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

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

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

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

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-

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-

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

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

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).

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
+

*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].

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

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.
References
1. Turner, A.J. & Hooper, N.M. (2002) The angiotensin-converting
enzyme gene family: genomics and pharmacology. Trends Phar-
macol. Sci. 23, 177–183.
2. Corvol, P., Michaud, A., Soubrier, F. & Williams, T.A. (1995)
Recent advances in knowledge of the structure and function of the
angiotensin I converting enzyme. J. Hypertension 13, S3–S10.
3. Oppong, S.Y. & Hooper, N.M. (1993) Characterization of a
secretase activity which releases angiotensin-converting enzyme
from the membrane. Biochem. J. 292, 597–603.
4. Hooper, N.M., Karran, E.H. & Turner, A.J. (1997) Membrane
protein secretases. Biochem. J. 321, 265–279.
5. Schlondorff, J. & Blobel, C.P. (1999) Metalloprotease -disin-
tegrins: modular proteins capable of promoting cell–cell inter-
actions and triggering signals by protein-ectodomain shedding.
J. Cell Sci. 112, 3603–3617.
6. Selkoe, D.J. (1998) The cell biology of b-amyloid precursor pro-
tein and presenilin in Alzheimer’s disease. Trends Cell Biol. 8,
447–453.
7. Hooper,N.M.&Turner,A.J.(2002)Thesearchforalpha-secre-
tase and its potential as a therapeutic approach to Alzheimer’s
Disease. Curr.Med.Chem.9, 1107–1119.
8. Mattson, M.P. (1997) Cellular actions of beta-amyloid precursor
protein and its soluble and fibrillogenic derivatives. Physiol. Rev.
77, 1081–1132.
9. Sisodia, S. (1992) b-amyloid precursor protein cleavage by a
membrane-bound protease. Proc. Natl Acad. Sci. USA 89, 6075–

talloproteinase 3 (stromelysin) by proteinases and (4-aminophe-
nyl) mercuric acetate. Biochemistry 29, 5783–5789.
17. Milla, M.E., Leesnitzer, M.A., Moss, M.L., Clay, W.C., Carter,
H.L., Miller, A.B., Su, J L., Lambert, M.H., Willard, D.H.,
Sheeley, D.M., Kost, T.A., Burkhart, W., Moyer, M., Blackburn,
R.K., Pahel, G.L., Mitchell, J.L., Hoffman, C.R. & Becherer, J.D.
(1999) Specific sequence elements are required for the expression
of functional tumor necrosis factor-a-converting enzyme (TACE).
J. Biol. Chem. 274, 30563–30570.
18. Merlos-Suarez, A., Ruiz-Paz, S., Baselga, J. & Arribas, J.
(2001) Metalloprotease-dependent protransforming growth fac-
tor-alpha ectodomain shedding in the absence of tumor necrosis
factor-alpha–convertingenzyme.J. Biol. Chem. 276, 48510–
48517.
19. Condon, T.P., Flournoy, S., Sawyer, G.J., Baker, B.F., Kishi-
moto, T.K. & Bennett, C.F. (2001) ADAM17 but not ADAM10
mediates tumor necrosis factor-alpha and L-selectin shedding
from leukocyte membranes. Antisense Nucleic Acid Drug Dev. 11,
107–116.
20. Hooper, N.M. & Turner, A.J. (1987) Isolation of two differentially
glycosylated forms of peptidyl-dipeptidase A (angiotensin con-
verting enzyme) from pig brain: a re-evaluation of their role in
neuropeptide metabolism. Biochem. J. 241, 625–633.
21. Parkin, E.T., Tan, F., Skidgel, R.A., Turner, A.J. & Hooper,
N.M. (2003) The ectodomain shedding of angiotensin-converting
enzyme is independent of its localisation in lipid rafts. J. Cell Sci.
116, 3079–3087.
22. Hooper, N.M., Keen, J., Pappin, D.J.C. & Turner, A.J. (1987) Pig
kidney angiotensin converting enzyme. Purification and char-
acterization of amphipathic and hydrophilic forms of the enzyme

29. Weskamp, G., Cai, H., Brodie, T.A., Higashyama, S., Manova,
K., Ludwig, T. & Blobel, C.P. (2002) Mice lacking the metallo-
protease-disintegrin MDC9 (ADAM9) have no evident major
abnormalities during development or adult life. Mol. Cell Biol. 22,
1537–1544.
30. Sadhukhan, R., Santhamma, K.R., Reddy, P., Peschon, J.J.,
Black, R.A. & Sen, I. (1999) Unaltered cleavage and secretion of
angiotensin-converting enzyme in tumor necrosis factor a-con-
verting enzyme-deficient mice. J. Biol. Chem. 274, 10511–10516.
Ó FEBS 2004 Ectodomain shedding of ACE and APP (Eur. J. Biochem. 271) 2547