PROTEIN

EXPRESSION

AND PURIFICATION

2, l-9

(1991)

Purification and Characterization of Recombinant Human Testis Angiotensin-Converting Enzyme Expressed in Chinese Hamster Ovary Cells Mario

R. W. Ehlers,

Center for Biochemical

Ying-Nan and Biophysical

P. Chen,

and James

Sciences and Medicine,

Received December 27, 1990, and in revised form January

Harvard

Medical School, Boston, Massachusetts

02115

X3,1991

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: PO-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which fulllength and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mglliter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either PO-ACE or pLEN-ACEVII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M, - 100 kDa. The native and recombinant proteins cross-reacted equally with antihuman kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investi-

i To whom correspondence should be addressed at Center for Biochemical and Biophysical Sciences and Medicine, Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115. Fax: (617) 5663137. 1046-5928/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

F. Riordanl

gating zyme.

structure-function

relationships

in

this

en-

0 1991 Academic Press, Inc.

Angiotensin-converting enzyme (ACE;’ EC 3.4.15.1) is a zinc-metallopeptidase that cleaves C-terminal dipeptides from oligopeptide substrates. The enzyme is best known for the conversion of angiotensin I (AI) to angiotensin II, a potent vasopressor, and for the degradation of bradykinin, a vasodilator (1,2). ACE is a component of the renin-angiotensin system and is thought to play a critical role in blood pressure regulation, as evidenced by the successful use of ACE inhibitors in the treatment of hypertension (3). There are two isoforms of ACE. The somatic isozyme is a widely distributed ectoenzyme, present most abundantly in endothelium and the renal and intestinal brush borders. The testis isozyme is uniquely present in developing spermatozoa (2). Both isozymes have been cloned and their structural relationship has been determined. The somatic isozyme consists of a 147-kDa polypeptide divided into two homologous domains, each containing a putative metal-binding site (4,5). The testis isozyme is a 75-kDa polypeptide, identical, except for the first 35 N-terminal residues, to the C-terminal domain of the somatic isozyme; it contains the second of the two metal-binding sites and the C-terminal hydrophobic sequence that likely constitutes the membrane anchor (6-8). The typical chloride-dependent, angio’ Abbreviations used: ACE, angiotensin-converting enzyme; ACEVII and ACE-615, ACE expressed in CHO cells transfected with pLEN-ACEVII and pLEN-ACE615, respectively; AI, angiotensin I; CHO, Chinese hamster ovary; hTACE, recombinant human testicular ACE; Fa-FGG, 2-furanacryloyl-L-phenylalanylglycylglycine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1

2

EHLERS,

CHEN,

tensin-converting activity of the two isozymes is similar or identical (Ref. (9); J. W. Harper, R. Shapiro, and J. F. Riordan, unpublished results), and thus the testis isozyme can be viewed as a simpler form of the somatic isozyme. Many questions remain regarding the molecular basis for the catalytic mechanism of ACE (1,2). One approach to their resolution is the construction and expression of recombinant mutant proteins designed to test hypotheses generated by chemical modification studies (10) and inferences from sequence homologies with better understood proteases (4). Such studies will likely be facilitated by use of the smaller testis isozyme. Toward this goal, we have subcloned a full-length human testis ACE cDNA into eukaryotic expression vectors, and have used these to express recombinant human testis ACE (hTACE) in stably transfected Chinese hamster ovary (CHO) cells. We report here that hTACE is expressed at high levels and can be purified to homogeneity by affinity chromatography. The recombinant enzyme is apparently identical to the native enzyme by physiocochemiCal, kinetic, and immunologic criteria.

