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The C-terminal Domain of Aminopeptidase A Is an Intramolecular Chaperone Required for the Correct Folding, Cell Surface Expression, and Activity of This Monozinc Aminopeptidase*

Aminopeptidase A (APA, EC 3.4.11.7) is a type II integral membrane glycoprotein responsible for the conversion of angiotensin II to angiotensin III in the brain. Previous site-directed mutagenesis studies and the recent molecular modeling of the APA zinc metallopeptidase domain have shown that all the amino acids involved in catalysis are located between residues 200 and 500. The APA ectodomain is cleaved in the kidney into an N-terminal fragment corresponding to the zinc metallopeptidase domain, and a C-terminal fragment of unknown function. We investigated the function of this C-terminal domain, by expressing truncated APAs in Chinese hamster ovary and AtT-20 cells. Deletion of the C-terminal domain abolished the maturation and enzymatic activity of the N-terminal domain, which was retained in the endoplasmic reticulum as an unfolded protein bound to calnexin. Expression in trans of the C-terminal domain resulted in association of the N- and C-terminal domains soon after biosynthesis, allowing folding rescue, maturation, cell surface expression, and activity of the N-terminal zinc metallopeptidase domain. We also show that the C-terminal domain is not required for the catalytic activity of APA but is essential for its activation. Moreover, we show that the C-terminal domain of aminopeptidase N (EC 3.4.11.2, APN) also promotes maturation and cell surface expression of the N-terminal domain of APN, suggesting a common role of the C-terminal domain in the monozinc aminopeptidase family. Our data provide the first demonstration that the C-terminal domain of an eukaryotic exopeptidase acts as an intramolecular chaperone.

Aminopeptidase A (APA, 1 EC 3.4.11.7) is a homodimeric membrane-bound zinc metallopeptidase that is activated by calcium and specifically cleaves the N-terminal glutamyl or aspartyl residue from peptide substrates such as angiotensin II (Ang II) and cholecystokinin-8 (1,2) in vitro. APA is present in many tissues, particularly in the renal and intestinal brush border epithelial cells and in the vascular endothelium (3). APA has been identified in several brain nuclei involved in the control of body fluid homeostasis and cardiovascular functions, together with other components of the brain renin-angiotensin system (4). Studies using specific and selective APA inhibitors (5) have demonstrated that, in vivo, APA is responsible for the conversion of brain Ang II to angiotensin III (Ang III) (6) and that brain Ang III, and not Ang II as established in the periphery, exerts a tonic stimulatory action in the central control of blood pressure in spontaneously hypertensive rats (7). Therefore, the inhibition of central but not peripheral APA with specific and selective inhibitors leads to a large decrease in arterial blood pressure (7), suggesting that central APA might be an interesting candidate target for the treatment of hypertension (8). Molecular cloning of mouse, rat, and human APA (9 -12) predicts a type II integral membrane protein of 945 residues composed of a short 17-residue N-terminal cytoplasmic tail, a 22-residue transmembrane domain, and an extracellular domain containing the active site, including the consensus sequence HEXXH . . .E found in the zinc metalloproteases family, the gluzincins (13). Site-directed mutagenesis studies, based on alignment of the APA sequence with the sequences of other monozinc aminopeptidases, were initially used to probe the organization of the APA active site (14 -19). We used the functional data collected in these studies and the recently resolved x-ray crystal structure of leukotriene A4 hydrolase/aminopeptidase (EC 3.3.2.6) (20), a bifunctional zinc metalloenzyme, to construct a three-dimensional model of the mouse APA extracellular domain from residues 79 to 559, corresponding to the zinc metallopeptidase domain (21). According to this model, the zinc metallopeptidase domain is folded into a flat triangle composed of an N-terminal subdomain consisting mostly of ␤-sheets, a globular catalytic subdomain, and a C-terminal helical subdomain. The active site is located at the interface of the N-and C-terminal subdomains and contains all the residues involved in zinc binding, substrate binding, and catalysis previously characterized by sitedirected mutagenesis.
It has been suggested that APA has two distinct extracellular domains in vivo. First, proteolytic fragmentation studies of purified pig APA (22) showed that this protein exists in vivo as two polypeptides of 107 and 45 kDa (22). N-terminal sequencing showed that the 107-kDa fragment was the N-terminal extracellular domain of APA from residues 43 to 602, corresponding to the active site-containing zinc metallopeptidase domain we have modeled, whereas the 45-kDa fragment corresponded to the C-terminal domain of APA, from residues 603 to 942. Second, structural studies based on electron microscopy of purified pig intestinal APN incorporated into lipid membranes also suggested the existence of a C-terminal globular domain different from the zinc metallopeptidase domain (23). Finally, in the rat hippocampus, a short variant of APA produced by alternative splicing and lacking the C-terminal domain has been cloned. This protein displayed no APA activity when produced in COS-1 cells (24). Similarly, in the absence of the C-terminal domain, the N-terminal zinc metallopeptidase domain of mouse APA, when produced alone in COS-1 cells, was an inactive enzyme, which remained blocked in the endoplasmic reticulum (25). This suggests that the C-terminal domain of APA may be involved in the correct folding of APA. We tested this hypothesis by constructing two truncated forms of recombinant mouse APA, one corresponding to the zinc metallopeptidase domain, from the first residue of APA to residue 598, tagged at its N terminus with the FLAG epitope (FLAG-N-APA), and the other corresponding to the C-terminal domain, from residues 595 to 945, tagged at its N terminus with the HA epitope (HA-C-APA). These proteins were expressed in CHO and AtT-20 cells, either separately or in trans, for studies of the influence of the C-terminal domain of APA on the maturation, subcellular localization, folding, and activity of the N-terminal metallopeptidase domain. To determine if there is a common role for the C-terminal domain of monozinc aminopeptidases, we have investigated the role of the C-terminal domain of APN. For this purpose, we produced the FLAG-tagged N-terminal domain of APN from residues 1 to 606 (FLAG-N-APN) and the HA-tagged C-terminal domain of APN from residues 607 to 966 (HA-C-APN) and studied if the role of the C-terminal domain of APN was similar to that of the C-terminal domain of APA.

EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs Inc. (Hitchin, England) and were used according to the manufacturer's instructions. The Expand high-fidelity Taq polymerase PCR system was purchased from Roche Applied Science (Mannheim, Germany). The liposomal transfection reagent LipofectAMINE 2000, the pSecTag2 vector, pcDNA3 vector, pcDNA3.1-His vector, and the monoclonal anti-Xpress antibody (anti-Xpress mAb) were purchased from Invitrogen. The polyclonal anti-HA antibody (anti-HA Ab) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the monoclonal anti-HA antibody (clone 12CA5, anti-HA mAb) was purchased from Roche Applied Science. The monoclonal M2 anti-FLAG antibody (anti-FLAG mAb) and the polyclonal anti-calnexin antibody (anti-calnexin Ab) were purchased from Sigma, and the monoclonal anti-protein disulfide isomerase antibody (anti-PDI mAb) was purchased from Streegen (Victoria, Canada). Immobilized cobalt affinity columns (Talon) were obtained from Clontech (Heidelberg, Germany). The synthetic substrate ␣-L-glutamyl-␤-naphthylamide (GluNA) was purchased from Bachem (Bunderdorf, Switzerland).
Plasmid Constructs-The subcloning of the cDNA encoding the mouse APA (9) and tagged at its N terminus with a polyhistidine tail (His 6 -WT-APA) has been described elsewhere (18). FLAG-WT-APA and FLAG-N-APA were constructed in pcDNA3 by PCR, using the mouse APA sequence as a template. A forward primer encoding the FLAG epitope and the seven first residues of APA and introducing an upstream HindIII restriction site was used for both constructs (primer A: T CAT CAA GCTT ATG AAG GAC TAC AAG GAC GAC GAT GAC AAG GCC ATG AAC TTT GCA GAG GAA GAG). A reverse primer encoding the last six residues of APA and introducing a downstream EcoRI restriction site was used for FLAG-WT-APA (primer B: ATC TGC AGA ATT CTA CGG CAG GCT AGC GAA CCA). A reverse primer encoding residues 592-598 of APA, replacing residue 599 by a stop codon and introducing a downstream EcoRI restriction site was used for FLAG-N-APA (primer C: ATC TGC AGA ATT CTA ATT CAG AGT GAT TCC TCC TTT GTC). HA-C-APA was constructed in pSec-tag2 by PCR, using FLAG-WT-APA as a template. A forward primer encoding the HA epitope and residues 596 -602 of APA and introducing an upstream HindIII restriction site was used (primer D: CGT ACG TAC GAA GCT  TCA TAC CCT TAC GAC GTT CCT GAT TAC GCT ATC ACT CTG AAT  GCT AAT CTT). The reverse primer used for this construct was primer B. N-APN was cloned from the mouse brain Marathon-Ready TM cDNA library (Clontech Laboratories, Inc.) using a forward primer encoding residues 1-7 of APN (primer E: ATG GCC AAG GGG TTC TAC ATT) and a reverse primer encoding residues 601-606 of APN, replacing residue 607 by a stop codon, and introducing a downstream EcoRI restriction site (primer F: ATC TGC AGA ATT CTA GTT TTT CTC GAC ATC CAG CCA). The resulting PCR product was used as a template to construct FLAG-N-APN in pcDNA 3 by PCR. A forward primer encoding the FLAG epitope, the first seven residues of APN and introducing an upstream HindIII restriction site was used (primer G: TC ATC AAG CTT ATG AAG GAC TAC AAG GAC GAC GAT GAC AAG GCC ATG GCC AAG GGG TTC TAC ATT). The reverse primer was primer F. C-APN was cloned from the mouse brain Marathon-Ready TM cDNA library using a forward primer encoding the residues 607-614 of APN (primer H: CAG AGT GCA AAG TTC CAG ACA), and a reverse primer encoding the residues 961-966 of APN and introducing a downstream EcoRI restriction site (primer I: ATC TGC AGA ATT CTA ACT GCT GTT CTC TGT GAA). The resulting PCR product was used as a template to construct HA-C-APN in pSec Tag-2 by PCR. A forward primer encoding the HA epitope, residues 607-614 of APN and introducing an upstream SfiI restriction site was used (primer J: GCG GCC CAG CCG GCC TCA TAC CCT TAC GAC GTT CCT GAT TAC GCT CAG AGT GCA AAG TTC CAG ACA). The reverse primer was primer I. The absence of nonspecific mutations was confirmed by automated sequencing on an Applied Biosystems 377 DNA Sequencer with dye deoxyterminator chemistry.
