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Gamete Biology |
Department of Applied Biological Chemistry,3 Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
The United Graduate School of Agricultural Science,4 Gifu University, Gifu 501-1193, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, gamete biology, granulosa cells, ovary, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nascent proteins translated in the endoplasmic reticulum (ER) receive posttranslational modification (e.g., removal of signal sequence, formation of disulfide bonds, glycosylation, and proteolytic cleavage) before maturation. Recent studies from several laboratories have shown that newly synthesized ZPC (proZPC) is cleaved at the consensus furin cleavage site (R-X-K/R-R) [10, 11] to generate mature ZPC prior to secretion in mice [12] and humans [13]. We also demonstrated that the proteolytic cleavage of the 43-kDa proZPC occurs at the consensus furin cleavage site, R359-F-R-R362, to generate the 35-kDa mature ZPC before secretion in quail [14]. In addition to these observations, it has been reported that the secretion of ZP3 (ZPC) and the subsequent assembly into the zona pellucida in mice require C-terminal proteolytic cleavage at a consensus furin cleavage site [15, 16]. On the contrary, Zhao et al. [17] demonstrated that the proteolytic processing at a consensus furin cleavage site of mouse ZP3 (ZPC) is not essential for either secretion or incorporation into the zona pellucida. To clarify the circumstances in quail, we examined the role of the proteolytic cleavage and the N-glycosylation of proZPC in the secretion of the mature ZPC by using site-directed mutagenesis of the furin cleavage site [18], or the N-glycosylation site [19]. The results of these investigations strongly suggest that the C-terminal proteolytic processing of proZPC is indispensable, but the absence of N-linked oligosaccharides does not affect quail ZPC secretion. Furthermore, the analysis of the nucleotide and the deduced amino acid sequences of quail ZPC (GenBank accession AB081506) revealed that the C-terminal domain that is removed after the proteolytic processing of proZPC contains the putative transmembrane domain (407–423) with a short cytoplasmic tail (424–437). On the basis of these observations, it is possible to hypothesize that these regions play a role in quail ZPC biosynthesis.
In the present study, we examined the role of the cytoplasmic tail in the membrane transport, the proteolytic processing, and the secretion of quail ZPC to extend our previous finding regarding intracellular ZPC trafficking in the cells. To achieve this, we produced a truncated mutant of ZPC lacking the cytoplasmic tail located in its C-terminus and expressing in the mammalian cell line. Our results in the present study suggest that this mutant could not exit from the ER and that this phenomenon leads to the failure of the later events that normally take place after proZPC is transported from the ER to the Golgi apparatus (i.e., the proteolytic processing and secretion of ZPC). Therefore, it is thought that the cytoplasmic tail of quail ZPC might play a role in the efficient transport of proZPC from the ER to the Golgi apparatus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Japanese quail, Coturnix japonica, 15–30 wk of age (Tokai-Yuki, Toyohashi, Japan), were maintained individually under a photoperiod of 14L:10D (with the light on at 0500 h) and provided with water and a commercial diet (Tokai-Kigyo, Toyohashi, Japan) ad libitum. Animals were decapitated, and the largest preovulatory follicles were dissected and placed in physiological saline. The granulosa layer was isolated as a sheet of granulosa cells sandwiched between the perivitelline membrane and the basal laminae, as previously described [20]. All experimental procedures for the use and the care of animals in the present study were approved by the Animal Care Committee of the Faculty of Agriculture, Shizuoka University.
Culture of Granulosa Cells
The isolated granulosa layer was cultured, as described previously [14]. A stock solution of the furin inhibitor, decanoyl-RVKR-chloromethylketone (RVKR, 50 mM; Bachem, King of Prussia, PA) was prepared in dimethylsulfoxide (DMSO) and stored at -80°C until use. When RVKR was added to the medium, the DMSO concentration never exceeded 0.1%. After culturing, the medium was recovered, and the granulosa cells were isolated by the procedure described by Sasanami et al. [14]. The isolated granulosa cells were dissolved in 1% SDS buffered at pH 6.8 with 70 mM Tris-HCl (SDS-Tris) with constant shaking at room temperature. Insoluble materials were precipitated by centrifugation at 10 000 x g for 10 min. The supernatants were served as total cell lysates. The protein concentration in each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL).
Generation of ZPC Expression Constructs
ZPC expression constructs containing the full-length quail ZPC cDNA (Fig. 1, qZPC) or the site-directed mutant at the consensus furin cleavage site (Fig. 1, qZPC-RFAA) were generated, as described previously [18]. The quail ZPC mutant lacking the cytoplasmic tail (from 424 to 437 of the deduced amino acid sequence of quail ZPC, Fig. 1, qZPC-
-CT) was generated by polymerase chain reaction (PCR) amplification of qZPC using a sense primer (5'-AAAAAAGCTTAGGATGCAAGGCAGCTGCG-3') and an antisense primer (5'-AAAATCTAGATCACACAGCCACCCCAACGG-3') (cycling conditions: 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min for 30 cycles) to introduce the chain termination codon at the upstream of the cytoplasmic tail. The PCR product was digested with HindIII and XbaI and ligated into mammalian expression plasmid vector pcDNA3.1(+) (Invitrogen, San Diego, CA) treated with the same restriction enzymes. The resulting construct was transformed into competent Escherichia coli, strain DH5
(Takara, Osaka, Japan), and the ampicillin-resistant clone containing qZPC--CT was selected after nucleotide sequence analysis was performed. The mutation was verified by DNA sequencing.
