Localization of an N-Domain Region of Angiotensin-Converting

Aug 9, 2004 - Cape Town, South Africa, and Department of Biology and ... Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h...
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Localization of an N-Domain Region of Angiotensin-Converting Enzyme Involved in the Regulation of Ectodomain Shedding Using Monoclonal Antibodies Irina V. Balyasnikova,† Zenda L. Woodman,‡ Ronald F. Albrecht, II,† Ramanathan Natesh,§ K. Ravi Acharya,§ Edward D. Sturrock,‡ and Sergei M. Danilov*,† Department of Anesthesiology, University of Illinois at Chicago, Division of Medical Biochemistry, University of Cape Town, South Africa, and Department of Biology and Biochemistry, University of Bath, United Kingdom Received August 9, 2004

ACE chimeric proteins and N domain monoclonal antibodies (mAbs) were used to determine the influence of the N domain, and particular regions thereof, on the rate of ACE ectodomain shedding. Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h. Deletion of the C domain of somatic ACE generated an N domain construct (ACE∆C) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACE∆N) dramatically increased shedding (212%). Testicular ACE (tACE) having 36 amino acid residues (heavily O-glycosylated) at the N-terminus of the C domain shows a 4-fold decrease in the rate of shedding (49%) compared to that of ACE∆N. When the N-terminal region of the C domain was replaced with the corresponding homologous 141 amino acids of the N domain (N-delACE) the rate of shedding of the ACE∆N was only slightly decreased (174%), but shedding was still 3.5-fold more efficient than wild-type testicular ACE. Monoclonal antibodies specific for distinct, but overlapping, N-domain epitopes altered the rate of ACE shedding. The mAb 3G8 decreased the rate of shedding by 30%, whereas mAbs 9B9 and 3A5 stimulated ACE shedding 2- to 4-fold. Epitope mapping of these mAbs in conjunction with a homology model of ACE N domain structure, localized a region in the N-domain that may play a role in determining the relatively low rate of shedding of somatic ACE from the cell surface. Keywords: angiotensin I-converting enzyme • monoclonal antibody • shedding • epitope mapping

Introduction Angiotensin I-converting enzyme (ACE)1 (kininase II, CD 143, EC 3.4.15.1) is a Zn2+ carboxydipeptidase, that cleaves two vasoactive peptides, angiotensin I and bradykinin. The enzyme is also involved in neuropeptide metabolism and reproductive and immune functions (for reviews, see refs 1-4). The somatic isoform is expressed widely at surface-fluid interfaces and plays an important role in blood pressure regulation, the development of vascular pathology, and endothelium remodeling in some disease states. ACE was assigned as a new CD markers CD 143.5 The testis isoform is limited to spermatozoa and is essential for male fertility.6 Somatic ACE has two homologous domains (N and C domains), of which the second (C-terminal) domain is identical to the single domain of testis ACE, except for a unique, 36residue, serine- and threonine-rich sequence at the N-terminus * To whom correspondence should be addressed. Sergei M. Danilov, MD, Ph.D., Anesthesiology Research Center, University of Illinois at Chicago, 1819 W. Polk St. (M/C 519), Chicago, IL 60612. Phone: (312) 413-7526. Fax: (312) 996-9680. E-mail: [email protected]. † Department of Anesthesiology, University of Illinois at Chicago. ‡ Division of Medical Biochemistry, University of Cape Town, South Africa. § Department of Biology and Biochemistry, University of Bath, United Kingdom.

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of testis ACE.7,8 Somatic and testis ACE, both Type 1 integral membrane proteins, are anchored to the plasma membrane near their C-terminus. Somatic ACE also exists as a soluble form, e.g., in plasma, cerebrospinal fluid and seminal plasma,1-3 that lacks the transmembrane domain responsible for membrane attachment.9-12 ACE is a member of the growing family of membrane proteins that are proteolytically cleaved in the juxtamembrane stalk region by secretases or sheddases that are membraneassociated. ACE secretase(s) are metalloproteases that colocalize with ACE in a number of tissues and havean absolute requirement for ACE to be membrane-anchored in order for cleavage to occur (for a review, see ref 13).13 However, the mechanism of production of soluble ACE in vivo, the components involved in this process, and the factors that regulate the generation of soluble ACE have not been specifically delineated. In addition to the soluble, full-length somatic ACE found in blood and in seminal fluid, shorter forms of ACE are found in biological fluids. Naturally occurring fragments of human ACE corresponding to regions of the N-terminal domain have been identified in ileal fluid14 and in human and animal urine.15-16 Previously it was shown that testis ACE was shed from the cell membrane more efficiently than somatic ACE and it is likely 10.1021/pr049859w CCC: $30.25

 2005 American Chemical Society

N-Domain Determines Rate of Somatic ACE Shedding

that the N domain is largely responsible for this downregulation.12,17-18 Recently, we demonstrated that certain monoclonal antibodies (mAbs) that influenced ACE shedding19 also blocked ACE dimerization in reverse micelles.20 More specifically, the dimeric interaction occurred between the N domains of somatic ACE. These results suggest a possible link between putative ACE dimerization and ACE cleavage from the cell surface. In this study, we used several chimeric constructs of ACE and a set of mAbs to different epitopes on the N domain of ACE to show that this domain determines the rate of somatic ACE shedding. Furthermore, the region of the N domain that is involved n the regulation of shedding was identified.

