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: 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) 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. References 1. Turner, A.J. & Hooper, N.M. 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(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 . 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. 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. 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