Báo cáo khoa học: The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein ppt

The role of ADAM10 and ADAM17 in the ectodomain shedding of
angiotensin converting enzyme and the amyloid precursor protein
Tobias M. J. Allinson
1
, Edward T. Parkin
1
, Thomas P. Condon
2
, Sylva L. U. Schwager
3
, Edward D. Sturrock
3
,
Anthony J. Turner
1
and Nigel M. Hooper
1
1
Proteolysis Research Group, School of Biochemistry and Microbiology, University of Leeds, UK;
2
Isis Pharmaceuticals, Carlsbad,
CA, USA;
3
Division of Medical Biochemistry, University of Cape Town, South Africa
Numerous transmembrane proteins, including the blood
pressure regulating angiotensin converting enzyme (ACE)
and the Alzheimer’s disease amyloid precursor protein
(APP), are proteolytically shed from the plasma membrane
by metalloproteases. We have used an antisense oligo-
nucleotide (ASO) approach to delineate the role of
ADAM10 and tumour necrosis factor-a converting enzyme

(TACE; ADAM17) in the ectodomain shedding of ACE and
APP from human SH-SY5Y cells. Although the ADAM10
ASO and TACE ASO significantly reduced (> 81%) their
respective mRNA levels and reduced the a-secretase shed-
ding of APP by 60% and 30%, respectively, neither ASO
reduced the shedding of ACE. The mercurial compound
4-aminophenylmercuric acetate (APMA) stimulated the
shedding of ACE but not of APP. The APMA-stimulated
secretase cleaved ACE at the same Arg-Ser bond in the
juxtamembrane stalk as the constitutive secretase but was
more sensitive to inhibition by a hydroxamate-based com-
pound. The APMA-activated shedding of ACE was not
reduced by the ADAM10 or TACE ASOs. These results
indicate that neither ADAM10 nor TACE are involved in
the shedding of ACE and that APMA, which activates a
distinct ACE secretase, is the first pharmacological agent
to distinguish between the shedding of ACE and APP.
Keywords: ADAM; antisense oligonucleotide; metallo-
protease; secretase; tumour necrosis factor-a converting
enzyme.
Angiotensin converting enzyme (ACE) is critically involved
in blood pressure regulation due to its action in generating
angiotensin II and in inactivating bradykinin [1]. The
enzyme also has a role in the development of vascular
pathology and endothelium remodelling in some disease
states [2]. Inhibitors of ACE have emerged as first-line
therapy for a range of cardiovascular and renal diseases,
including hypertension, congestive heart failure, myocardial
infarction and diabetic nephropathy. The transmembrane
protein ACE is proteolytically shed from the cell surface

by its cognate secretase with the resulting soluble form
circulating in the blood and present in other body fluids [3].
In addition to ACE, a number of other integral
membrane proteins are shed from the cell surface by a
post-translational proteolytic cleavage event mediated by
zinc metalloproteases [4,5]. Another such shedding process
is the nonamyloidogenic processing of the Alzheimer’s
disease amyloid precursor protein (APP) [6]. Cleavage of
APP within the neurotoxic amyloid b region by a-secretase
precludes the deposition of intact amyloid b [7] and releases
the large soluble ectodomain of APP, sAPPa, which has
been shown to have neuroprotective and memory
enhancing properties [8]. The APP a-secretase is a mem-
brane-associated metalloprotease [9] that is inhibited by
hydroxamic acid-based compounds such as batimastat [10].
Members of the ADAMs (a disintegrin and metallo-
protease) family have been put forward as candidate
a-secretases, in particular ADAM10 and ADAM17
(tumour necrosis factor-a converting enzyme; TACE)
([11,12] and reviewed in [13]). Although the ACE secretase
has not yet been identified, studies with a range of
hydroxamic acid-based inhibitors have shown that it has a
remarkably similar inhibition profile to the APP a-secretase
[10,14], leading us to conclude that the two secretases are,
at the very least, closely related.
The organomercurial compound 4-aminophenylmercuric
acetate (APMA) activates latent metalloproteases by indu-
cing autocatalytic cleavage and removal of the enzyme
prodomain inhibitory region [15]. In matrix metallopro-
teases APMA acts by disrupting the cysteine-zinc bond that