Materials and Methods Chemicals. Hippuryl-L-histidyl-L-leucine (Hip-His-Leu), phorbol 12-myristate 13 acetate (PMA), 3,4-dichloroisocoumarin (DCI), 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanosulfonate (CHAPS), 4-methylumbelliferyl-β-D-xyloside and Nbutyldeoxynojirimycin (NB-DNJ), were obtained from Sigma (St. Louis, MO), Z-Phe-His-Leu from Bachem (King of Prussia, PA). Expression of Human ACE Mutants in CHO Cells. Stable cell lines of CHO cells expressing wild type human somatic ACE, testis ACE or only the N domain of ACE (ACE∆C) was obtained and cultured as described previously.8,21-22 NdelACE,23 was generated by the replacement of Pro604 to Pro739 of the C domain with the corresponding protein sequence from the N domain of somatic ACE (Leu1 to Pro141). To construct this chimera, advantage was taken of the sequence identity between the N and C domains of somatic ACE, which results in BglII occurring at corresponding positions in the two domains. Somatic ACE cDNA sub-cloned into pBluescipt II SK(() was digested with BglII, to excise the intermediate sequence. The flanking N and C domains were re-ligated to give the deletion mutant comprising the first 141 amino acids of the N domain (and the signal peptide of somatic ACE) fused to the C domain fragment, Asp740 to Ser1277. This was then sub-cloned into the mammalian expression vector, pLEN.24A stable cell line of CHO cells expressing only the C domain was obtained as follows. pcDNA3-ACE21 was used as template for amplification with the following primers: forward 5′-GGGCCCCGGGCTGGTGACTGATGAGGCTGAG-3′ and reverse 5′-AGTGTTCCCATCCCAGTCTCT-3′. The PCR product coding Leu613 to Arg1213 was subcloned into pGEM-T (Promega, USA). The ACE fragment was excised with XmaI and NotI and subcloned into pEX116, containing -24 to +116 nt (coding signal peptide and first two amino acids). Resulting plasmid was digested with EcoRI and NotI restriction enzymes and subcloned directly into pcDNA3.1-A expression vector (Invitrogen). Thus, expression vector psCfr contains ACE C-domain (amino acid residues 1-4, 612-1213). Then plasmid pcDNA3 with ACE cDNA was digested with XhoI and XbaI restriction enzymes and subcloned into psCfr vector digested with the same restriction enzymes. Thus, pcDNA-Cfr expression vector contained C-domain (coding amino acid residues 1-4, 613-1277). Resulting mutant (ACE∆N) have glycine (G) in 616 position instead of asparagine (D) of original template, probably due to mutation which occurred during PCR. However, it is highly unlikely that this mutation at the N-terminus of the construct would affect ectodomain shedding. CHO cells were transfected by these plasmids, and a stable cell line was generated as described above.

research articles Antibodies. Properties of a set of monoclonal antibodies directed to different epitopes located on the N domain of ACE were described in detail elsewhere.19,20,25 ACE Activity Measurements. Membrane-bound and soluble somatic ACE activity was assayed in detergent extracts and conditioned medium of transfected cells, respectively, using a fluorimetric assay with the substrates Hip-His-Leu and Z-PheHis-Leu as described.25-26 ACE Shedding Assay. Cells were fed with “complete culture medium” (Mediatech, Inc, Herndon, VA) without fetal bovine serum. In the experiments where glycosylation was inhibited, the cells were cultured for at least one passage before seeding them on a 35 mm dish with “complete culture medium” containing 2 mM 4-methylumbelliferyl-β-D-xyloside (O-glycosylation inhibitor) or N-butyl-deoxynojirimycin (NB-DNJ), a N-glycosylation inhibitor. Basal shedding of ACE was calculated after 24 h of culturing with fresh “complete medium” containing the glycosylation inhibitors. In the experiments investigating the effects of PMA (100 nM) and DCI (30 µM) on ACE shedding, CHO-ACE cells were grown in 96 well microtiter plates. Once confluent, they were washed 3 times with complete medium and incubated for 4 h with PMA or inhibitor, diluted in the same medium. To determine cell-associated ACE activity as well as to quantify the rate of ACE release (by relating the levels of ACE in the culture medium to those on the cell surface), cells were lysed with 100 µL of 8 mM 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanosulfonate (CHAPS). Both culture medium and cell lysates were centrifuged and aliquots (10-50 µL) were added to 5 mM Hip-His-Leu or 2 mM Z-Phe-His-Leu (200 µL) and incubated for the appropriate time at 37 °C.19,25-26 Cell ELISA. CHO-ACE cells were grown in 96-well plates until confluent, chilled on ice for at least 30 min, and washed several times with cold PBS. Control mouse IgG or 10 µg/mL anti-ACE mAbs in PBS/casein (0.2%) was added and incubated for 2 h on ice or at 37 °C. After washing, cells were fixed with 4% PFA for 15 min at room temperature and washed several times with PBS, before the bound mAbs (that reflect membrane-bound ACE) were quantified by incubation with alkaline phosphataseconjugated anti-mouse Ab followed by spectrophotometric assay at 405 nm. Binding of ACE by anti-ACE mAbs in Solution. Microtiter plates, bound with goat-anti-mouse IgG were coated with different anti-ACE mAbs and were incubated with either serumfree culture medium obtained from CHO-ACE cells as a source of ACE or with serum or plasma from tested animals. The precipitated ACE activity was estimated directly in the wells using Hip-His-Leu as substrate as described previously.25 Negligible background hydrolysis of the substrate in the wells coated by nonimmune mouse IgG was subtracted from each value obtained with the specific anti-ACE mAbs. Sequencing of ACE Gene in Monkeys. Total RNA was isolated from white blood cells of baboon, macaque Rhesus, macaque Bear, macaque Cynomolgus, and macaque Snow. cDNA was prepared using Qiagen kit. PCR amplification of the 2nd exon of the baboon ACE gene as well as exons 11-12 of the ACE gene of all the monkeys studied was performed using primers designed by Dufour et al.27 PCR products were sequenced at the DNA facility of the University of Illinois at Chicago. Homology Modeling of N Domain of Somatic ACE. The N domain of somatic ACE shares 55% amino acid sequence identity (65% sequence similarity) with tACE (except for the Journal of Proteome Research • Vol. 4, No. 2, 2005 259