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Transient Expression of ZPC Constructs by CHO-K1 Cells
Chinese hamster ovary cells (CHO-K1 cells; generously provided by Dr. Kazuhiko Imakawa, Department of Animal Breeding, University of Tokyo, Tokyo, Japan) were grown in a Petri dish in Hams F-12 (Dainippon Seiyaku, Osaka, Japan) supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere with 5% CO2. Two micrograms of plasmid DNA (qZPC, qZPC--CT, or qZPC-RFAA) were introduced into CHO-K1 cells as described previously [18, 19]. The cells were then cultured in fresh medium (3 ml/well) for 48 h for expressing recombinant ZPC. After the culture, the conditioned medium was collected and centrifuged at 10 000 x g for 10 min to remove cellular debris. Cells were washed twice with PBS (pH 7.4) and scraped from the plates into SDS-Tris (500 µl/well) and solubilized by vigorous shaking. After centrifugation at 10 000 x g for 10 min, clear supernatants were stored as the total cell lysates. The protein concentration was determined as described above.
Endoglycosidase H Digestion
The culture medium and cell lysates from the CHO-K1 cells that had been transfected with qZPC, qZPC--CT, or qZPC-RFAA were digested with endoglycosidase H (Endo H, New England Biolabs, Beverly, MA) according to the manufacturer's instructions. Briefly, the culture medium (50 µl; 1/60 of the total culture medium) and the cell lysate (1/10 of the total cell lysates; approximately 15 µg of protein) mixed with 1/10 volume of denaturing buffer containing 5% SDS and 10% ß-mercaptoethanol were boiled for 15 min, and 1/10 volume of 10 x G5 buffer (0.5 M sodium citrate, pH 5.5) was added. The mixture was incubated in the presence or absence of 500 U of Endo H at 37°C for 3 h. The reaction was terminated by boiling the sample for 10 min. The conditioned medium (50 µl; 1/20 of the total culture medium) and the cell lysates (1/10 of the total cell lysates; approximately 3 µg of protein) from the granulosa cell culture with RVKR (0 or 30 µM) were treated using the procedure as that regarding CHO-K1 cells.
Electrophoresis and Western Blot Analysis
One-dimensional SDS-PAGE under a nonreducing or a reducing condition was carried out as described previously [21], using 12% and 5% polyacrylamide for resolving and stacking gels, respectively. To confirm the expression of each construct in CHO-K1 cells, the conditioned medium (8 µl) or the cell lysate (5 µg of protein) were separated on SDS-PAGE under a nonreducing condition. For the analysis of Endo H digests, 1/10 of each reaction from granulosa cell culture (equivalent to 1/200 of the total medium, and 1/100 of the total cell lysates; 0.3 µg of protein) was applied onto the gels. In the case of the CHO-K1 cells culture, one fifth of these reactions (corresponding to 1/50 of the total cell lysates; 3 µg of protein) were loaded. They were then separated on SDS-PAGE under a reducing condition.
For the Western blot analysis, proteins separated on SDS-PAGE were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) [22]. The membranes were reacted with rabbit anti-ZPC antiserum (1:2000) [23] or rabbit anti-proZPC-derived peptide antiserum (1:1000, see Fig. 1) [14] and visualized using a chemiluminescent technique (Amersham Pharmacia Biotech, Piscataway, NJ) employing horseradish peroxidase-conjugated anti-rabbit IgG (Cappel, Durham, NC) as a secondary antibody.
Immunofluorescence Microscopy
The CHO-K1 cells transfected with qZPC--CT were cultured for 48 h on 0.1% gelatin-coated coverslips and fixed in 3.7% formaldehyde in PBS at room temperature for 30 min and then permeabilized in cold acetone (-20°C) for 5 min. After being washed with PBS, the cells were incubated with PBS containing 3% gelatin for 1 h and then incubated with rabbit anti-proZPC-derived peptide antiserum (1:100) or mouse anti-KDEL antibody raised against the ER (1:100, StressGen Biotech, San Diego, CA) [24] for 2 h at 4°C. After several washings with PBS, the specimens were reacted with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:50, Cappel) in combination with Texas red-conjugated sheep anti-mouse IgG (1:50, Cappel) for 1 h at 4°C. After several washings with PBS, the samples were embedded in glycerol and examined under a laser-scanning microscope equipped with an HeNe laser (543 nm) and argon laser (488 nm) to determine multicolor fluorescence (LSM410, Carl Zeiss, Oberkochen, Germany).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the potential roles of the cytoplasmic tail in the biosynthesis of quail ZPC, qZPC--CT and qZPC-RFAA were introduced into CHO-K1 cells, and Western blot analyses using the anti-ZPC antiserum were performed to examine the expression, proteolytic processing, and secretion of recombinant ZPCs. The results are shown in Figure 2. As reported previously [18, 19], the conditioned medium from the CHO-K1 cells transfected with qZPC construct contained a 35-kDa immunoreactive ZPC band (Fig. 2A, lane 2). The lysates of these cells were shown to contain three immunoreactive bands of 35-kDa ZPC and 43- and 94-kDa proZPC (Fig. 2B, lane 2). On the other hand, a trace amount of immunoreactive band in the medium was detected when the cells were transfected with qZPC--CT construct (Fig. 2A, lane 3) as in the case of the cells carrying qZPC-RFAA (Fig. 2A, lane 4). In comparison with that of the qZPC-expressing cells, the faster mobility shift of the proZPC bands (41 and 90 kDa) that tended to decrease in intensity were observed in the qZPC--CT–transfected cells (Fig. 2B, lane 3). It is not likely that the insubstantial amount of the immunoreactive band in the medium was caused by the failure of the expression of this construct because the cell lysates expressing qZPC--CT contained proZPC. These results suggest that the ZPC variant lacking the cytoplasmic tail is not efficiently secreted by CHO-K1 cells.