exists between the critical cysteine of the prodomain and the
zinc atom of the active site, the so-called Ôcysteine switchÕ
[16]. ADAMs also contain a cysteine switch in their
prodomain and APMA has been shown to activate
recombinant TACE [17]. More recently it has been shown
that APMA could induce the shedding of APP and the
Correspondence to N. M. Hooper, Proteolysis Research Group,
School of Biochemistry and Microbiology, University of Leeds, Leeds,
LS2 9JT, UK. Fax: + 44 113343 3167, Tel.: + 44 113343 3163,
E-mail:
Abbreviations: ACE, angiotensin converting enzyme; ADAM, a
disintegrin and metalloprotease; APMA, 4-aminophenylmercuric
acetate; APP, amyloid precursor protein; CHO, Chinese hamster
ovary; HB-EGF, heparin-binding epidermal growth factor-like factor;
sAPPa, soluble APP cleaved by a-secretase; TACE, tumour necrosis
factor-a converting enzyme; TGFa, transforming growth factor-a
ASO, antisense oligonucleotide; QRT, quantitative reverse transcrip-
tion; IC
50
, 50% inhibitory concentration.
(Received 2 March 2004, revised 21 April 2004,
accepted 26 April 2004)
Eur. J. Biochem. 271, 2539–2547 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04184.x
transmembrane growth factors pro-heparin-binding epi-
dermal growth factor-like factor (pro-HB-EGF) and pro-
transforming growth factor-a (pro-TGFa) from Chinese
hamster ovary (CHO) cells [18]. In fibroblasts derived from
TACE knockout mice the APMA-induced shedding of
APP and pro-HB-EGF was removed, however, the APMA-
induced shedding of pro-TGFa in these cells was not

affected. This led the authors to conclude that APMA-
induced activation of TACE was responsible for the
shedding of APP and pro-HB-EGF, but that an alter
native metalloprotease was responsible for the shedding of
pro-TGFa [18].
In this study we have investigated the role of ADAM10
and TACE in the shedding of ACE using an antisense
oligonucleotide (ASO) approach to selectively reduce the
expression of each ADAM. Although we show that both
ADAM10 and TACE are involved in the shedding of APP,
neither ADAM is involved in the shedding of ACE.
Furthermore we show that APMA can distinguish between
the shedding of ACE and APP.
Materials and methods
Materials
Isis 16337 (5¢-
CCTAGTCAGTGCTGTTATCA-3¢; under-
lined residues indicate 2¢-O-methoxyethyl modifications)
and Isis 100750 (5¢-
GGTCTGAGGATATGATCTCT-3¢)
(TACE and ADAM10 ASOs, respectively) [19] were
synthesized at Isis Pharmaceuticals (Carlsbad, CA, USA).
Lisinopril)2.8 nm-Sepharose was prepared as described
previously [20]. Antibody 6E10 was from Signet Pathology
Systems (Dedham, MA, USA). Antibody 22C11 was from
Roche Diagnostics (Lewes, UK). The polyclonal antibody
RH179 that recognizes human ACE has been described
previously [3]. The anti-TACE Ig was a gift from R. Black
(Immunex, Seattle, Washington, USA), and the anti-
ADAM10 Ig was a gift from W. Annaert (Vlaams

Interuniversitair Institut voor Biotechnologie, Gent, Bel-
gium). Compound 24 [14] was a gift from GlaxoSmithKline
Pharmaceuticals (Harlow, UK). All other materials were
from Sigma (Poole, UK) or from sources previously noted.
Cell culture
CHO cells stably expressing ACE [21] and SH-SY5Y cells
stably expressing ACE [14] or APP
695
(E. T. Parkin, A. J.
Turner and N. M. Hooper, unpublished data) were
established as described previously. CHO cells were
cultured in Ham’s F-12 medium (Cambrex, Wokingham,
UK) supplemented with 10% (v/v) foetal bovine serum
(Invitrogen, Paisley, UK), penicillin (100 UÆmL
)1
), strepto-
mycin (100 lgÆmL
)1
) and Amphotericin B (2.5 lgÆmL
)1
)
(all from Cambrex). SH-SY5Y cells and HeLa cells were
cultured in Dulbecco’s modified Eagle’s medium (Camb-
rex) supplemented as above. Cells were maintained in a
humidified incubator at 37 °C in 5% (v/v) CO
2
in air.
When the cells were confluent, the medium was changed
to Opti-MEM (Invitrogen), and the cells incubated with
the indicated compounds. The medium was then harves-