MATERIALS

AND

METHODS

Recombinant DNA materials and methods were as described previously (6). In general, DNA manipulations were performed in the cloning vector pBluescript (PBS, Stratagene). Vectors were propagated in the Escherichia coli strain XLl-Blue (Stratagene). Restriction fragments were purified and ligated in low-melting agarose (NuSieve GTG, FMC BioProducts). Oligonucleotides were synthesized on a Biotix Model 102 DNA synthesizer. All new constructs were analyzed by restriction mapping and by double-stranded sequencing of miniprep plasmid DNA by the dideoxynucleotide chain termination method (11) using Sequenase (USB). Construction of expression vectors. The full-length testis ACE cDNA sequence previously reported (6) was a composite of incomplete 3’ fragments cloned from a human testis cDNA library in Xgtll and 5’cDNAs generated from human testis mRNA by anchored PCR. A full-length testis ACE cDNA for expression purposes was generated from these fragments as follows (see Fig. 1). Clone lOA, a 2.1-kb ACE cDNA that is incomplete by -400 bp at its 5’end and that extends up to the polyadenylation signal at its 3’ end (6) was cloned into the EcoRI site of PBS. This construct, PBS-lOA, was digested with the restriction endonucleases SmaI and KpnI, which cut at unique sites 5’ and 3’ to the cDNA insert, respectively. The SmaIIKpnI restriction fragment was then ligated into the similarly digested polylinker of PJ4Q, a pBR322-based eukaryotic expression vector containing the Moloney murine leukemia virus (MO-MuLV) promoter, and SV40 splice sites and termi-

AND

RIORDAN

nation signals (a gift from C. L. Cepko, Harvard Medical School). This construct was termed p%lOA (Fig. 1). A second ACE cDNA clone, RI.2, consists of a 650-bp fragment corresponding to the 5’ end of the testis ACE cDNA, and its 3’ end overlaps the 5’end of 10A by -200 bp (6); this region of overlap contains a unique DraIII site (Fig. 1). RI.2, generated by anchored PCR, possesses a synthetic 5’ linker sequence containing the restriction sites XbaI, BgZII, XhoI, and ScaI. RI.2 was digested with XbaI and ligated into PBS cut with XbaI and HincII. The PBS-RI.2 construct (Fig. 1) was then digested with ScaI and &a111 and the released insert was cloned into p&lOA cut with SmaI and DraIII, to generate the full-length ACE cDNA in pJ49, termed p&-ACE (Fig. 1). A second ACE expression cassette, pLEN-ACEVII, was constructed in the vector pLEN (a gift from P. J. Kushner, University of California, San Francisco). pLEN (Fig. 1) is a pUC8-based eukaryotic expression vector containing, from 5’to 3’, the SV40 enhancer element present within the 1118-bp HindIIIC fragment of SV40 (12), the human metallothionein IIA (hMTIIA) promoter (841-bp fragment of the hMTIIA gene from nt -770 to +71; Ref. (13)), and a 300-bp 3’ fragment of the human growth hormone gene (hGH3’; P. J. Kushner, personal communication); pLEN is based on the reported vector pMThGHSV402 (( 14); see also Refs. (15,16)). pLEN was digested with EcoRI and the cut ends were filled in with Klenow. A second digestion with BamHI released the hGH3’ fragment. Similarly, pQ-ACE was digested with PuuI (cutting at a site downstream from the SV40 polyadenylation signal; Fig. l), blunt-ended with Klenow, and cut with BamHI to release the entire ACE insert with the downstream SV40 splice sites and termination signals from pat; this was ligated into the cut pLEN vector. This construct, pLEN-ACEVII, thus contains, from 5’ to 3’, the SV40 enhancer, hMTIIA promoter, full-length testis ACE cDNA (RI.2/10A), and the SV40 splice sites and termination signal (Fig. 1). pLEN-ACEVII was modified to produce a construct, designated pLEN-ACE6/5, that will direct the expression of testis ACE lacking the putative C-terminal membrane anchor. For this purpose, PBS-1OA (see above) was digested with Not1 and HindIII, thereby removing a 450-bp fragment from the 3’ end of the ACE cDNA which includes the region encoding the hydrophobic sequence (see diagram of pLEN-ACEVII, Fig. 1). This fragment was replaced by a synthetic oligoduplex engineered with appropriate Not1 and Hind111 ends, and containing a stop codon immediately after the codon (nt 2105) encoding the last residue prior to the putative membrane-spanning sequence (Gln 654). This construct, PBS-lOA3’, was restricted with NhcI (a unique site at nt 1363 in the ACE cDNA) and CZaI (a site 3’ to the Hind111 site in the PBS polylinker) and the