Cell Culture, Transfection of CHO-K1 and AtT-20 Cells, and Purification of Recombinant His 6 -WT-APA and His 6 -N-APA-CHO-K1 (American Type Culture Collection, Manassas, VA) cells were maintained in Ham's F-12 medium (Invitrogen) supplemented with 7% fetal bovine serum (Invitrogen), 0.5 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (all from Roche Applied Science). AtT-20 cells were maintained in high glucose Dulbecco's modified Eagle's medium (Invitrogen), supplemented with 10% NuSerum IV (BD Biosciences, Franklin Lakes, NJ), and 2.5% fetal bovine serum (Invitrogen). CHO and AtT-20 cells were maintained at 37°C in an atmosphere containing 5% CO 2 . Cells were transfected with LipofectAMINE 2000, according to the manufacturer's protocol (Invitrogen). For stable expression of FLAG-WT-APA, FLAG-N-APA, and FLAG-N-APAϩHA-C-APA, transfected CHO cells were selected for resistance to Zeocin (10 days, 200 g/ml) or zeocin plus hygromycin (10 days, 200 g/ml and 100 g/ml, respectively). Clones were screened by immunoblotting and immunofluorescence analysis, to select cells with homogeneous expression. For transient expression, CHO and AtT-20 cells were transfected in 35-mm dishes and treated for biochemical or immunofluorescence studies 24 -36 h later. His 6 -WT-APA and His 6 -N-APA were purified from membrane preparations by metal affinity chromatography, with a metal chelate resin column (Talon Co 2ϩ ), as previously described (18). The purity of the final preparation was assessed by SDS-PAGE in 7.5% polyacrylamide gels, as described by Laemmli (26). Proteins were stained with Coomassie Brilliant Blue R-250. Protein concentrations were determined with the Bradford assay (27), using bovine serum albumin as the standard.
Metabolic Labeling and Immunoprecipitation-300,000 stably transfected CHO cells (for FLAG-WT-APA, FLAG-N-APA, and FLAG-N-APAϩHA-C-APA) or transiently transfected CHO cells (for HA-C-APA, FLAG-N-APN, HA-C-APN, and FLAGN-APNϩHA-C-APN) were seeded in 6-well plates. The cells were grown for 36 h and then incubated for 30 min in methionine-, cysteine-, and serum-free medium (Ham's F-12) supplemented with 100 Ci/ml [ 35 S]methionine/cysteine (pulse). The cells were then incubated for various periods of time in Opti-MEM medium (chase). The medium was discarded, the cells were harvested, and proteins were solubilized by incubation at 4°C with 500 l of 50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% (v/v) Triton X-100 (lysis buffer). The resulting lysate was centrifuged at 20,000 ϫ g for 15 min at 4°C to remove the insoluble material. The supernatant was incubated with 1 g of anti-FLAG mAb or 1 g of anti-HA mAb, and protein A-Sepharose (Amersham Biosciences) (10% v/v) for 2 h at 4°C for immunoprecipitation. The immune complexes were collected by centrifugation and washed four times with solubilization buffer and once with 20 mM Tris-HCl buffer, pH 6.8. Proteins were eluted by boiling in 30 l of Laemmli buffer and resolved by 5%, 7.5%, or 10% SDS-PAGE. The gel was dried and placed next to a K-screen (Kodak) for FX Molecular Imaging and QuantityOne software analyses (Bio-Rad).
Peptide N-Glycosidase F and Endoglycosidase H Treatment-A population of 300,000 stably transfected CHO cells producing FLAG-WT-APA and FLAG-N-APA, either alone or with HA-C-APA, or of transiently transfected CHO cells producing HA-C-APA, was subjected to 5 h of [ 35 S]methionine/cysteine labeling. Cell extracts were produced, and FLAG-tagged proteins were immunoprecipitated. The samples were washed and centrifuged as described above. The immune complexes were then eluted by boiling for 15 min in 25 l of denaturing buffer (0.25% SDS). The samples were diluted 1:4 in 20 mM Tris-HCl buffer, pH 6.8, and 1 l of Triton X-100 was added to prevent the denaturation of glycosidases by SDS. The samples were then incubated with or without peptide N-glycosidase F (PNGase F) (Roche Applied Science) (5 milliunits at 37°C for 18 h), or endoglycosidase H (Endo H) (Roche Applied Science) (1 unit at 37°C for 18 h). The reaction was stopped by adding Laemmli buffer, and the samples were subjected to SDS-PAGE in 5%, 7.5%, or 10% acrylamide gels, which were then dried and analyzed with a phosphorimaging device (Bio-Rad).
Trypsin Treatment-The immunoprecipitates from 300,000 stably transfected [ 35 S]methionine/cysteine-labeled CHO cells were treated with 25 g of trypsin for the indicated times at 37°C. The reaction was terminated by adding 5 l of 57 mM PMSF.
Co-immunoprecipitation and Western Blotting-Transiently transfected CHO or AtT-20 cells (300,000 cells) were harvested and lysed by incubation for 1 h in lysis buffer. For immunoprecipitation, lysates were incubated with 1 g of anti-HA Ab or anti-calnexin Ab, and 10% v/v protein A-Sepharose for 2-3 h. The beads were washed once with lysis buffer, then three times with PBS and once with 20 mM Tris-HCl buffer, pH 6.8, resolved by 7.5% SDS-PAGE, and subjected to Western blotting using an anti-FLAG mAb.
Immunodepletion of HA-C-APA-300,000 CHO cells were transiently transfected with FLAG-N-APA and HA-C-APA. The cells were grown for 36 h and then extracted by incubation in lysis buffer overnight. Lysates were then immunoprecipitated by incubation with 1 g of anti-HA mAb, and 10% v/v protein A-Sepharose for 4 h. Supernatants were separated from the bead complex and subjected to two more rounds of immunoprecipitation. Clearing of the samples from HA-C-APA was monitored by immunoblotting, with an anti-HA mAb, the beads and the supernatants at each round of immunoprecipitation. The FLAG-N-APA levels in each sample were compared by anti-FLAG mAb (1:1000) immunoblotting of the samples, for equivalent amounts of protein. Immunoreactive material was detected with an alkaline phosphatase-coupled anti-mouse antibody, using ATTOPHOS as the chemifluorescent substrate. Chemifluorescence was measured using a phosphorimaging device (Bio-Rad), allowing direct quantification of the bands.