ted, centrifuged at 1000 g, for 5 min and concentrated
 50-fold using Vivaspin centrifugal concentrators (10 000
molecular mass cut-off; Vivascience Ltd, Cambridge,
UK).
Transfection of cells with ASOs
Pre-confluent SH-SY5Y cells were washed with NaCl/P
i
and trypsinized. The cells were centrifuged at 1000 g for
5 min and the pellet resuspended in Opti-MEM. ASO was
added to a final concentration of 15 l
M
and the mixture
incubated for 1 min before electroporation at 250 V,
1650 lF and infinite resistance. The cells were immediately
decanted into complete medium. After 24 h, the cells were
incubated in fresh Opti-MEM for 7 h. HeLa cells were
seeded at 10 000 cellsÆcm
)2
andallowedtogrowfor
3 days. ASO (200 n
M
final concentration) and Lipofectin
(6 lgÆmL
)1
)wereaddedto8mLOpti-MEMina
polystyrene tube, mixed and incubated at room tempera-
ture for 20 min. The cells were washed three times with
Opti-MEM prior to addition of the ASO/lipofectin
complexes and subsequent incubation for 4 h at 37 °C.
The medium was then aspirated, the cells washed twice

with NaCl/P
i
and 10 mL complete medium added. After a
further 20 h incubation the cells were incubated in Opti-
MEM for 7 h. For both cell lines, after incubating in
Opti-MEM the medium was harvested and concentrated
as described above. The cell monolayers were washed
twice with NaCl/P
i
and trypsinized. One-tenth of the cell
suspension was removed to a microfuge tube and
centrifuged at 13 000 g for 1 min. The supernatant was
aspirated and the cell pellets lysed by vortexing in 350 lL
of the RNA extraction buffer RLT (with 1% 2-merca-
ptoethanol added before use) (Qiagen). Samples were
frozen at )70 °C prior to quantitative reverse transcription
(QRT)-PCR analysis.
QRT-PCR
QRT-PCR analysis of ADAM10 and TACE mRNAs in
SH-SY5Y and HeLa cells following ASO treatment were
carried out as described previously [19]. Total RNA was
purified after ASO transfection using the RNeasy Mini kit
(Qiagen). All primers and probes were synthesized by IDT
Inc. (Coralville, IA). The 25 lL PCR reaction contained
2.5 lL10· PCR buffer (Perkin Elmer), 5 m
M
MgCl
2
,
0.3 m

M
each dNTP (Pharmacia), 10 U RNase inhibitor
(Perkin Elmer), 0.625 U Taq (Perkin Elmer), 6.25 units
murine leukaemia virus reverse transcriptase (Perkin
Elmer), 0.1 l
M
primers and 0.1 l
M
5-amino methyl
fluorescein-probe (Fam-probe) and  50 ng total RNA
(10 lL). First strand cDNA synthesis was carried out at
48 °C for 30 min followed by a 10 min heat inactivation
step at 95 °C. PCR denaturation was at 95 °Cfor15s,and
annealing/extension was at 60 °C for 1 min for 40 cycles.
ADAM17 PCR primers: 5¢-GAAGAAGTGCCAGGAG
GCGATT-3¢,5¢-CGGGCACTCACTGCTATTACCT-3¢
and the fluorescent probe 5¢-ATGCTACTTGCAAA
GGCGTGTCCTACTGC-3¢, ADAM10 primers: 5¢-TCC
ACAGCCCATTCAGCAA-3¢,5¢-GCGTCTCAGTGGT
CCCATTTG-3¢ and the fluorescent probe 5¢-CGTCA
GCGGCCCCGAGAGAGT-3¢ and b-actin primers: 5¢-AT
TGCCGACAGGATGCAGAA-3¢,5¢-GCTGATCCAC
ATCTGCTGGAA-3¢ and the fluorescent probe 5¢-CA
2540 T. M. J. Allinson et al. (Eur. J. Biochem. 271) Ó FEBS 2004
AGATCATTGCTCCTCCTGAGCGCA-3¢.ADAM10
and ADAM17 RNA levels were normalized to b-actin
expression.
SDS/PAGE and immunoblot analysis
Concentrated conditioned medium (20 lgprotein)was
resolved on 7–17% polyacrylamide/SDS gels and electro-