RECOMBINANT

CONVERTING

released insert was ligated into the similarly cut pLENACEVII vector, to generate pLEN-ACE615 Cell culture and gene transfer. CHO Kl cells obtained from the American Type Culture Collection (CCL 61 CHO-Kl) were maintained in Ham’s F12/ DME medium supplemented with 15 mM Hepes buffer (pH 7.0) and 10% fetal calf serum. For gene transfer, the cells (- lo6 cells/lOO-mm plate) were grown to 60-70% confluence and cotransfected with plasmid DNA, 10 pg of each expression plasmid plus 2 pg of pSV2NEO (encoding the neomycin resistance gene, see Ref. (17); a gift from C. L. Cepko, Harvard Medical School), in the form of a calcium phosphate precipitate (18). After 4 h, the DNA was removed and the cells were subjected to a 2-min glycerol shock in medium without serum. Twenty-four to thirty-six hours later, cells were split 1:20 and allowed to grow for 24 h prior to addition of the neomycin analogue G418 at 0.4 mg/ml. After 2 weeks, colonies from eight plates (-100 colonies) were confluent. In the case of pLEN-ACEVII and pLEN-ACEG/ 5, the metallothionein promoter was induced by growing cells in media containing 80 PM ZnCl, and 2% fetal calf serum for 2-3 days. At approximately weekly intervals, cells were incubated for 2 days in F-12/DME/5% fetal calf serum to allow recovery from metal exposure and were then reinduced by metal-supplemented media. Both the conditioned media and solubilized membrane fractions were collected and assayed for enzyme activities. Membrane fractions were prepared by extracting transfected cells with 1% Triton X-100 in 50 mM Hepes, pH 7.5,l mM phenylmethylsulfonyl fluoride at 4°C for 2 h. Genomic DNA blot analysis. High-molecular-weight DNA was isolated (19) and 10 pg was digested with Hind111 overnight at 37°C. The digested fragments were electrophoresed on a 0.8% agarose gel and analyzed by Southern blot transfer (20). A 32P-labeled 0.5-kb 5’fragment of the ACE cDNA clone 10A (6) was used as hybridization probe. Purification of recombinant ACE. hTACE was purified from conditioned media and detergent-extracted CHO cell pellets by affinity chromatography on a Sepharose-28-lisinopril affinity resin (6). Pooled conditioned media, 0.5-1.0 liter, were concentrated (Amicon) to 100 ml, dialyzed against 2 X 4 liters of 20 mM Hepes, pH 7.0, 0.3 M NaCl, and applied to the affinity column, equilibrated with the same buffer, at a flow rate of 20 ml/h. The column was washed with 300-500 ml buffer, and enzyme was eluted with 50 mM borate, pH 9.50. CHO cells were washed with phosphate-buffered saline and incubated for 10 min at 37°C in the same buffer containing 0.01% trypsin (0.5 ml per 3 X lo7 cells), and the suspended cells were centrifuged at 1OOOgfor 5 min. The supernatant contained no activity and was discarded. The pellets were resuspended in 20 mM Hepes, pH 7.5,1% Triton X-100,1 mM PMSF (3 ml per 3 X lo7