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Figure 1. Schematic diagram of different ACE constructs. Wildtype human somatic ACE is comprised of two homologous domains, N (open box) and C (black box) domains, joined by an interdomain region of approximately 16 amino acids.44 A stalk region (SR), from residue 1192 to 1227,45 links the ectodomain and transmembrane (TM) domains. The TM, residues 1228-1249, is indicated by a hatched box. In the construct ACE∆C the N-terminal domain of somatic ACE (Leu1 to Pro601) is fused to the SR, TM, and cytosolic domains. Deleted sequences are shown by dashes. ACE∆N contains the first four amino acid residues (LDPG) from the N domain, the C-terminal domain, stalk region, TM and cytosolic domains. Recombinant human testicular ACE has a unique 36-amino acid sequence and the rest of the molecule is identical to the C domain of somatic ACE. NdelACE was constructed by fusing the first 141 amino acid residues from the N domain to the ID region, C-terminal domain, SR, TM, and cytosolic domains.

36 residues at the N-terminus). The 3D structure of tACE;28 PDB code 1O86) with all the water molecules removed was used as a template to model the N domain structure. On the basis of the sequence alignment, ‘in silico’ mutations were performed to the tACE structure using the graphics package ‘O’.29 The best conformer for the side chain was selected at each mutation site. The resultant model for the N domain was minimized for 30 cycles using conjugate gradient minimization with the CNS suite of programs without the experimental energy terms.30

Results Construction and Expression of ACE Chimeric Constructs. To investigate the role of the N and C domains of ACE in ectodomain shedding, five ACE constructs were used (Figure 1). Wild-type human somatic ACE and human testis ACE constructs were described previously.10,21,24 ACE∆C comprises the N domain of somatic ACE (Leu1 to Pro601) fused to the juxtamembrane stalk, transmembrane and cytosolic domains).22 NdelACE mutant, which contains the first 141 amino acids from N domain fused to the C domain (Asp740 to Ser1277), was described previously.23 ACE∆N was constructed by adding the first four amino acids of the N domain to the C domain (Leu613 to Ser1277). All constructs were transfected into CHO cells and stable cell lines were established. Each of the cell lines analyzed exhibited a comparable level of ACE expression. The molecular mass of soluble and membranebound ACE constructs were determined by Western blotting with novel anti-ACE mAbs 3C5 and 2E2.31 The size of ACE secreted as a result of the basal shedding was the same as in the case of 3A5-induced ACE shedding (not shown). Rate of Basal Shedding of ACE Constructs. Previously, it was shown that the testicular isoform of ACE was shed from 260

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Figure 2. Rate of basal shedding of ACE for different ACE constructs and the effects of PMA (100 nM) and DCI (30 µM) on the rate of ACE shedding. The CHO-ACE cell lines were grown on 35 mm Petri dishes until confluence. When they reached confluence they were washed 3 times with serum-free medium and incubated with fresh medium during 24 h (A) or with tested compounds (PMA or DCI), diluted in the same medium during 4 h. After indicated time of incubation at 37 °C, the culture medium was collected, whereas cells were washed several times and were lysed with 8 mM CHAPS. The rate of ACE shedding was calculated as described in Materials and Methods.

the cell membrane more efficiently than the somatic isoform.12,17,18 We compared the rate of shedding of five different ACE constructs (Figure 1) under identical conditions and found that the rate differed dramatically between constructs (Figure 2A). The C domain (ACE∆N) demonstrated the highest rate of shedding (212%/24 h). Addition of the first 141 amino acids of the N domain to this construct only slightly decreased the rate of proteolytic cleavage (174%), whereas the rate of shedding of testicular ACE (which also contains a unique, heavily O-glycosylated 36 amino acid sequence) was approximately 4-fold less than ACE∆N (49%). Iincomplete O-glycosylation of tACE significantly increased the rate of tACE shedding. When CHO-cells were grown in the presence of 2 mM 4-methylumbelliferyl-β-D-xyloside (O-glycosylation inhibitor), the rate of tACE shedding increased 2.3fold (p < 0.05). Growing of tACE cells in the presence of NB-