blotted onto Hybond P poly(vinylidene) difluoride mem-
branes (Amersham) [20]. Membranes were probed for
TACE using a monoclonal anti-TACE antibody (1 : 2000
dilution), ADAM10 using a polyclonal anti-ADAM10 Ig
(1 : 5000 dilution), sAPPa using antibody 6E10 (1 : 2500
dilution), which detects a-secretase cleaved human APP, or
antibody 22C11 (1 : 5000 dilution), which detects soluble
APP [10]. ACE was detected with the polyclonal antibody
RH179 (1 : 2000 dilution) [3]. Bound antibody was detected
with the enhanced chemiluminescent detection system
(Amersham). Blots were quantified by densitometric analysis.
ACE assay
Equal amounts of concentrated conditioned medium pro-
tein were assayed for ACE activity with BzGly-His-Leu
(5 m
M
)assubstrateat37°Cin0.1
M
Tris/HCl pH 8.3,
0.3
M
NaCl, 10 l
M
ZnCl
2
. Reactions were terminated by
heating at 100 °C for 4 min, and the substrate and reaction
products were resolved and quantified by RP-HPLC [20].
Determination of the secretase cleavage site
in soluble ACE

Soluble ACE shed upon APMA stimulation of the cells was
purified from the conditioned medium by affinity chroma-
tography on lisinopril)2.8 nm Sepharose as described
previously [22,23]. Purified soluble ACE was reduced and
protected with vinyl pyridine prior to digestion with
endoproteinase Lys-C. The total digest was analysed
directly by MALDI-TOF MS [23,24].
Statistical analysis
Significance of results was determined using a two-tailed
nonparametric Mann–Whitney U test on the SPSS software
package. A P value < 0.05 was considered significant.
Results
Neither ADAM10 nor TACE are responsible
for the shedding of ACE
Although there are remarkable similarities between the
a-secretase and ACE secretase [10,14,25], the enzyme
responsible for the shedding of ACE has yet to be identified.
We therefore used ASOs directed against either ADAM10
or TACE [19] to transiently knock-down the expression of
their respective mRNAs in the human neuroblastoma
SH-SY5Y cell line and examined the effect on the shedding
of ACE and APP (Fig. 1). The TACE ASO reduced TACE
mRNA by 93% while the ADAM10 ASO reduced
ADAM10 mRNA by 81% in the SH-SY5Y cells (Fig. 1A).
Neither ASO significantly affected the level of the mRNA
for the other ADAM, confirming the specificity of these
ASOs [19]. The ASOs reduced the level of their respective
proteins in cell lysates but had little effect on the other
protein (Fig. 1B,C). The activity of a-secretase was monit-
ored by immunoblotting for the soluble ectodomain frag-

ment of APP, sAPPa, in the cell medium with antibody
6E10. In medium from SH-SY5Y cells sAPPa appears as
a doublet due to the presence of the different isoforms of
APP. The TACE ASO reduced the shedding of sAPPa from
the SH-SY5Y cells by 30%, whereas the ADAM10 ASO
reduced sAPPa levels by 60% (Fig. 1D,E). Similar results
were obtained with another human cell line, HeLa, where
the ASOs (0.2 l
M
) reduced the mRNA levels of their
respective protease by 74%, the TACE ASO reduced
sAPPa shedding by 20% and the ADAM10 ASO reduced
sAPPa levels by 60% (data not shown). In contrast with
their effect on sAPPa shedding, neither ASO had a
significant effect on the levels of soluble ACE in the
conditioned medium (Fig. 1F). These data show that
although both ADAM10 and TACE play a role in the
a-secretase shedding of APP, neither protease was respon-
sible for the shedding of ACE.
APMA stimulates the shedding of ACE but not APP
The effect of the organomercurial compound APMA on
the shedding of ACE and APP in the same cell line was
compared. SH-SY5Y cells stably expressing ACE were
incubated with either APMA or the muscarinic agonist
carbachol which is known to stimulate the shedding of both
APP and ACE from these cells [10,14] (Fig. 2). Although
the shedding of both proteins was stimulated by carbachol
(Fig. 2C,D), only the shedding of ACE was stimulated by
APMA (Fig. 2A,B). We examined further this differential
effect of APMA on the shedding of APP and ACE in