Immunofluorescence-Transiently or stably transfected CHO cells and AtT-20 cells were seeded (25,000 cells) on 14-mm diameter coverslips. The cells were cultured for 48 h, then fixed and permeabilized by incubation for 5 min in 100% ice-cold methanol. The cells were rinsed three times in 0.1 M phosphate-buffered saline, pH 7.4 (PBS), then saturated by incubation with 5% BSA for 30 min at room temperature. They were then incubated with a 1:1000 dilution of rabbit polyclonal anti-(rat APA) serum (28), or of anti-FLAG mAb, and/or of anti-HA Ab, and/or 1:500 dilution of anti-PDI mAb in PBS, 2% BSA for 2 h at room temperature. The coverslips were washed three times with cold PBS and then incubated with a 1:1000 dilution of cyanin 3-conjugated polyclonal anti-rabbit antibody and/or of Alexa 488-conjugated polyclonal anti-mouse antibody, and/or cyanin 5-conjugated anti-rabbit antibody, in PBS, 2% BSA for 2 h at room temperature. The coverslips were washed four times with PBS and mounted in Mowiol (Sigma-Aldrich) for confocal microscopy analysis. Cells were examined with a Leica TCS SP II (Leica Microsystems, Heidelberg, Germany) confocal laser scanning microscope equipped with an argon/krypton laser and configured with a Leica DM IRBE inverted microscope. All the double-labeling experiments were analyzed in sequential scanning mode. Images (1024 ϫ 1024 pixels) were obtained with a 63ϫ magnification oilimmersion objective. Each image corresponds to a cross-section of the cell.
Enzyme Assay of Solubilized Cells-CHO cells stably expressing FLAG-WT-APA and FLAG-N-APA were solubilized in lysis buffer. The levels of each protein were compared by anti-FLAG (1:1000) immunoblotting of the samples, as described above. The APA activity of the various samples was determined by monitoring the rate of hydrolysis of a synthetic substrate, GluNA, as previously described (5). Equivalent amounts of recombinant FLAG-N-APA were incubated at 37°C in the presence of 5 ϫ 10 Ϫ4 M GluNA, 4 mM CaCl 2 in a final volume of 100 l of 50 mM Tris-HCl buffer, pH 7.4. Bestatin, a nonspecific aminopeptidase inhibitor, was used at a concentration of 1 M, which did not inhibit APA but prevented degradation of the substrate by aminopeptidases such as B, N, and W, and cytosolic aminopeptidases (29,30). Enzyme assays were performed in the presence or absence of the APA inhibitor glutamate phosphonate (GluPO 3 H 2 , 1 M).
Enzyme Assay of Purified APA-The enzymatic activities of His 6 -WT-APA and His 6 -N-APA were determined in a microtiter plate, by monitoring the rate of hydrolysis of GluNA, as previously described (18). The kinetic parameters (K m and k cat ) were determined from Lineweaver-Burk plots, with a final concentration of GluNA of 0.025-2 mM.
All enzymatic assays were performed under initial velocity conditions. Spontaneous hydrolysis of the substrate was corrected by subtracting the absorbance of blank incubations without enzyme. The sensitivity of His 6 -WT-APA and His 6 -N-APA to inhibition by glutamate phosphonic acid (L-GluPO 3 H 2 (31)) was determined by establishing dose-dependent inhibition curves for a final GluNA concentration of 0.5 mM and calculating K i values with GraphPad Prism 2 software. The sensitivity of His 6 -WT-APA and His 6 -N-APA to calcium was determined by establishing dose-dependent activation curves and calculating ED 50 values of CaCl 2 with GraphPad Prism 2 software. Statistical comparisons were performed with Student's unpaired t test. Differences were considered significant if p was Ͻ0.05.

RESULTS AND DISCUSSION
APA is composed of a cytoplasmic domain, a transmembrane domain, and an extracellular domain divided into two parts: an N-terminal zinc metallopeptidase domain up to residue 598, including the active site of the enzyme, and a C-terminal domain corresponding to the last 347 amino acid residues of the extracellular domain of APA. We investigated the impact of the C-terminal domain of APA on the generation of a correctly membrane-bound active APA enzyme.
To this end, we generated several cDNA constructs ( Fig. 1) encoding either the entire mouse APA or one of the individual domains, which we then expressed in CHO cells. We assessed the folding, trafficking, subcellular localization, and enzymatic activity of each of the corresponding recombinant proteins. One construct corresponded to the entire sequence of the mouse wild-type APA (WT-APA), tagged at its N terminus either with the FLAG epitope (FLAG-WT-APA), or with a polyhistidine (His 6 ) tail (His 6 -WT-APA) to allow purification of the enzyme. A second construct (N-APA) included the mouse APA sequence from residues 1 to 598 (which corresponds to Asn-602 of pig APA) containing the cytoplasmic domain, the transmembrane domain functioning as a signal peptide, the N-terminal zinc metallopeptidase domain, tagged at its N terminus either with the FLAG epitope (FLAG-N-APA), or with the polyhistidine tail (His 6 -N-APA). A third construct was also generated, corresponding to the C-terminal domain of APA from residues 596 to 945 (C-APA). Because the C-terminal domain does not possess a signal sequence for translocation into the ER, the cleavable signal sequence of mouse Ig (32) was fused to the mouse APA C-terminal domain sequence allowing ER translocation and secretion of the fusion protein. This third construct was tagged at its N terminus with the HA epitope (HA-C-APA). The cDNA constructs encoding FLAG-WT-APA, FLAG-N-APA, and HA-C-APA were used to transfect CHO cells, and the expression of the corresponding recombinant proteins was studied.