N-Domain Determines Rate of Somatic ACE Shedding

DNJ, an inhibitor of canonical N-glycosylation, did not change the rate of tACE shedding significantly. Somatic ACE (having both N and C domains) and the N domain construct were shed least efficiently (20% and 12%, respectively). However, incomplete N-glycosylation of somatic ACE in the presence of NBDNJ increased the rate of somatic ACE shedding by 1.8-fold (36%, p < 0.05). Therefore, the underglycosylated ACE is shed more effectively and this might be due to the reduced steric hindrance of the hypoglycosylated form and increased accessibility, not only of the sheddase cleavage site, but also of the ACE ectodomain. We also tested the effect of PMA and DCI, both known inducers of ACE shedding.32-34 Interestingly, PMA had the most marked effect on the shedding of testis ACE (more than 3-fold), whereas the effect on the shedding of the other ACE constructs was similar -around 150% (Figure 2B). Moreover, the effect of DCI was construct-specific. DCI increased shedding of testis ACE (160%) and somatic ACE (140%), whereas the shedding of other ACE constructs was unaltered (Figure 2C). A characteristic of ACE shedding is its inhibition by the peptide hydroxamate TAPI. Addition of 50 µM TAPI resulted in inhibition of the release of ACE∆N and NdelACE to 10% of control levels (data not shown). These data are similar to previous results for the ectodomain shedding of wild-type tACE and somatic ACE, which are also inhibited to between 10 and 20% of control levels.34 Thus, both ACE∆N and NdelACE are likely released from the membrane by one of the ACE sheddases. Epitope-Dependent Antibody-Induced Shedding of ACE from the Cell Surface. Recently, we demonstrated that various mAbs specific for the N domain of ACE have a striking effect on shedding of somatic ACE from the cell surface.19 To compare the pattern of antibody-induced shedding of somatic ACE with that of the N domain (ACE∆C), we investigated the binding of this set of anti-ACE mAbs to the surface of cells expressing these two constructs. We also tested the binding of these mAbs with NdelACE which contains the first 141 amino acids of the N domain. The binding patterns of all seven mAbs, directed to different epitopes on the N domain,25 were almost identical to that seen in somatic ACE and ACE∆C (Figure 3). Binding of six of the mAbs was significantly decreased at 37 °C in comparison with binding at 4 °C, which reflects shedding and internalization of these mAbs. However, mAb 3G8 binding, which effectively inhibited somatic ACE shedding,19-20 to both somatic ACE and ACE∆C cells was significantly higher at 37 °C. Three of the mAbs bound to cells expressing NdelACE suggesting that the epitopes for these mAbs (3G8, 5F1, and i2H5), or at least some stretches of their epitopes, are localized on the N-terminal region (first 141 amino acids) of the N domain. We compared the rate of antibody-induced ACE shedding for two mAbs, 3G8, and 5F1, that bound to all three ACE constructs (Figure 4). Shedding of ACE in the presence of mAb i2H5 was not analyzed because the binding with NdelACE was very weak. Furthermore, i2H5 strongly inhibited catalytic activity of the N domain25 preventing quantification of the proteolytic release of ACE∆C using a plate precipitation assay based on the determination of enzymatic activity. mAb 5F1 caused a small, but significant, increase in shedding of wildtype somatic ACE. However, binding of this mAb to the surface of ACE∆C and NdelACE resulted in a 2-fold increase in the rate of shedding. The most intriguing effect was demonstrated by the binding of mAb 3G8. Shedding of somatic ACE and ACE∆C was

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Figure 3. Binding of anti-ACE mAbs to the surface of the CHO cells expressing different ACE constructs. The CHO-ACE cell lines were grown on 96 well plates until confluence. Anti-ACE mAbs or control mouse IgG at the concentration of 10 µg/mL, were added to the cells in serum-free medium (containing 2% BSA) and incubated 2 h at 4 °C or 37 °C for mAb binding assay. Bound anti-ACE mAbs were revealed with goat-anti-mouse IgG, conjugated with alkaline phosphatase. *p < 0.05 for the differences of mAb 3G8 binding at 4 °C and 37 °C.

inhibited in the presence of mAb 3G8, whereas the shedding of NdelACE, comprised mainly of C domain, increased 2-fold. One explanation, for the effect of mAb 3G8 on shedding, may be that the epitope for this particular antibody consists of several stretches with one localized in the N-terminal part of the N domain and another more distal (see below). Thus, binding of mAb 3G8 to all stretches in the intact N domain (as in somatic ACE and ACE∆C) results in inhibition of ACE shedding, whereas binding with only one stretch (as in NdelACE) has an opposite effect. To substantiate this hypothesis, we carried out epitope mapping of this particular mAb as well as overlapping mAbs 3A5 and 9B9 which influenced shedding of somatic ACE.19 Epitope Mapping of mAbs to ACE. The binding of mAbs 3G8, 9B9, and 3A5 influenced ACE shedding in CHO cells19 as well as ACE dimerization in reverse micelle.20 Furthermore, they recognized overlapping epitopes on the N domain of human ACE.25 To localize the epitopes for these mAbs, we examined the reported cross-reactivity of these mAbs to ACE from different species.25 Previously, we showed that mAb 9B9 Journal of Proteome Research • Vol. 4, No. 2, 2005 261

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416 contains the epitopes for mAbs i2H5, 1G12, and 6A12 (Danilov et al. unpublished work), which do not overlap with mAbs 9B9, 3G8, and 3A5.25 The most likely region for the 9B9 epitope would be in the vicinity of Lys535 because it is close to Gln568, the most probable epitope for 3A5, which is common for human, chimpanzee, and rabbit ACE.

