aß (see Figure F7 7), and the particular cell shown exhibited relatively high levels of internalized Aß, allowing us to highlight the overlap with BACE2.
1750-1326-7-46-6
BACE2 degrades Aß at intracellular sites
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.
Figure 7BACE2 degrades Aß at intracellular sites
BACE2 degrades Aß at intracellular sites. A, CHO cells expressing GFP-BACE2 (green, left), but not those expressing GFP alone (green, right), exhibit marked reductions in intracellular Aß (red). For these experiments, cells were loaded for 6 h with 400 nM fluorescently labeled Aß40, washed, then incubated at 37°C for 1 h prior to imaging by conventional fluorescence microscopy. B,C, BACE2 overexpression significantly lowers intracellular Aß. B, Quantification of intracellular pools of fluorescently labeled Aß40 in CHO cells 0 and 2 hours after loading. C, Relative levels of intracellular (unmodified) Aß40 in CHO cells 0 and 2 hours after loading, as quantified by ELISA. Data are mean ± SEM of 3 replicates, normalized to vector-only controls. *P <0.05 by Tukey’s multiple comparisons test.
1750-1326-7-46-7
Conclusions
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
21
. 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
12
13
14
15
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
3
. For instance, cultured neurons from BACE2 knockout mice did not show reductions in Aß following transfection with APP
B30 30
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
12
13
14
15
16
. This activity, dubbed θ-secretase
17
, 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
12
13
14
15
16
.As confirmed by previous work
24
, 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
24
. 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
B31 31
B32 32
B33 33
B34 34
B35 35
B36 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
30
. 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
31
. 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
B37 37
, 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
9
, 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
B38 38
. 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
B39 39
, 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
B40 40
B41 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.
Methods
cDNA screening
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)
B42 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.
AP activity
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 ELISA
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
B43 43
. 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.
Mass spectrometry
The cleavage sites within Aβ40 and Aβ42 hydrolyzed by BACE2 and BACE1 were determined essentially as described
B44 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
B45 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).
In vitro analyses of Aβ degradation by BACE2
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.
Fluorescence microscopy
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.).
Abbreviations
Aß: Amyloid ß-protein; AßDP: Aß-degrading protease; AD: Alzheimer disease; AP: Alkaline phosphatase; APP: Amyloid precursor protein; BACE1: ß-site APP-cleaving enzyme-1; BACE2: ß-site APP-cleaving enzyme-2; ECE1: Endothelin-converting enzyme-1; IDE: Insulin-degrading enzyme; NEP: Neprilysin.
Competing interests
The authors declare they have no competing interests.
Authors' contributions
SA-H contributed to the design of experiments, executed the screen and all follow up experiments, analyzed data, and drafted the manuscript. TS assisted with the execution of the primary screen. MM and DK assisted with the maintenance of cell cultures. ML conceived of the experimental approach, designed experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
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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Abstract
Background
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.
Results
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.
Conclusions
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.
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Abdul-Hay, Samer O
Sahara, Tomoko
McBride, Melinda
Kang, Dongcheul
Leissring, Malcolm A
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BioMed Central Ltd
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Samer O Abdul-Hay et al.; licensee BioMed Central Ltd.
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Molecular Neurodegeneration. 2012 Sep 17;7(1):46
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