Comparison of the Expression of the N-terminal Monozinc Metallopeptidase Domain of APA with That of WT-APA-We investigated whether deletion of the C-terminal domain of APA affected the maturation of the N-terminal domain, by performing metabolic labeling and pulse-chase experiments with FLAG-WT-APA and FLAG-N-APA. For FLAG-WT-APA, we detected a single immunoprecipitated band with an apparent molecular mass of 140 kDa after 30 min of pulse and a second band of 160 kDa after 90 min of chase ( Fig. 2A). In contrast, for FLAG-N-APA, we detected only an 85-kDa immunoprecipitated band, even after 5 h of chase ( Fig. 2A). In addition, the intensity of the band corresponding to FLAG-N-APA decreased after 3 h of chase, probably due to degradation, because the protein was not secreted (Fig. 2C).
We further investigated the pattern of processing of FLAG-WT-APA and FLAG-N-APA by treating immunoprecipitates with PNGase F or Endo H. The treatment of glycoproteins with PNGase F results in the removal of all N-linked oligosaccharide side chains and was used as a positive control for the deglycosylation of recombinant APAs. Treatment with Endo H removes immature, but not medial Golgi-processed N-linked oli-gosaccharide side chains. The treatment of FLAG-WT-APA with PNGase F led to the disappearance of both the 160-and 140-kDa forms of the protein, resulting in a single band 110 kDa in size. FLAG-N-APA was also sensitive to this treatment and yielded a 75-kDa band. For wild-type APA, the 140-kDa form was Endo H-sensitive and shifted to 110 kDa upon treatment, whereas the 160-kDa form was Endo H-resistant (Fig.  2B). For FLAG-N-APA, the 85-kDa form was Endo H-sensitive and shifted to 75 kDa upon treatment (Fig. 2B). Thus, the high molecular mass form of APA corresponded to the mature glycosylated complex sorting from the Golgi apparatus. In contrast, the 85-kDa form of FLAG-N-APA corresponded to an immature protein that was not transported to the Golgi apparatus. We then investigated the subcellular localization of FLAG-WT-APA and FLAG-N-APA by performing double-labeling immunofluorescence experiments with a rabbit polyclonal anti-(rat APA) antibody and a monoclonal antibody raised against the endogenous protein disulfide isomerase (PDI), a chaperone protein resident in the ER.
Confocal microscopy analysis of CHO cells expressing either FLAG-WT-APA or FLAG-N-APA showed that WT-APA was located at the plasma membrane, whereas FLAG-N-APA and PDI were co-localized in the ER (Fig. 2D). This retention in the ER, in contrast to the membrane location of WT-APA, is consistent with the incorrect maturation of the truncated form of APA, deleted of its C-terminal domain.
We investigated the basis of this impaired maturation and transport block by comparing the folding patterns of FLAG-WT-APA and FLAG-N-APA in a trypsin sensitivity assay. FLAG-WT-APA was trypsin-resistant even after 90 min of treatment, demonstrating that the correct folding of native WT-APA prevents its degradation by trypsin (Fig. 3A). Changes in the sensitivity of APA to trypsin may therefore reflect changes in folding pattern. Consistent with this hypothesis, the unfolded APA mutant Ala-220 (21) was totally degraded after 15 min of trypsin treatment (not shown). FLAG-N-APA was entirely degraded by 15 min of trypsin treatment, whereas FLAG-WT-APA was resistant to trypsin, suggesting that FLAG-N-APA is retained in the ER as an unfolded protein (Fig. 3A).
Analysis of the functional role of a protein may also provide information about its folding status. Unlike FLAG-WT-APA, FLAG-N-APA was completely devoid of specific enzymatic activity (Fig. 3B), consistent with the incorrect folding of this protein.
The biosynthetic labeling, confocal microscopy, and trypsin assay data together demonstrate that expression of the Nterminal zinc metallopeptidase domain of APA alone in CHO cells results in the production of an unfolded, transport-incompetent protein that has no enzyme activity, is rapidly degraded, and is unable to pass the ER quality control (for review see Ref. 33). These findings are consistent with those of Ofner et al. (25) and shed light on the potentially critical role of the C-terminal domain of APA in facilitating the folding, trafficking, and plasma membrane expression of APA.
Expression of a Secreted Form of C-APA-We then analyzed the biosynthetic and structural features of C-APA expressed alone, to determine the effect of the C-terminal domain of APA on the folding and transport behavior of the N-terminal zinc metallopeptidase domain of APA. C-APA does not possess a signal sequence for translocation into the ER. We therefore generated a construct encoding the entire C-APA sequence, tagged at its N terminus with the HA epitope (HA-C-APA), fused to the cleavable Ig leader sequence, allowing secretion of the protein.
We transfected CHO cells with this construct. Following metabolic labeling, immunoprecipitation with an anti-HA mAb, and endoglycosidase digestion analysis, we detected in the cell lysate, after a 30-min pulse, a single Endo H-sensitive immunoprecipitated band with an apparent molecular mass of 45 kDa (Fig. 4, A and B). After 1 h of chase, two larger Endo H-resistant species with apparent molecular masses of 50 and 55 kDa were detected in the culture medium. Thus, the Cterminal domain of APA, unlike the N-terminal domain of APA, exited from the ER, was processed by the Golgi apparatus and was secreted into the culture medium as a mature protein. This secreted protein was resistant to trypsin digestion, because this treatment gave less than 50% degradation after 90 min (Fig.  4C). Consistently, confocal microscopy analysis of CHO cells transfected with the HA-C-APA construct showed C-APA expression, as detected with an anti-HA Ab, in vesicles probably corresponding to secretory vesicles (Fig. 4D). These data suggest that the C-terminal domain of APA was correctly folded and secreted independently of the N-terminal domain.