3

ENZYME

cells) and incubated overnight at 4’C. The cell suspensions were then homogenized with a glass-Teflon homogenizer and centrifuged at 15,000g for 10 min. Supernatants were pooled and -20 ml was dialyzed and applied to the affinity column as described above for the conditioned media. Enzyme purity was assessed by specific activity and by SDS-PAGE on 7% homogeneous or 4-20% gradient gels. Protein concentrations (molarity) were determined by amino acid analyses; methods for the latter and for N-terminal sequence analyses are described elsewhere (21). Immunoreactivity was determined by Western blotting (method as described in Ref. (22)). Polyclonal anti-human kidney ACE antisera were raised in rabbits by standard procedures and used at a titer of l/500. Enzyme assays, kinetics, and inhibition studies. Assays for ACE activity in culture media, column fractions, etc., and determinations of specific activities were performed with the kinetic method established by Holmquist et al. (23) under standard conditions: 0.1 mM Fa-FGG (2-furanacryloyl-r.,-phenylalanylglycylglycine) in 50 mM Hepes, pH 7.5, 0.3 M NaCl, at 25°C; 1 unit of activity is defined as AA334of 1 min-’ (24). Kinetic constants for Fa-FGG hydrolysis at pH 7.5,0.3 M NaCl, [E] = l-2 X lo-’ M, were determined from LineweaverBurk plots of initial rates at [S] lo-fold below and above K,,,. Chloride dependence was estimated under firstorder conditions with 0.07 mM Fa-FGG ([S] 4 K,) at pH 7.5 and [E] = 0.2-2.5 X lo-‘M over the [Cl-] range O-800 mM, and the apparent activator constants (K,) determined from plots of l/u vs l/[Cl-1. Enzyme used in these studies was first washed extensively with Cl--free buffer (5 mM Hepes, pH 7.5) in a Centricon 30 microconcentrator (Amicon). Inhibition by the tight-binding inhibitor lisinopril (a gift from A. A. Patchett, Merck) was evaluated as follows: ACE, at either [E] N Ki or [E] > Ki, was incubated with lisinopril for 4 h in 50 mM Hepes pH 7.5, 0.3 M NaCl, at room temperature, and residual activity was then measured by the addition of 0.15-0.25 mM Fa-FGG ([S] < K,) in the same buffer. Apparent inhibitor constants (KJ were determined by the graphical method of Dixon (25) for tight-binding inhibitors (see also Ref. (26)). Briefly, a graph of u vs [I] is plotted from the starting value u0 at [I] = 0. A family of lines is drawn from u0 to intersect the curve at v,/2, u,/3, v,l4, etc., giving equally spaced intersection points on the [I] axis, with the expression for the distance between these points (K) given by K = Ki(l

+ [S]/K,).

Hence, if K,,, is known Ki can be calculated directly, and the method is independent of [S]. Kinetic constants for the hydrolysis of AI in 50 mM Hepes, pH 7.5, 30 mM NaCl, were derived from initial

4

EHLERS,

CHEN,

AND

RIORDAN

PBS-RI.2 3527 bp

5710 bp

ACE cDNA

\\

Pblytinker

pQ-ACE 6130 bp

pLEN

Nhe I 5622

-7

-~-

Sites

\

Hindrbd Ill 4352

LiY 3159

SVACI

, 2347

r’ a mm,. ,lwu

559

“Nhe .I

Hvdroahabic .I -- -r-----eequence nC

M&lice

Dmmmttn.

Hind Ill 7715

pBS Poltiinker . Eco RI - 2495

pLEN-ACEVII 8567 bp B

Sites 2549

Hpa I3159 SV40 Poly-A Site xl-lo II 3402

FIG. 1. Construction of expression vectors for hTACE. A full-length testis ACE cDNA was derived from overlapping, incomplete clones 10A and RI.2 (6) and assembled in the vector pJ4R to generate pR-ACE, as outlined under Materials and Methods. p&ACE contains, in addition to the full-length testis ACE cDNA, the Moloney murine leukemia virus (MO-MuLV) promoter (LTR, long-terminal repeats) and two fragments of SV40 DNA: SV40 positions 4038-4648 containing splice donor and acceptor sites, and SV40 positions 2461-2704 containing the SV40 T

RECOMBINANT

CONVERTING

rates measured with the fluorometric method of Friedland and Silverstein (27) adapted for kinetic analyses as previously described (28). RESULTS