Figure 4. Effect of anti-ACE mAbs on ACE shedding from the surface of CHO-ACE cells.The CHO-ACE cell lines were grown on 96 well plate until confluence. Anti-ACE mAbs or control mouse IgG at the concentration of 10 µg/mL, were added to the cells in serum-free medium (containing 2% BSA) and incubated 4 h at 37 °C for ACE shedding assay. The culture fluids were collected, centrifuged (for the precipitation of the detached cells) and ACE activity shed from the plasma membrane of cells was measured fluorimetrically with Hip-His-Leu as a substrate. Antibody-induced shedding is expressed as % from the basal shedding of ACE (in the presence of control mouse IgG only). The data represent result of several independent experiments and expressed as mean ( SD. (*p < 0.05)

cross-reacts with rat ACE and mAb 3A5 with rabbit ACE, whereas mAb 3G8 is human ACE specific.25 Figure 5 demonstrates the amino acid sequence alignment of the N domain of ACE of human, chimpanzee, rabbit, bovine, rat, and mouse. On the basis of the mAb 9B9 cross-reactivity with chimpanzee and rat ACE, the epitope or part thereof for mAb 9B9 might be localized in four putative regions on the N domain of ACE: around Phe228, Ala323, Arg413, or Lys535. The region that includes Arg413 can be excluded, as the motif including 403-

The binding of mAbs 3G8, 9B9, and 3A5 to chimpanzee ACE35 provides additional information about the possible localization of epitopes for these mAbs. Binding of mAbs 9B9 and 3A5 was essentially unaltered when compared to their binding to human ACE (86 and 88%, respectively), whereas binding of 3G8 to chimpanzee ACE decreased to 40% (Figure 6). Interestingly, binding of mAb i1A8 (overlaps with 3G8, 9B9, and 3A5) to chimpanzee ACE is further decreased to 18% (Figure 6). The protein sequence of chimpanzee somatic ACE has very few substitutions in the N domain in comparison with human ACE: Ile79/Val, Arg381/Gly, Glu403/Ala, Arg413/Asn, Pro456/ Asp, and Arg532/Gln (27, see also Figure 5, in green). The residues between Glu403 and Arg413 define the epitopes for i2H5, 1G12, and 6A12 (Danilov et al. unpublished work), which do not overlap with 3G8.25 Out of the remaining amino acid residues, Ile79, Arg381, and Arg532 may participate in the epitope for 3G8; however, inspection of the N-domain homology model (Figures 7 and 8) suggests the involvement of Ile79 and Arg532 in the epitope for this mAb is unlikely. To further delineate the epitopes for these functionally important mAbs, previous data on the cross-reactivity of these mAbs to 10 primates species was used (Figure 1 in ref 35). First, mAb 3G8 does not bind to ACE from any of the monkeys. Second, the binding of mAb 9B9 decreased by 98% in all species excluding apes (higher primatessin that case, chimpanzee). Third, binding of mAb 3A5 differed among the primates studied. Binding was almost completely abolished in the case of baboon ACE, whereas binding with other macaque species decreased by 70% (Figure 6). Interestingly, binding of mAb i1A8,

Figure 5. Alignment of the N-domains of mammalian ACE.Amino acid residues unique for human and chimpanzee ACE are highlighted by red. Amino acid residues in chimpanzee ACE, which are differ from human ACE are highlighted by green. The following amino acid residues were highlighted according to their cross-reactivity: 9B9-yellow, 3A5, and i1A8- blue. 262

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Figure 6. Binding of mAbs to ACE of chosen species and the alignment of the sequences of ACE from these species. Binding of each particular mAbs to ACE from studied species was expressed in % to that for binding to human somatic ACE.Amino acid residues unique for human and chimpanzee ACE are highlighted by red. The following amino acid residues were highlighted according to their cross-reactivity: 9B9-yellow, 3A5, and i1A8- blue. The amino acid residues which were common in all monkeys, but different to that in other species were highlighted by green (one exception -564Thr in baboon). The amino acid residues which were common in monkeys (not apes) and in other species, but different to that in human were highlighted by magenta.