Expression in Trans of the C-terminal Domain of APA Rescues the Functions of the N-terminal Zinc Metallopeptidase
Domain-A growing number of membrane and secretory proteins have been shown to have pro-domains at their N-terminal ends that undergo post-translational proteolytic cleavage in the late secretory pathway after the acquisition of transport competence. These domains, known as intramolecular chaperones, are responsible for the correct folding of their cognate catalytic domain. The presence of such intramolecular chaperone domains, or pro-domains, has been reported in enzymes such as subtilisin (34) and ␣-lytic protease (35). Studies with these bacterial enzymes have shown that deletion of the Nterminal propeptides results in inactive enzymes maintained in an unfolded state. Results from the in vitro refolding of bacterial subtilisin (36) and in vivo studies with several proteases, such as bacterial ␣-lytic protease (37), subtilisin (38), thermolysin (39), Saccharomyces cerevisiae proteinase A (40), and Kex2p (41), have all indicated that pro-domains can activate the protease domain in trans. The presence of functional pro-domains in the eukaryotic membrane type 1 matrix metalloproteinase and in mammalian endoproteases of the subtilisin family has also been reported (42)(43)(44). Recently, an intramolecular chaperone domain located at the C terminus of the intestinal enzyme, sucrase-isomaltase has been described. The sucrase domain is autonomous and folds independently (45). However, to our knowledge, no such intramolecular chaperone domain has ever been reported in the monozinc aminopeptidase family.
We investigated whether the correct folding of the N-terminal zinc metallopeptidase domain of APA required the presence of the C-terminal domain of APA, functioning as an intramolecular chaperone via a direct interaction between the two domains. To this end, we co-expressed the FLAG-N-APA and HA-C-APA constructs in CHO cells and assessed the matura- FIG. 2. FLAG-N-APA is not matured and is rapidly degraded within the cell. A, CHO cells stably expressing wildtype and truncated FLAG-tagged APAs were labeled for 30 min with [ 35 S]methionine/cysteine and subjected to chase periods with serum-free medium for various periods of time. Proteins were immunoprecipitated from cell lysates with an anti-FLAG mAb, and resolved by SDS-PAGE. B, after 5 h of [ 35 S]methionine/cysteine labeling, proteins were immunoprecipitated from cell lysates with an anti-FLAG mAb and treated with PNGase F or Endo H. Samples were subjected to SDS-PAGE. C, cells were incubated overnight in Opti-MEM medium, and secreted proteins were precipitated with trichloroacetic acid. Celllysate proteins and secreted proteins were resolved by SDS-PAGE and immunoblotted with an anti-FLAG mAb. D, CHO cells stably expressing FLAG-WT-APA or FLAG-N-APA were fixed in cold methanol. APA co-immunolocalization was performed using polyclonal rabbit anti-(rat APA) serum and monoclonal anti-protein disulfide isomerase (PDI) antibody. FLAG-WT-APA (blue) was present at the plasma membrane, whereas FLAG-N-APA (blue) was co-localized in the ER with PDI (red). Immunolabeled cells were analyzed by confocal microscopy. Bar ϭ 20 m. tion, folding, trafficking, and enzymatic activity of the corresponding recombinant proteins, together with interactions between them.
Maturation and Folding-Metabolic labeling and endoglycosidase digestion in CHO cells co-expressing both the N-and C-terminal domains of APA (Fig. 5A) showed the presence, after a 30-min pulse, of FLAG-N-APA, immunoprecipitated with an anti-FLAG mAb, in an 85-kDa form. A second band, 100 kDa in size, was detected after 90 min of chase, and remained the principal band present after 5 h. The 100-kDa form of FLAG-N-APA, unlike the 85-kDa form, was Endo Hresistant (Fig. 5B). Moreover, whereas FLAG-N-APA expressed alone (85 kDa) was totally degraded after 15 min of trypsin treatment, such treatment gave less than 50% degradation of the 100-kDa form of FLAG-N-APA, even after 90 min (Fig. 5C). Thus, the co-expression of these two proteins resulted in a 100-kDa FLAG-N-APA form exhibiting all the characteristics of a matured, correctly folded protein.
Trafficking-Confocal microscopy analysis of double-labeled CHO cells co-expressing FLAG-N-APA and HA-C-APA showed both proteins to be present at the plasma membrane (Fig. 5D). This membrane localization was also observed in AtT-20 cells (a cell line derived from adenohypophyseal corticotropes (46)) expressing both the N-and C-terminal domains of APA (see Fig. 7B). In contrast, in AtT20 cells, FLAG-N-APA expressed alone was located in the ER, and HA-C-APA expressed alone was accumulated in cell process, probably corresponding to secretory vesicles (see Fig. 7A). This demonstrates that the pattern of expression of N-APA and C-APA does not depend on cell type. In cells expressing both the N-and C-terminal domains of APA, the membrane localization of the N-APA suggested that, in the presence of the C-terminal domain of APA, the N-terminal zinc metallopeptidase domain of APA displayed a similar pattern of trafficking to WT-APA. Furthermore, the presence of both proteins at the plasma membrane suggests that they may be associated.