Expression of recombinant human testis ACE in CHO cells. CHO cells were transfected with p&ACE, pLEN-ACEVII, and pLEN-ACE615 using the calcium phosphate procedure and cotransformation with pSV2NEO. In the case of pLEN-ACEVII and -6/5, the cells were treated with zinc after confluence in order to fully induce the metallothionein promoter. Both the conditioned media and detergent-solubilized membrane fractions were assayed for ACE activity (Table 1). No activity was detected in control cells transfected with pSV2NEO only. In comparing the G418-resistant pools, the protein expressed under control of the pLEN plasmids after induction reached about the same level as with pQ-1OA. However, when individual colonies of CHO cells transfected with pLEN-ACEVII or -6/5 were assayed for activity, wide variations were observed. About 5 of 100 colonies were found to express high levels of hTACE. These clones were selected and grown in the presence of 80 PM zinc, which further increased the production four- to five-fold (Table 1). Surprisingly, CHO cells transfected with either POACE or pLEN-ACEVII, both of which code for recombinant ACE complete with its putative membrane anchor, secreted significant amounts of soluble hTACE, in addition to expressing the expected membrane-bound hTACE (Table 1). Construct pLEN-ACE6/5 gave slightly higher expression levels after induction than pLEN-ACEVII. pLEN-ACE615 was constructed to direct the expression of ACE lacking its C-terminal hydrophobic anchor, and almost all the ACE activity was found to be present in the conditioned medium; the detergent-solubilized membrane fraction, as expected, contained very little activity (Table 1). Throughout this paper, hTACE expressed in CHO cells transfected with pLEN-ACEVII and pLEN-ACE6/5 is referred to as ACE-VII and ACE-6/5, respectively. Stability of hTACE production in CHO cells. Highproducing clones isolated as described above have been studied for their production over prolonged periods in culture. The stability of production was compared through 10 population doublings in the presence or absence of the selective agent G418 (data not shown). The various cultures behaved identically, indicating that pro-

duction lines.

5

ENZYME

is a stable feature

of these recombinant

cell

Southern blot analysis of DNA from CHO lines. To assess whether stably transfected CHO cells contained the ACE cDNA, DNA from CHO cells transfected with PO-ACE was digested with Hind111 and subjected to Southern blot analysis. A 2.5-kb fragment corresponding to the entire ACE coding sequence (see Fig. 1) was identified in transformed cells, while nothing was detected in the control cells. The intensity of this band indicated that more than one copy of the ACE cDNA was likely incorporated into the CHO genome (data not shown). Purification of hTACE. Recombinant ACE expressed by metal-induced, high-producing clones (Table 1) was purified from conditioned media (soluble form) and Triton X-100 extracts of CHO cell pellets (membrane-bound form) by single-step affinity chromatography on Sepharose-28-lisinopril. In general, -85% of the total activity in conditioned media was eluted from the affinity column as electrophoretically homogeneous hTACE, to give a yield of -1.8 mg purified soluble ACE-VII and ACE-6/5 per liter of conditioned media. From the detergent-extracted cell pellets, -70% of the total activity was eluted as homogeneous enzyme from the column, to give -16 pug of membrane-bound ACE-VII per 3 X lo7 cells. All forms of hTACE had a specific activity of 122 Ulmg for the hydrolysis of FaFGG under standard conditions. SDS-PAGE revealed that the recombinant protein from all sources migrated with the same mobility as the native human testis enzyme and was >98% pure, although it ran as broad, diffuse bands (Fig. 2A). Immunological charadterization. As shown by Western transfer blotting (Fig. 2B), both ACE-VII and ACE6/5 were specifically recognized by polyclonal antiserum raised against human kidney ACE. The reactivity of native and recombinant testis enzyme toward the antiserum was less prominant than that of the somatic isozyme. Amino acid and N-terminal sequence analysis. Amino acid compositions were determined for membrane-bound ACE-VII, soluble ACE-VII, and ACE-6/5, and compared to the predicted compositions (Table 2). The compositions of the latter two are similar and differ from that of membrane-bound ACE-VII. In all cases the compositions were in reasonable agreement with the

