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
Trang 1The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein
Tobias M J Allinson1, Edward T Parkin1, Thomas P Condon2, Sylva L U Schwager3, Edward D Sturrock3, Anthony J Turner1and Nigel M Hooper1
1
Proteolysis Research Group, School of Biochemistry and Microbiology, University of Leeds, UK;2Isis Pharmaceuticals, Carlsbad,
CA, USA;3Division 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 cysteine switch [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: n.m.hooper@leeds.ac.uk
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)
Trang 2transmembrane growth factors pro-heparin-binding
epi-dermal growth factor-like factor (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 APP695(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 37C in 5% (v/v) CO2 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/Pi 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 lM 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 and allowed to grow for
3 days ASO (200 nMfinal concentration) and Lipofectin (6 lgÆmL)1) were added to 8 mL Opti-MEM in a 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 37C The medium was then aspirated, the cells washed twice with NaCl/Piand 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/Piand 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 lL 10· PCR buffer (Perkin Elmer), 5 mM MgCl2, 0.3 mM 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 lM primers and 0.1 lM 5-amino methyl fluorescein-probe (Fam-probe) and 50 ng total RNA (10 lL) First strand cDNA synthesis was carried out at
48C for 30 min followed by a 10 min heat inactivation step at 95C PCR denaturation was at 95 C for 15 s, and annealing/extension was at 60C 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
ATCTGCTGGAA-3¢ and the fluorescent probe 5¢-CA
Trang 3AGATCATTGCTCCTCCTGAGCGCA-3¢ ADAM10
and ADAM17 RNA levels were normalized to b-actin
expression
SDS/PAGE and immunoblot analysis
Concentrated conditioned medium (20 lg protein) was
resolved on 7–17% polyacrylamide/SDS gels and
electro-blotted onto Hybond Ppoly(vinylidene) difluoride
mem-branes (Amersham) [20] Memmem-branes 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 mM) as substrate at 37C in 0.1M Tris/HCl pH 8.3,
0.3M NaCl, 10 lM ZnCl2 Reactions were terminated by
heating at 100C 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 lM) 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 lM), 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 lM 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 cells were used and no increase in the level of sAP P a 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
Trang 4the 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).
Trang 5was 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 APP695 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 APP695-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/z¼ 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 (IC50) of
50 nMfor the inhibition of the APMA-induced shedding of ACE by compound 24, compared with an IC50of 1.06 lM 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 IC50 of 80 nM [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 transfected with the ASOs and then exposed to AP MA and 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).
Trang 6(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
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 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).
Trang 7in 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, to sAP P a 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).
Trang 8the 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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