Interaction between the N-and C-terminal Domains-We investigated whether N-APA and C-APA interacted with each other by means of co-immunoprecipitation experiments. Cell lysates from CHO cells expressing FLAG-N-APA, either alone or together with HA-C-APA, were subjected to immunoprecipitation with antibodies recognizing HA-C-APA (anti-HA mAb) but not FLAG-N-APA. Then, the presence in the immunoprecipitate of FLAG-N-APA was detected with an anti-FLAG mAb. FLAG-N-APA was detected only in immunoprecipitates from cells co-expressing both domains, as the 85-kDa and 100-kDa forms (Fig. 6A). Thus, in CHO cells co-expressing the N-and C-terminal domains of APA, C-APA is associated with both the immature and the mature form of N-APA, because these two forms of N-APA are co-immunoprecipitated with C-APA. This suggests that the N-and C-terminal domains of APA interact shortly after their biosynthesis.
In addition, metabolic labeling of CHO cells co-transfected with the FLAG-N-APA and the HA-C-APA constructs, followed by immunoprecipitation of cell lysates and culture media with an anti-HA mAb, showed that the immature form of HA-C-APA was interacting with the immature 85-kDa form of N-APA after 30 min of pulse, in agreement with the association between both proteins shortly after their biosynthesis. HA-C-APA was present in only very small amounts in the medium after 3 h (Fig. 6B). In contrast, HA-C-APA was found as a mature protein in the cell lysate, interacting with the mature form of FLAG-N-APA, as shown by the presence of a 100-kDa band (Fig. 6B), even after 6 h of incubation (not shown). Thus, the co-expression of both domains in CHO cells induced retention of the mature form of C-APA at the plasma membrane. This finding is consistent with the observed change in the transport behavior of C-APA in the presence of N-APA resulting from a specific interaction between the two proteins.
Finally, the purification by metal affinity chromatography of His 6 -N-APA (Fig. 6C) from CHO cells expressing both HA-C-APA and His 6 -N-APA led to the detection in the eluate of both N-APA and C-APA, identified with the anti-Xpress and anti-HA antibodies as 100-kDa and 55-kDa proteins, respectively. This finding confirms the close association between the two proteins.
Enzymatic Activity of the N-APA⅐C-APA Complex-We investigated whether the membrane-bound N-APA⅐C-APA complex had recovered the enzymatic properties of WT-APA. Using the purified preparation from CHO cells co-transfected with His 6 -N-APA and HA-C-APA, as described above, we showed that this complex had similar enzymatic characteristics to His 6 -WT-APA (Table I). His 6 -N-APA had kinetic parameters and calcium activation patterns similar to those of His 6 -WT-APA. We also assessed the ability of a specific and selective inhibitor of APA, glutamate phosphonate (a transition state pseudo-analog), to inhibit His 6 -WT-APA and the His 6 -N-APA⅐HA-C-APA complex. This inhibitor had similar inhibitory potencies toward both enzymes. In conclusion, the expression of the C-terminal domain in trans led to the N-and C-terminal domains of APA becoming associated soon after biosynthesis, restoring the folding, processing, plasma membrane expression, and activity of FLAG-N-APA and HA-C-APA, by incubating cell lysates three times for 4 h each on an anti-HA column and measuring APA enzymatic activity (Fig. 6D). We found that the immunodepletion of co-transfected CHO cell extracts did not affect the activity of the N-terminal domain after complete dissociation from the C-terminal domain, as shown by comparison with the original extracts containing N-APA⅐C-APA complexes. The disappearance of the C-terminal domains was assessed by anti-HA immunoblotting of the samples (not shown) and beads (Fig. 6D). We cannot completely rule out the possibility that the lysate still contains some C-terminal domain that cannot be detected by Western blot analysis but that could be sufficient for activating the N-terminal domain. It should, however, be emphasized that to dismiss this possibility we have performed three rounds of immunodepletion and that the level of the N-terminal domains remains constant after the first one. Thus, the C-terminal domain was required only for APA activation, acting as an intramolecular chaperone ensuring the correct folding of the N-terminal zinc metallopeptidase domain, but not for the enzymatic activity.
Interaction of the N-terminal Zinc Metallopeptidase Domain of APA and Calnexin-Given that the N-terminal monozinc metallopeptidase domain expressed alone is retained in the ER, we examined its interaction with calnexin, a molecular chaperone involved in the proper folding of proteins exiting the from culture medium and cell lysates with an anti-HA mAb and, then were resolved by SDS-PAGE. C, His 6 -N-APA co-expressed with HA-C-APA was purified on a Talon column. Immunoblotting of the purification eluate was performed with the anti-Xpress mAb and anti-HA Ab. D, CHO cells were co-transfected with the FLAG-N-APA and HA-C-APA constructs. Cell lysates were subjected to three rounds of incubation with anti-HA mAb immobilized on protein A-Sepharose. The presence of HA-C-APA in the immunoprecipitates was monitored by immunoblotting, using an anti-HA mAb. The APA activity of FLAG-N-APA was compared in native cell lysates (S0), or cell lysates resulting from one (S1), two (S2), or three (S3) incubations. Activity measurements were performed on equivalent quantities of FLAG-N-APA and in the presence of bestatin, to prevent nonspecific hydrolysis of the substrate, and in the presence or absence of the APA inhibitor glutamate phosphonate, to confirm that hydrolysis was indeed due to APA activity. Activity is expressed as a percentage of APA activity in S0. Results are representative of four separate experiments.   (47). Expression of FLAG-N-APA alone in AtT-20 cells, followed by immunoprecipitation with an anti-calnexin Ab, resulted in the detection of FLAG-N-APA with an anti-FLAG mAb, as a band with an apparent molecular mass of 85 kDa (Fig. 7C). This band corresponded to the immature form of FLAG N-APA, which remained in the ER and, as demonstrated by these data, interacted with the ER chaperone calnexin. When AtT-20 cells were co-transfected with FLAG-N-APA and HA-C-APA, the immunoprecipitation of cell lysates with an anticalnexin Ab, followed by immunoblotting with an anti-FLAG mAb did not result in the detection of FLAG-N-APA. The same behavior of N-APA was observed in pulse-chase experiments. When expressed alone, FLAG-N-APA was associated to calnexin after 30 min of pulse (Fig. 7D), and the two proteins were still associated after 2 h of chase (Fig. 7D). When co-expressed with HA-C-APA, the N-terminal domain of APA was associated to calnexin early after biosynthesis, but was rapidly dissociated from calnexin, as shown by the absence of interaction between the two proteins after 2 h of chase (Fig. 7D). Thus, in the presence of C-APA, N-APA dissociates from calnexin in the ER, and the C-terminal domain of APA allows correct folding of the N-terminal zinc metallopeptidase domain of APA via an association of the two domains. This confirms that the C-terminal domain of APA acts as an intramolecular chaperone.