another cell line. CHO cells, which endogenously express
APP, were stably transfected with ACE and exposed to
APMA (Fig. 3). APMA did not stimulate the shedding of
APP from the CHO cells (Fig. 3A,C). Indeed at the highest
concentration (500 l
M
), APMA significantly down-regula-
ted the shedding of APP, although the mechanism for this is
not apparent. In contrast, APMA caused a dose-dependent
increase in the shedding of ACE (Fig. 3B,C), with a 12-fold
increase in the amount of soluble ACE in the CHO cell
medium observed with 500 l
M
APMA. The effect of
APMA on the shedding of ACE was not due to a direct
stimulatory effect on enzyme activity because ACE protein
levels as determined by immunoblotting (Fig. 3B), paral-
leled the increase in enzyme activity (Fig. 3C) and APMA
had no effect on the activity of purified porcine kidney ACE
(data not shown). Thus, in both SH-SY5Y and CHO cells,
APMA stimulated the shedding of ACE but not the
shedding of APP.
As APMA has been shown previously to stimulate the
shedding of APP [18], we carried out a number of other
experiments to confirm the above result. APMA did not
stimulate the shedding of APP when exponentially growing
cellswereusedandnoincreaseinthelevelofsAPPa was
detectable when a comprehensive protease inhibitor cocktail
was added to the cell medium immediately after the APMA
incubation (data not shown). These experiments show that

the confluency state of the cells did not affect the response of
Ó FEBS 2004 Ectodomain shedding of ACE and APP (Eur. J. Biochem. 271) 2541
the a-secretase to APMA, and that sAPPa shed in response
to APMA was not being degraded during the concentration
of the medium. To assess if APMA was causing the release
or activation of a protease that was capable of rapidly
degrading sAPPa during the time course of the experiment,
CHO cells were incubated in the presence of phorbol
12-myristate 13-acetate which stimulates the shedding of
APP. The resulting conditioned medium containing sAPPa
Fig. 2. Effect of APMA and carbachol on the shedding of APP and ACE from SH-SY5Y cells. SH-SY5Y cells stably expressing ACE were incubated
in the absence or presence of either 10 l
M
APMA (A and B) or 20 l
M
carbachol (C and D) in Opti-MEM. The conditioned medium was then
harvested and concentrated. Equal amounts of protein were subjected to electrophoresis on a 7–17% polyacrylamide/SDS gel before immuno-
blotting for sAPPa with antibody 6E10 (A and C) followed by densitometric analysis (B and D, closed bars). ACE activity in the conditioned
medium was determined using BzGly-His-Leu as substrate (B and D, open bars). The results are the mean ± SD of three separate experiments.
*Significantly different (P £ 0.05).
Fig. 1. The effect of antisense-mediated ADAM10 and TACE knockdown on APP and ACE shedding. SH-SY5Y cells stably expressing ACE were
either mock transfected or transiently transfected with 15 l
M
of either ADAM10 or TACE ASOs. The levels of ADAM10 and TACE mRNA in
cell lysates were analysed by QRT-PCR (A). Equal amounts of cell lysate protein were electrophoresed on 7–17% polyacrylamide/SDS gels before
immunoblotting for TACE (B) or ADAM10 (C). After incubation in Opti-MEM for 7 h the medium was harvested, concentrated and equal
volumes subjected to electrophoresis on a 7–17% polyacrylamide/SDS gel before immunoblotting for sAPPa with antibody 6E10 (D) followed by
densitometric analysis (E). ACE activity in the conditioned medium was assayed with BzGly-His-Leu as substrate (F). The results are the
mean ± S.D. of three separate experiments. *Significantly different (P £ 0.05).
2542 T. M. J. Allinson et al. (Eur. J. Biochem. 271) Ó FEBS 2004

was applied to fresh cells in the absence or presence of
APMA. Following this second incubation, the level of
sAPPa in the medium was examined (Fig. 4A,B). There was
no significant difference in the level of sAPPa in the medium
of cells exposed, or not, to APMA, indicating that there did
not appear to be a protease released or activated upon
APMA stimulation that was rapidly degrading sAPPa.
To ascertain that the effect of APMA on ACE shedding
was not an artefact of the transfection process, SH-SY5Y
cells were stably transfected with APP
695
using the same
method as had been used for the stable transfection of ACE.
These cells were then exposed to APMA and the level of
sAPPa in the medium examined (Fig. 4C,D). sAPPa levels
were not increased upon APMA exposure in either the
untransfected SH-SY5Y cells or in the APP
695
-transfected
cells, indicating that the effect of APMA on ACE shedding
was not as a result of over-expression of the protein.
Together these data confirm that APMA induces the
shedding of ACE but not the shedding of APP from two
different cell lines.
The APMA-stimulated secretase cleaves ACE at the same
Arg-Ser bond as the constitutive secretase
As a point mutation in the juxtamembrane stalk of ACE
invoked the action of a distinct protease that cleaved ACE
at a different peptide bond [23], we examined whether the
soluble ACE shed upon APMA stimulation was cleaved at