antigen polyadenylation signal. The remaining sequence corresponds to nt 2066-4286 from pBR322. The second expression vector for hTACE, pLEN-ACEVII, was based on the vector pLEN, which contains the HindIIIC fragment from SV40 containing the enhancer element, the human metallothionein IIA (hMTIIA) promoter, the 3’ end of the human growth hormone gene (hGHB’), and the complete pUC8 sequence. pLEN-ACEVII was constructed by inserting the ACE cDNA and SV40 sequences from pQ-ACE into pLEN from which the hGH3’ had been removed (see Materials and Methods). pLEN-ACE6/5 (not shown) is identical to pLEN-ACEVII, except that the fragment between Not1 (nt 2046) and Hind111 (nt 2507) that contains the sequence encoding the putative membrane anchor (solid black) was replaced by a synthetic oligoduplex that introduces a stop codon at nt 2105. Unique restriction sites are indicated by asterisks; others are shown as landmarks. Open bars, testis ACE cDNAs; darkly stippled bars, promoter sequences; lightly stippled bar, hGH3’ sequence; cross-hatched bars, SV40-derived sequences; hatched bars, ampicillin-resistance gene (Amp’).

6

EHLERS, TABLE Expression

CHEN,

AND

RIORDAN

1

of hTACE

TABLE in CHO

Cells”

Amino

Acid

Composition

G418’ High-producing G418’ Pool Plasmid

Uninduced

0.36 0.15 0.11 0 Membrane-bound

pa-1OA pLEN-ACEVII pLEN-ACE615 Control cells

of hTACE”

Residues/m01

ChSS

Induced b

Uninduced

Induced*

Soluble ACE (pglml)’ p&ACE pLEN-ACEVII pLEN-ACE615 Control cells

2

0.41 0.36

0.63 0.55

1.8 2.4

ACE (fig/3 x 10’ cells)’

4.8 4.7 0

23.3 0.4

0

a CHO cells were cotransfected with pSV2NEO and one of the folp&ACE, pLEN-ACEVII, and pLEN-ACE6/5. Control cells were transfected with pSVPNE0 alone. G418-resistant (G418’) pools and clones were analyzed for ACE activity in the conditioned media (soluble ACE) and in Triton X-loo-extracted cell pellets (membranebound ACE). * Induced with 80 PM ZnCl,. ’ Protein quantities were deduced from the specific activity of 122 U/mg. lowing:

predicted values. Automated Edman degradation of hTACE failed to yield an N-terminal sequence, suggesting that the N-terminus is blocked, as is that of native human testis ACE (6).

FIG. 2. Recombinant and native testis ACE. (A) SDS-polyacrylamide gel electrophoresis on a 4-20% gradient gel stained with Coomassie brilliant blue. Lanes a and c, native rabbit and human testis ACE, respectively; note that human testes contain equimolar quantities of the somatic and testis isozymes. Lanes d-f, membrane-bound ACE-VII, soluble ACE-VII, and ACE-6/5, respectively. Lane b, molecular weight markers. (B) Western blot. Enzymes were electrophoresed on a 4-20% gradient SDS-gel, electroblotted onto nitrocellulose, and incubated first with a l/500 dilution of anti-human kidney ACE antiserum and then with a l/200 dilution of alkaline phosphatase-labeled goat anti-rabbit IgG. Lanes a-c, native human testis ACE, soluble ACE-VII, and ACE-615, respectively.

Amino acid Asx Glx Ser GUY His Aw Thr Ala Pro Tyr Val Met Ile Leu Phe LYS

Membrane-bound ACE-VII 63.1 72.2 49.3 45.9 23.0 34.7 42.2 57.9 37.6 25.5 34.4 17.9 27.4 76.4 27.6 36.8

(64) (87) (49) (39) (28) (34) (43) (53) (37) (26) (34) (17) (27) (77) (29) (30)