Figure 7. Schematic view of the modeled structure of N domain of ACE.A. Ribbon presentation. The antigen recognition residues 519-589 and 80-96 are colored in coral and pink on the outer plane. The colors yellow, blue, red, magenta, and green on the inner plane of helices R3, H7, R18, R19, and R20 helices, respectively follow the coloring scheme as in B. The Secondary structure assignment for the N-domain model- alpha helices (R) and 310 helices (H) are as follows: R1(13-44); R2(47-73); H1(73-80); R3(86-96); H2(98-104); R4 (104-125); R5(141-150); R6 (152-168); R6′(171-189); R7(193-201); H3 seem to appear as loop; R8(206-238); H4(261-264); R9(264-269); R10(278-286); R11(289-304); R12(310-317); R13(352-372); H5(376-380); R14(384-408); R15(417-451); R16(458-472); H6(484-490); R16′(490495); R17(498-519); H7(524-528); R18 (533-545); R19(551-561); R20(567-589) and strands (β) are as follows: β1(126-127); β2(137-139); β3(248-250); β4(333-337); β5(342-346); β6(473-474) (Note: secondary structural element numbering in the N-domain ACE model corresponds to tACE structure; numbering not shown in the figure for clarity). B. Surface presentation. Orientation is similar to as in A. Epitope recognition regions 519-589 and 8096 are colored with loops in dark gray and the helices R3, H7, R18, R19, and R20 are in yellow, blue, red, magenta and green, respectively. Rest of the surface are in gray color.

which overlaps with mAbs 3G8, 9B9, and 3A5, with monkey ACE was unchanged (Figure 6). To define the structural rationale for the above-mentioned pattern of mAbs binding with ACE from the different monkeys and to assign epitopes for these mAbs, we sequenced the exons 11 and 12 of the monkeys used in this study. Figure 6 demonstrates the alignment of the N-domain sequences encoded by these exons. Analysis of this alignment led to the following conclusions: mAb 3A5. The binding of mAb 3A5 to rabbit ACE is 10-fold weaker than to human ACE and 3-fold weaker in the case of macaque ACE (Figure 6). There is only one difference between

Figure 8. Epitope mapping of the N-domain of human ACE. The various amino acid residues belonging to the putative epitopes for 3A5, 9B9, 3G8, and i1A8 are color coded as red, green, blue, and magenta bullets. The areas spanned by individual residues are marked by dashed lines. Circles with 25 Å diameter shows the putative surface on ACE covered by mAb 3G8, 9B9, and 3A5.

rabbit and macaque ACE in this region, namely the replacement of Lys572 with Asp in rabbit ACE and Asn in macaque ACE. Thus, it is likely that the conversion of Lys572 to a Ser in bovine ACE or a Glu in rat and mouse ACE, and the replacement of Gln568 with other amino acid residues (Arg, Ser, Lys), completely abolishes binding of mAb 3A5 to ACE from these species. Thr 564 in baboon ACE further decreased binding of mAb 3A5 by 20-fold. Leu562 does not make a significant contribution to the epitope for 3A5, despite the fact that it is close to Ala564 and Gln568, for the following reasons: First, in rabbit, bovine and rodent ACE, which do not bind to the antibody, it is replaced by a serine, as in monkey ACE (which binds to mAb 3A5). Second, substitution of only a serine for the Leu 562 in the N domain construct (D629, ref. 31) did not alter 3A5, 9B9, 3G8, and i1A8 binding (data not shown). Therefore, the epitope for mAb 3A5 in human ACE minimally consists of a combination of Ala564, Gln568, and Lys572. mAb 9B9. The epitope for this mAb consists of combination of Lys535 (chimpanzee and rat specific) and amino acid residues from another unidentified stretch(s) on the N-domain. Journal of Proteome Research • Vol. 4, No. 2, 2005 263

research articles There are two substitutions in monkey ACE in this regions Leu562Ser and Lys572Asn and, furthermore, 9B9 binding with monkey ACE decreased dramatically (Figure 6). However, Leu562 may not participate in the epitope for 9B9 because in monkey ACE, which lost 90% of binding to 9B9, it is replaced by a serine. This is the same in rat ACE which retains more than 30% of binding to mAb 9B9. Examination of the surface of the N-domain model (Figures 7 and 8) ruled out the participation of Lys572 in the 9B9 epitope. mAb 3G8. On the basis of the cross-reactivity data, one of the stretches of the epitope for this mAb might consists of Arg381 (see data from chimpanzee ACE), Lys535 and Lys542 due to the overlap of the epitopes for 3G8, 9B9, and i1A8. Another stretch of the 3G8 epitope could be situated between Leu1 and Pro141 (Figures 3 and 4). mAb i1A8. The epitope for mAb i1A8 which does not affect ACE shedding includes Lys542 because binding of i1A8 was unaltered with monkey ACE. Moreover, Lys542 is common for all monkey sequences in contrast to other species such as rabbit, bovine, rat, and mouse. Rabbit ACE, which binds to i1A8 10-fold weaker than to human or any of the monkey ACE forms, has only one substitution in this region, namely Ala542 instead of Lys542. Finally, participation of the dipeptide Tyr521-Glu522 could not be excluded due to its close proximity to Arg532. Description of the Modeled N Domain 3D Structure. To confirm our hypothesis about the putative epitopes for mAbs which influenced ACE shedding and dimerization, homology modeling of the N domain of human ACE was carried out (Figure 7A,B), using the crystal structure of testis ACE.28 The loop between Gly561 and Gln568, comprising the putative epitope for mAb 3A5, is located in a close proximity with helix R18 containing Lys542, which is proposed to be crucial for epitope i1A8. This part of the helix contains four additional positively charged amino acid residues Arg532, Lys535, Lys539, and Arg541, which are located on the surface of the molecule (Figure 8), and likely participate in the epitopes for i1A8, and 9B9. On the basis of the sequence alignment (Figure 5,6) and ribbon and surface presentation (Figure 7A,B), we suggest that the amino acid residues Tyr521 and Glu522 may also participate in the epitope for at least mAb 3A5. Experiments examining the effect of mAb 3G8 on the shedding of different ACE constructs (Figures 3 and 4) suggest that an epitope for this mAb might be created by at least two stretches on the ACE molecule localized far from each other. One stretch was likely created by the amino acid residues, localized on the loop between Gly547 and Pro551 and by amino acid residues on the surface of the helices R18 and/or R19. The second stretch was likely created by amino acid residues, localized on the R3 helix and the loop prior to R3 (Figure 7A,B). Since the interface between a protein antigen and a specific antibody can extend over an area ranged between 534 and 901 Å2,36-38 it is not surprising that amino acid residue deriving from sequentially disparate regions of a protein molecule can form a single antibody-binding site. For example, it was shown that the epitope on tissue factor for Fab 5G9 consists of 18 amino acid residues from four strands. However, only four amino acid residues out of 18 played a major role in this antibody recognition.38 Circles, 25 Å in diameter, in Figure 8 show the putative surface, covered by mAb 3G8, 9B9, and 3A5. This figure demonstrates the possibility that the different stretches on ACE might be masked by one antibody (as in the case of 3G8). The surface representation of the N-domain model allows us to 264