the same peptide bond as constitutively shed ACE. CHO
cells stably transfected with ACE were exposed to APMA
and the soluble ACE purified from the conditioned medium
by affinity chromatography on lisinopril-Sepharose [20].
The purified soluble ACE was digested with endoproteinase
Lys-C and subjected to MALDI-TOF MS. Mass spectro-
metric analysis of the soluble ACE released from the
APMA-stimulated cells revealed several peptides which
correspond to those of somatic ACE (Table 1). In partic-
ular, a peak at m/z ¼ 1690.1 was observed which is in close
agreement with the calculated mass of the peptide
LGWPQYNWTPNSAR (1690.8). This peptide corres-
ponds to the C terminus of constitutively shed somatic
ACE [24] and shows that ACE shed upon APMA
stimulation of the cells was cleaved at the same Arg-Ser
bond.
The APMA-induced secretase can be distinguished
from the constitutive ACE secretase by its sensitivity
to a hydroxamic acid inhibitor
To determine whether the APMA-induced shedding of
ACE was mediated by the same protease as the constitutive
shedding of ACE, CHO cells stably transfected with ACE
were exposed to a number of hydroxamic acid-based
metalloprotease inhibitors [14] in the presence or absence
of APMA. In the majority of cases there was little difference
between the effect of inhibitors on the constitutive shedding
of ACE and the APMA-induced shedding of ACE (data
not shown). However, compound 24 was found to be
significantly more potent on the APMA-induced shedding
of ACE than on the constitutive shedding. Dose–response

curves revealed a 50% inhibitory concentration (IC
50
)of
50 n
M
for the inhibition of the APMA-induced shedding of
ACE by compound 24, compared with an IC
50
of 1.06 l
M
for the constitutive shedding [14]. These data indicate that
this inhibitor can distinguish between the constitutive and
APMA-induced shedding of ACE, and suggest that the
APMA-induced activity is distinct from the constitutive
ACE secretase.
Neither TACE nor ADAM10 is responsible
for the APMA-induced shedding of ACE
As APMA has been shown to activate TACE [18] and
compound 24 has previously been shown to inhibit TACE
with an IC
50
of 80 n
M
[14], we considered whether the
APMA-induced shedding of ACE was mediated by TACE.
The effect of the TACE ASO, along with the ADAM10
ASO, on the APMA-induced shedding of ACE was
therefore investigated. SH-SY5Y cells expressing ACE were
transfectedwiththeASOsandthenexposedtoAPMAand
the levels of soluble ACE in the medium examined (Fig. 5).

APMA caused a twofold increase in the shedding of ACE
from the mock transfected SH-SY5Y cells, as seen before
Fig. 3. Effect of APMA on the shedding of APP and ACE from CHO
cells. CHO cells stably expressing ACE were incubated in Opti-MEM
in either the absence or presence of the indicated amount of APMA for
30 min. The conditioned medium was harvested, concentrated and
equal amounts of protein subjected to electrophoresis on 7–17%
polyacrylamide/SDS gels before immunoblotting for either sAPP with
antibody 22C11 (A) followed by densitometric analysis (C, closed bars)
or soluble ACE with antibody RH179 (B). Soluble ACE activity in
equal volumes of medium was assayed with BzGly-His-Leu as sub-
strate (C, open bars). Results are the mean ± S.D. of three separate
experiments. *Significantly different (P £ 0.05).
Ó FEBS 2004 Ectodomain shedding of ACE and APP (Eur. J. Biochem. 271) 2543
(see Fig. 2). The ASOs significantly reduced the expression
of their target ADAMs, the TACE ASO reducing TACE
expression by 93% and the ADAM10 ASO reducing
ADAM10 expression by 50% (Fig. 5A). However, the
levels of soluble ACE in the medium of the ASO-treated
cells exposed to APMA were not significantly different to
the level of soluble ACE from APMA-exposed mock
transfected cells (Fig. 5B), indicating that neither TACE,
nor ADAM10, was responsible for the APMA-induced
shedding of ACE.
Discussion
Various members of the ADAM family of metalloproteases
have been implicated in the a-secretase shedding of APP
(reviewed in [13]. In the present study we used an ASO
approach to reduce the expression of ADAM10 and TACE
in the human neuroblastoma SH-SY5Y cell line that has