Is the Role of the C-terminal Domain of APA Extendable to Other Monozinc Aminopeptidases?-We studied if the role of intramolecular chaperone of the C-terminal domain of APA could be extended to other membrane-bound monozinc aminopeptidases. To this end, we generated constructs corresponding to the FLAG-tagged N-terminal domain of APN from residues 1 to 606 and to the HA-tagged C-terminal domain of APN from residues 607 to 966. We studied the maturation of FLAG-N-APN, HA-C-APN expressed alone, and FLAG-N-APN coexpressed with HA-C-APN. For this purpose, we performed pulse-chase experiments on transiently transfected CHO cells. FLAG-N-APN, when expressed alone, was detected only as an 85-kDa immunoprecipitated band, even after 3 h of chase (Fig.  8A). HA-C-APN was detected in the cell lysate as a 40-kDa protein after the pulse and was progressively secreted as a higher molecular mass form of 50 kDa (Fig. 8B). When co- FIG. 7. Expression in trans of HA-C-APA rescues its subcellular localization in AtT-20 cells and releases FLAG-N-APA from calnexin. A, AtT-20 cells transiently expressing either FLAG-N-APA or HA-C-APA were fixed in cold methanol. FLAG-N-APA was immunolocalized with anti-FLAG mAb, and HA-C-APA was immunolocalized with anti-HA Ab. FLAG-N-APA (red) was localized within the cell, and HA-C-APA (green) displayed vesicular labeling, with accumulation in the cell processes. B, AtT-20 cells transiently co-expressing FLAG-N-APA and HA-C-APA were fixed in cold methanol. FLAG-N-APA and HA-C-APA were immunolocalized as described above. FLAG-N-APA (red) and HA-C-APA (green) were co-localized at the plasma membrane. Bar ϭ 20 m. C, AtT-20 cells were transiently transfected with the FLAG-N-APA construct, either alone or with HA-C-APA. Proteins were immunoprecipitated from cell lysates with anticalnexin Ab. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting with anti-FLAG mAb. Cell lysates were also subjected to anticalnexin immunoblotting and anti-FLAG immunoprecipitation followed by anti-FLAG immunoblotting, to check the amount of each protein. D, AtT-20 cells were transiently transfected with the FLAG-N-APA construct, either alone or with HA-C-APA and were labeled for 30 min with [ 35 S]methionine/cysteine and subjected to a 2-h chase period with Opti-MEM medium. Cell lysates were divided into two equal aliquots for immunoprecipitation with anti-FLAG mAb or anticalnexin Ab. The immunoprecipitates were analyzed by SDS-PAGE and phosphorimaging.
expressed with the C-terminal domain, the N-terminal domain, immunoprecipitated with an anti-FLAG mAb, was detected as an 85-kDa form after a 30-min pulse. A second band, 100 kDa in size was detected after 60 min of chase (Fig. 8C). Thus, co-expression of these two proteins resulted in the production of a high molecular mass form of 100 kDa of FLAG-N-APN.
Confocal microscopy analysis of AtT-20 cells (Fig. 8D) or CHO cells (not shown) expressing FLAG-N-APN showed the retention of this protein in the ER. In AtT-20 cells (Fig. 8D) or in CHO cells (not shown), HA-C-APA expressed alone was present in vesicles and accumulated in the cell process. In cells expressing both the N-and C-terminal domains of APN, both domains were often co-localized at the plasma membrane (Fig. 8E).
Data from metabolic labeling and immunofluorescence experiments strongly suggest that the C-terminal domain of APN has the same function as the C-terminal domain of APA and promotes maturation and cell surface expression of the Nterminal domain of APN. These results suggest that membrane-bound aminopeptidases share a common structural organization with a C-terminal domain acting as an intramolecular chaperone responsible for the activation of their cognate N-terminal zinc metallopeptidase domain.
Concluding Remarks-The C-terminal domain of APA fulfills all the requirements for an intramolecular chaperone. We show here that activation can take place in trans in eukaryotic cells for two members of the monozinc aminopeptidase family. These data provide, to our knowledge, the first evidence for the presence of an intramolecular chaperone domain in a mammalian exopeptidase. Furthermore, all the endoproteases for which intramolecular chaperone activity has been reported, whether cytosolic or membrane-associated, share a common structure, with an N-terminal prodomain. The recent expression in trans of the C-terminal domain of sucrase-isomaltase provided evidence that sucrase acts as an intramolecular chaperone for this enzyme (45). The data presented here further demonstrate that the C-terminal domain of APA acts as an intramolecular chaperone that is absolutely required for APA activation, but not involved in the enzymatic activity of APA.