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propose other amino acid residues which are likely to participate in the epitope for mAb 3G8: (i) Gln87 creates a protrusion in the center of putative epitope for 3G8 (Figure 8). Sequence analysis of the second exon of baboon ACE (encoding the amino acid residues from Glu55 to Gln110) demonstrated that baboon ACE, which is not recognized by mAb 3G8, has three substitutions, namely Gln87Glu, Arg90Lys, and Arg96Gly. It is likely that Gln87 is a crucial residue for the second stretch of the 3G8 epitope, because in rabbit, bovine, rat, and mouse ACE (which does not bind to mAb 3G8), Gln87 was replaced by Glu, Thr, Lys, and Lys, respectively (Figure 5). (ii) This region also contains Gln22 which is ape-specific (Figure 5) and Asn25, a putative glycosylation site in human somatic ACE.39 We demonstrated that underglycosylation of human somatic ACE and N-domain ACE, expressed in CHO cells in the presence of NB-DNJ, led to a significant increase in mAb 3G8 and i1A8 binding [1.63 ( 0.24 and 1.48 ( 0.16 fold, respectively in comparison with untreated ACEs (p < 0.05)], whereas binding of mAb 9B9 and 3A5 was unaffected. (iii) Leu 379 is an amino acid residue located on the surface of the protein that may also participate in the epitope for 3G8 (Figure 8). Thus replacement of the nearby, positively charged Arg381 in human ACE with a nonpolar Gly in chimpanzee ACE27 may cause a change in conformation of the epitope resulting in decreased binding of mAbs 3G8 and i1A8 to chimpanzee ACE (Figure 6). Therefore, epitope mapping based on the N-domain modeling suggests that the binding of mAbs 3G8, 9B9, and 3A5 to this particular region of the N-domain of ACE may be responsible for their effect on the process of antibody-induced ACE shedding investigated in this study. Moreover, it seems that the results of this study support the notion that this particular region on the N-domain of ACE may play a role in the lower rate of somatic ACE shedding in comparison with testicular ACE or C-domain.

Discussion Ectodomain shedding is a universal phenomenon that affects a wide range of ectoproteins including enzymes, receptors, and cell adhesion molecules. This process is likely regulated by the marshalling of the secretase and its substrate within the lipid bilayer as well as interactions between the ectodomains of these proteins. However, the fine-tuning of this important posttranslational modification is poorly understood. The N domain appears to influence the shedding of somatic ACE,12,17,18 and we have used a panel of monoclonal antibodies together with various chimeric proteins to examine further the effect of this domain on the proteolytic cleavage of ACE. Previously, we showed that only two monoclonal antibodies, 9B9 and 3A5, out of a panel of seven directed to the N domain of ACE, induce shedding of somatic ACE.19 Thus, binding of these mAbs to the region of the N-domain defined by the overlapping regions of the two epitopes might cause conformational changes in the ACE ectodomain that result in a more accessible stalk region and thus increased shedding. Indeed, similar conformational changes in the juxtamembrane have resulted in altered ectodomain shedding and even the action of different secretases.40-42 However, it is unclear to what extent conformational changes, on one hand, and possible interaction(s) between the ACE secretase and a region of the ectodomain distal to the cleavage site, on the other, are responsible for these modulations in shedding. Discreet changes