been used extensively to study APP processing. Our data
clearly show that ADAM10 is responsible for the majority
of the constitutive a-secretase shedding of APP in both the
SH-SY5Y cells and in another human cell line, HeLa. This
is consistent with an earlier study in which overexpression
of ADAM10 in HEK293 cells increased the a-secretase
cleavage of APP, while expression of a dominant negative
form of ADAM10 with a point mutation in the zinc
binding site inhibited a-secretase activity [12]. ASO knock-
down of TACE resulted in only a slight decrease in
a-secretase activity in the SH-SY5Y and HeLa cells,
implying that this protease has only a minor role to play
Table 1. Observed [M + H
+
] ions of ACE peptides generated by endoproteinase Lys-C digestion. The peptides observed by MALDI-TOF MS
following endoproteinase Lys-C digestion of soluble ACE purified from the medium of APMA-stimulated cells are compared to those previously
observed for the constitutively shed human somatic ACE. The C-terminal peptide representing cleavage at Arg1203-Ser bond has a predicted mass
of 1690.8 and is observed in the APMA shed soluble ACE sample showing that cleavage occurs at this bond. Amino acid numbering corresponds to
human somatic ACE.
Peptide no. Amino acid residue
Mass M + H
+
(calculated)
APMA-shed soluble ACE
mass M + H
+
(observed)
Human somatic ACE
mass M + H
+

(observed)
a
32 695–713 2217.4 2217.8 2217.2
34 751–764 1868.0 1867.9 1868.1
43 972–1001 3075.5 3076.3 3076.1
44 1002–1025 2695.0 2696.3 2695.0
47 1055–1067 1766.9 1766.9 1767.3
51 1133–1143 1176.4 1176.3 1175.6
53 1174–1189 1951.1 1951.3 1950.1
54 1190–1203 1690.8 1690.1 1691.1
a
Data from [24].
Fig. 4. The effect of APMA on exogenously added sAPPa and on APP
695
-transfected SH-SY5Y cells. CHO cells were incubated in Opti-MEM in the
presence of 1 l
M
phorbol 12-myristate 13-acetate to stimulate the shedding of sAPPa. The medium was harvested, centrifuged at 2000 g to remove
cell debris and either APMA (250 l
M
) or an equal volume of dimethyl sulfoxide added before applying the conditioned medium to fresh cells for
30 min. The medium was then harvested, concentrated and equal amounts of protein subjected to electrophoresis on a 7–17% polyacrylamide/SDS
gel before immunoblotting for sAPPa with antibody 22C11 (A) followed by densitometric analysis (B). Untransfected SH-SY5Y cells and SH-
SY5Y cells stably transfected with APP
695
were incubated in Opti-MEM in the presence or absence of 10 l
M
APMA. The medium was harvested,
concentrated and equal volumes of conditioned medium subjected to electrophoresis on a 7–17% polyacrylamide/SDS gel before Western blotting
for sAPPa with antibody 6E10 (C) followed by densitometric analysis (D). The results are the mean ± SD of three separate experiments.

*Significantly different (P £ 0.05).
2544 T. M. J. Allinson et al. (Eur. J. Biochem. 271) Ó FEBS 2004
in the shedding of APP. Previously we have shown that the
a-secretase shedding of APP has a distinct inhibitory profile
with a battery of hydroxamic acid-based inhibitors to
recombinant TACE and that a potent inhibitor of TACE
failed to reduce the a-secretase cleavage of APP in SH-
SY5Y cells [14,26]. Thus, this ASO approach confirms and
extends previous observations, providing additional evi-
dence for the central role of ADAM10 and confirming that
TACE has a minor role in the a-secretase cleavage of APP
in human cells.
During the course of the present study it was reported
using an RNA interference approach that ADAM10,
TACE and ADAM9 all contributed equally (30%) to the
shedding of APP in human glioblastoma A172 cells [27].
What appears to be emerging from these studies is that there
is a team of metalloproteases contributing to the a-secretase
cleavage of APP. In different cell types, and possibly under
particular conditions, different members of this team
contribute to a greater or lesser extent to the shedding of
APP. Studies with transgenic mice deficient in a particular
ADAM support this idea. In primary embryonic fibroblasts
derived from TACE knockout mice, although the phorbol
ester-induced a-secretase cleavage of APP was deficient, the
constitutive activity was unaffected [11]. In fibroblasts
derived from ADAM10 knockout mice a-secretase activity
was preserved [28] and in cultured hippocampal neurons
from ADAM9 knockout mice a-secretase activity was also
unaltered [29].