Soluble ACE-VII

ACE-615

62.8 77.3 46.2 35.9 23.3 29.7 42.3 54.3 35.8 25.3 32.2 17.2 24.5 66.8 26.4 30.9

64.4 75.8 46.5 35.1 23.3 29.7 41.6 54.4 36.8 25.1 33.6 17.4 25.1 67.3 26.0 30.5

(64) (83) (43) (35) (23) (29) (42) (51) (36) (26) (32) (17) (25) (66) (26) (30)

(64) (83) (43) (35) (23) (29) (42) (51) (36) (26) (32) (17) (25) (66) (26) (30)

‘Samples were hydrolyzed for 18-24 h at 110°C in 6 M HCl and analyzed by reverse-phase high-performance liquid chromatography after derivatization with phenylisothiocyanate, as described (21). The results are the average of two, five, and four analyses for membranebound ACE-VII, soluble ACE-VII, and ACE-6/5, respectively. The values are presented as residues per mole and integer (in parentheses), the latter based on the inferred polypeptide sequence of the mature human testis ACE (6) for membrane-bound ACE-VII, and from the sequence with the membrane anchor and cytoplasmic tail deleted for soluble ACE-VII and ACE-615.

Kinetic characterization. The recombinant proteins ACE-VII (all kinetic experiments were performed with the soluble form) and ACE-615 were examined kinetically and compared to the native enzymes from human kidney (HK-ACE; somatic isozyme) and rabbit testis (RT-ACE; testis isozyme) (these enzymes were from previous studies; see Refs. (6,lO)). Due to its limited availability and greater difficulty of purification (6), native human testis ACE was not used in these studies. A summary of all the kinetic parameters is given in Table 3. Fa-FGG hydrolysis. No kinetic anomalies were apparent over the [S] range tested (lo-fold above and below K,,,), and the kinetic constants determined for the four enzymes agreed with one another (Table 3) and with published values (23,29). AI hydrolysis. Linear Lineweaver-Burk plots were generated by all four enzymes, and the derived kinetic constants for HK-ACE and the recombinant human testis enzymes were in good agreement and similar to values for human kidney ACE estimated previously (28). The RT-ACE K,,, for AI was 1.5 to 2-fold higher, possibly because the assays were performed in 30 InM Cl-, previously shown to be optimal at pH 7.5 for human

RECOMBINANT

CONVERTING TABLE

7

ENZYME

3

Kinetic Parameters for Native and Recombinant ACE”

Fa-FGG

hydrolysis’

Cl- activation (M)

AI hydrolysisd

Kb Lisinopril (X10-”

KIT2 Enzyme * HK VII 615 RT

(X10-‘M)

2.0 2.9 2.4 2.8

k cat

k cat (mini) 21,000 21,000 18,000 19,000

(&M)

2.3 3.2 3.0 4.8

(min-i)

Low [cl-]e

High [cl-]’

1960 2050 2440 1420

0.36 0.50 0.52 0.11

0.08 0.20 0.07

inhibition

K:

M)

[E] z K,”