research articles

N-Domain Determines Rate of Somatic ACE Shedding

in conformation resulting from the mutation of a single residue in the juxtamembrane region can regulate shedding,40-42 whereas more substantive alterations, such as the fusion of two tandem C-domain repeats, have little effect on the efficiency of the proteolytic cleavage of the membrane-anchored ectoprotein (Woodman et al., unpublished data). In this study, we show that mAb interaction with a region within the N domain inhibits ACE shedding. The rapid shedding of a C domain chimera, comprising N domain sequence containing these epitopes, further suggests that this region is involved in the regulation of shedding of ACE (Woodman et al., unpublished data). The apparent conundrum of mAb 3G8 inhibiting shedding of somatic ACE and ACE∆C, while increasing the shedding of a construct that was predominantly comprised of C domain and the first 141 residues of the N domain (NdelACE), may be explained by the fact that the epitope for this particular antibody consists of several stretches with one localized in the N-terminal part of the N domain and another more distal. Thus, binding of mAb 3G8 to all stretches in the intact N domain (as in somatic ACE and ACE∆C) results in inhibition of ACE shedding, whereas binding with only one stretch (as in NdelACE) has the opposite effect. However, NdelACE has more N domain-like enzymatic activity23 (Woodman et al., unpublished data) despite its predominantly C domain sequence. Thus, the altered effect that mAb binding has on shedding could be due to its inherently C domain structure. Recently, Kost et al.20 demonstrated that dimerization of somatic ACE in reverse micelles was inhibited by two mAbs, 9B9 and 3G8, out of the eight studied. It is likely that the putative carbohydrate recognition domain, localized on the N-terminal domain of ACE and responsible for the observed dimerization, was shielded by these two mAbs.20 On the basis of the effects of mAbs to different ACE functions, we have a complex picture: (1) the binding of mAbs to the region on the N domain, defined as an overlapping surface of the epitopes 9B9 and 3A5, significantly induced ACE shedding; (2) the binding of mAbs to the region on the N domain, defined as an overlapping surface of the epitopes 9B9 and 3G8, inhibited dimerization of the somatic ACE in the reverse micelles. Therefore, using a set of mAbs, we identified a region on the N-domain of ACE, which is very sensitive to the binding of monoclonal antibodies. mAb 3G8 (as highlighted in Figures 7,8) results in inhibition of ACE shedding on the cells surface or preventing of dimerization in reverse micelles. Binding of mAb 3A5 to its epitope on the N-domain induced gross conformational changes on ACE molecule, which led to prevention of binding of other mAbs to the N domain, inhibition of ACE catalytic activity,25 and induction of ACE shedding (ref 19 and present study). However, binding of mAb 9B9 covered the region on the N domain, which seems to have two different (or opposite directed) functions: prevention of dimerization on reverse micelles (as 3G8) and induction of ACE shedding (as 3A5). Surprisingly, the 3D structure of the N domain of ACE, obtained by homology modeling, supports this dichotomy (Figures 7,8). It is interesting that the results of the present study and two recently published papers19-20 provide a possible link between ACE dimerization and ACE shedding. Although the role of ACE dimerization in vivo is uncertain, it is possible that this interaction could facilitate cross talk between ACE and other important components of the renin angiotensin system. A number of studies have provided evidence for bradykinin-

potentiating effects of ACE inhibitors that are independent of bradykinin hydrolysis implicating some interaction between ACE and the bradykinin B2 receptor (reviewed in ref 43). Thus, it is conceivable that dimerization of ACE could effect this type of interaction or, alternatively, the co-localiztion of ACE and the B2 receptor in the membrane micro-environment harboring these two proteins. The fine epitope mapping of various monoclonal antibodies has substantiated earlier structure-function analysis, which provided some of the first evidence for structural differences between the N and C domains.25 Further mutagenesis of amino acid residues within these epitopes will elucidate the exact surface topography of these antigen-binding sites and provide important insights into the relationship between the structure of ACE, and its catalysis and post-translational processing.

Acknowledgment. This work was supported by the Wellcome Trust (United Kingdom) grants to E.D.S. and K.R.A. We thank Dr. V. Gavrilyuk (University of Illinois at Chicago, USA) for the preparation of ACE∆N construct. The technical assistance of Zhu-Li Sun is greatly appreciated. Abbreviations. ACE, angiotensin converting enzyme; CRD, carbohydrate-recognizing domain; Hip-His-Leu, hippuryl-Lhistidyl-L-leucine; Z-Phe-His-Leu, benzyloxycarbonyl-phenylalanyl-L-histidyl-L-leucine; CHO, Chinese Hamster ovary; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; CD markers, cluster designation markers; NBDNJ, N-butyldeoxynojirimycin; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13 acetate; DCI, 3,4-dichloroisocoumarin; (CHAPS), 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanosulfonate; The nucleotide sequences for the exon 2 baboon ACE gene and exons 12 baboon and macaque Rhesus ACE gene have been deposited in the GenBank database under GenBank Accession No. sAY344231, AY348176, AY348177, respectively.

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N-Domain Determines Rate of Somatic ACE Shedding (43) Tom, B.; Dendorfer, A.; Danser AH Bradykinin, angiotensin-(17), and ACE inhibitors: how do they interact? Int. J. Biochem. Cell. Biol. 2003, 35, 792-801. (44) Sturrock, E. D.; Danilov, S. M.; Riordan, J. F. Limited proteolysis of human kidney angiotensin-converting enzyme and generation of catalytically active N- and C-terminal domains. Biochem. Biophys. Res. Commun. 1997, 236, 16-19.

(45) Chubb, A. J.; Schwager, S. L.; Woodman, Z. L.; Ehlers, M. R.; Sturrock E. D. Defining the boundaries of the testis angiotensinconverting enzyme ectodomain. Biochem. Biophys. Res. Commun. 2002, 297, 1225-1230.

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