As ACE is shed by a metalloprotease that has a
remarkably similar inhibition profile to that of the
a-secretase [10,14], we investigated whether ADAM10 was
responsible for the shedding of ACE. However, ASO
directed knock-down of ADAM10 had no effect on the
level of the soluble form of ACE from SH-SY5Y cells,
clearly indicating that this ADAM is not involved in the
shedding of ACE. Likewise ASO directed knock-down of
TACE also had no effect on the shedding of ACE,
consistent with previous studies showing that the inhibition
profile of the ACE secretase was distinct to that of TACE
[26] and that the shedding of ACE was preserved in
fibroblasts derived from TACE knock out mice [30]. Thus it
would appear that ADAM10 and TACE are not critically
involved in the shedding of ACE.
APMA has been reported to induce the shedding of a
number of protein ectodomains, including APP, from
CHO cells [18]. However, we failed to observe an effect of
APMA on the shedding of APP from either SH-SY5Y or
CHO cells. This lack of effect was not due to APMA
inducing the shedding and/or activation of a protease
capable of rapidly degrading sAPPa,tosAPPa being
rapidly taken up by the APMA-stimulated cells, the
confluency state of the cells or an artefact of over-
expression of the substrate protein. The reason for the
discrepancy between our study and that of Merlos-Suarez
et al. [18] is not readily apparent. As APMA is known to
activate TACE [17,18], the lack of effect of APMA on the
a-secretase cleavage of APP in the human SH-SY5Y cells
again underlines that TACE is not critically involved in

APP shedding, at least in this cell line.
Although we failed to see an increase in the shedding of
APP upon incubation of the cells with APMA, the
shedding of ACE was increased several-fold. The soluble
form of ACE shed upon APMA stimulation was cleaved at
the same Arg-Ser bond in the juxtamembrane stalk as the
constitutively cleaved form of ACE. However, the APMA-
induced shedding was significantly more sensitive to the
hydroxamic acid-based compound 24 than the constitutive
shedding of ACE, suggesting that a distinct metallo-
protease was being activated. This is in contrast with a
previous study where we observed that a point mutation in
the juxtamembrane stalk of ACE invoked the action of a
mechanistically distinct protease that cleaved ACE at a
different peptide bond [23]. As the inhibitory potency of
compound 24 towards the APMA-induced secretase was
similar as that towards TACE [14], we considered the
possibility that APMA was activating TACE as shown
previously for the APMA-induced shedding of APP and
pro-HB-EGF [18]. However, ASO knock-down of TACE
failed to reduce the APMA-induced shedding of ACE
indicating that this ADAM is not involved. ASO knock-
down also revealed that ADAM10 was not involved in
the APMA induced shedding of ACE. It remains to be
determined whether the APMA-induced metalloprotease
that cleaves ACE is the same as the one that cleaves pro-
TGFa [18].
In conclusion, we have shown that in the human SH-
SY5Y and HeLa cells ADAM10 is the major a-secretase
cleaving APP, with TACE playing a minor role. In

contrast, neither ADAM is involved in the shedding of
ACE. Furthermore, we show that APMA activates a
metalloprotease activity distinct from ADAM10 and
TACE that cleaves ACE at the normal Arg-Ser bond in
Fig. 5. The effect of antisense-mediated ADAM10 and TACE knock-
down on APMA-induced ACE shedding. SH-SY5Y cells stably
expressing ACE were either mock transfected or transiently transfected
with 15 l
M
of either ADAM10 or TACE ASOs. After incubation in
Opti-MEM in the presence or absence of 10 l
M
APMA the medium
was harvested and concentrated. The levels of ADAM10 and TACE
mRNA in cell lysates were analysed by QRT-PCR (A). ACE activity in
the conditioned medium was assayed with BzGly-His-Leu as substrate
(B). The results are the mean ± SD of three separate experiments.
*Significantly different (P £ 0.05).
Ó FEBS 2004 Ectodomain shedding of ACE and APP (Eur. J. Biochem. 271) 2545
the juxtamembrane stalk. In the same cells APMA failed
to activate the shedding of APP, revealing that this is the
first pharmacological agent to clearly differentiate between
the shedding of APP and ACE. What is emerging from
this and other studies is that multiple metalloproteases
are involved in the shedding of individual membrane
proteins and that further work is required to elucidate
the physiological role played by each enzyme in the
increasingly complicated process of protein ectodomain
shedding.
Acknowledgements

T.M.J.A. was in receipt of a studentship from, and we gratefully
acknowledge the financial support of, the Medical Research Council of
Great Britain.
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