[E] > Ki

0.25

1.7 2.0 1.3 1.0

D Derived from initial rates measured in 50 mM Hepes, pH 7.5,25”C. * Enzymes: HK, native human kidney ACE; VII and 615, recombinant human testis ACE expressed in CHO cells transfected with pLENACEVII and PLEN-ACE6/5, respectively; RT, native rabbit testis ACE. c-h K,,, and kc,, values were determined from Lineweaver-Burk plots at ‘300 mM and d30 mM NaCl. Kh values were determined from l/v vs l/[Cl-] plots over the [Cl-] range e low, 20-200 mM and/high, 80-800 mM, with [Fa-FGG] 2 pg/ml after Zn induction were identified. These are remarkably high expression levels for a glycosylated ectoenzyme of this size (-100 kDa), and are in accord with the prior observation that the inducible hMTIIA promoter has considerable strength in CHO cells and can be further enhanced by the SV40 enhancer (14). Moreover, we show here that insertion of SV40 splice sites and termination signals into pLEN after removal of the hGH3’ gene fragment (Fig. 1) generates a vector competent for expression of mammalian cDNAs. We were surprised by the observation that CHO cells transfected with vectors containing a full-length testis ACE cDNA (pR-ACE and pLEN-ACEVII) expressed both the expected membrane-bound enzyme and large amounts of a secreted, soluble form (Table 1). Further investigation of this phenomenon revealed that the soluble hTACE likely derives from the membrane-bound enzyme after proteolytic cleavage of the C-terminal membrane anchor (35). The soluble and membranebound forms of hTACE are catalytically indistinguishable (data not shown). A similar soluble form could be expressed in CHO cells transfected with pLEN-ACEG/ 5, a vector that contains an “anchor-minus” ACE cDNA; indeed, levels of secreted hTACE were even higher than with pLEN-ACEVII (Table 1). The ready production of large quantities of fully active soluble hTACE in this expression system considerably facilitates the harvesting of wild-type hTACE and of any prospective mutant enzymes. The recombinant enzymes could be purified from the conditioned media and from Triton X-100 extracts of CHO cell pellets by single-step lisinopril-affinity chromatography in good yield, and analysis by SDS-PAGE revealed them to be >98% homogeneous (Fig. 2A). The M, of all forms of hTACE was similar to that of the native human testis ACE isozyme (-100 kDa), although the recombinant enzymes ran as broad, diffuse bands, likely due to different patterns of glycosylation produced by the CHO cells. Complete enzymatic deglycosylation by sequential digestion with N-Glycanase, neuraminidase, and 0-Glycanase results in the conversion of all forms of hTACE and of native human testis ACE to a single polypeptide of -70 kDa in each case (35), close to a predicted M, of 75 kDa (6). The amino acid composition of hTACE was within experimental limits of the predicted values (Table 2). Further evidence for the physicochemical similarity or identity be-

AND

RIORDAN

tween hTACE and the native enzyme was provided by the Western blot: recombinant and native human testis ACE cross-reacted with similar affinity with an antihuman kidney ACE antiserum (Fig. 2B). The catalytic activity of the recombinant enzyme ACE-VII (soluble form) and ACE-6/5 was examined kinetically in terms of several well-established criteria: hydrolysis of the best-known physiological substrate, AI, and of the commonly used synthetic N-blocked tripeptide substrate Fa-FGG; Cl- activation of Fa-FGG hydrolysis; and inhibition of Fa-FGG hydrolysis by the widely used ACE inhibitor lisinopril. Comparison with the native enzymes from human kidney and rabbit testis indicates that by these criteria the recombinant enzymes ACE-VII and ACE-615 possess typical angiotensin-converting activity similar or identical to that of the native enzymes (Table 3). We and others have previously observed that the somatic and testis isozymes of rabbit’ ACE appear to be kinetically identical (J. W. Harper, R. Shapiro, and J. F. Riordan, unpublished results; Ref. (9)). The present work indicates that the same is true for the human isozymes and lends support to the hypothesis that the classical angiotensin-converting active site resides in the C-terminal domain of somatic ACE, the domain that is identical to the testis isozyme (6). Thus, while the somatic isozyme consists of two homologous domains, each containing a putative metal-binding site, only the C-terminal domain that constitutes the testis ACE polypeptide is apparently required for full activity. For these reasons, structure-function studies directed at elucidating the mechanism of the classical angiotensin-converting site are best undertaken with the testis isozyme that is unencumbered by a second domain. Therefore, whereas the testis isozyme likely serves a specialized function unrelated to blood pressure regulation, it is well suited as a model to study the angiotensin I-converting catalytic mechanism. ACKNOWLEDGMENTS We thank Drs. P. J. Kushner and C. L. Cepko for providing the vectors pLEN and pJ4s2, respectively. We are indebted to Drs. Frank S. Lee, Daniel J. Strydom, and Edward A. Fox for advice, Dr. Stana Weremowicz for assistance with the cell cultures and transfections, Dr. Werner Dafeldecker for the synthetic oligonucleotides, and Wynford Brome for technical help. This work was supported in part by Grant HL 34704 from the National Institutes of Health.

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