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Despite the presence of immunoreactive proZPC in the cells, we did not detect 35-kDa ZPC in the qZPC--CT–expressing cells (Fig. 2B, lane 3). This result demonstrated that the truncated form, i.e., that lacking the cytoplasmic tail, did not undergo C-terminal proteolytic processing in CHO-K1 cells. This argument gains support from the result of Western blot analysis using anti-proZPC-derived peptide antiserum showing the absence of the 12-kDa fragment, a by-product derived from the proteolytic processing of proZPC [14], in the lysate of the qZPC--CT-expressing cells (Fig. 2C, lane 3). On the other hand, both the medium and the lysates from the cells into which the vector alone had been introduced did not contain ZPC bands reacted with neither anti-ZPC antiserum (Fig. 2A, lane 1 and Fig. 2B, lane 1) nor anti-proZPC derived peptide antiserum (Fig. 2C, lane 1).
Endo H Digestion of Native ZPC From Quail Granulosa Cells
From the analysis of the deduced amino acid sequence, quail ZPC possesses one potential N-glycosylation site (N at position 159) in its molecule [18]. To investigate the status of the N-linked oligosaccharide modification of ZPC, we treated the native ZPC with Endo H, which specifically cleaves the high-mannose N-linked oligosaccharides but does not affect the complex oligosaccharides. These were then probed with anti-ZPC antiserum (Fig. 3A), or anti-proZPC–derived peptide antiserum (Fig. 3B). The secreted 35-kDa ZPC band was shifted to migrate to the position at 44 kDa in the molecular mass on the SDS-PAGE gel under the reducing condition (Fig. 3A, panel 1, lane -), which was not reacted with anti-proZPC derived-peptide antiserum (Fig. 3B, panel 1, lane -). This was consistent with the results of a previous study [25] showing the presence of intramolecular disulfide bonds in the 35-kDa ZPC. Treatment of the medium with Endo H did not affect the molecular mass of the secreted ZPC (Fig. 3A, panel 1, lane +). On the other hand, the intracellular proZPC, which was detected as 43- and 94-kDa bands on the SDS-PAGE gel without reducing reagent [14], shifted to the single 52-kDa band under the reducing condition that reacted with both anti-ZPC antiserum (Fig. 3A, panel 2, lane -) and anti-proZPC-derived peptide antiserum (Fig. 3B, panel 2, lane -). This 52-kDa proZPC band was apparently shifted to that with a lower molecular mass by the Endo H digestion, whereas the intracellular 44-kDa ZPC resisted this glycosidase treatment (Fig. 3A, panel 2, lane +). These results demonstrate that the 44-kDa ZPC had passed through the Golgi apparatus and possesses the complex-type oligosaccharide, but proZPC remains in the ER with the high mannose type of sugar moiety. This finding is consistent with our previous observations that proZPC mainly localizes in the ER and receives C-terminal proteolytic processing after exiting from the ER [18]. In contrast, proZPC derived from the granulosa cells cultured with RVKR, a specific inhibitor for furin, displays Endo H resistance (Fig. 3A, panel 3, lane +). This result indicates that the inhibition of the proteolytic cleavage does not interfere with the proZPC transport from the ER to the Golgi apparatus in quail granulosa cells.
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Deletion of Cytoplasmic Tail Affects the Intracellular Trafficking of ProZPC
To investigate the intracellular transport of the mutated ZPCs from ER to Golgi apparatus, we treated the lysates from the qZPC-, qZPC--CT-, or qZPC-RFAA-transfected cells with Endo H. The results are shown in Figure 4. When the mock-transfected cell lysates were digested with Endo H, some bands reacted weakly with our antibodies (data not shown). As in the case of the native ZPC, the qZPC-expressing cells contained either the Endo H-sensitive 52-kDa proZPC or the Endo H-resistant 44-kDa ZPC that reacted with ant-ZPC antiserum (Fig. 4A, panel 1). When the cells were introduced with qZPC-RFAA, the digestion with Endo H of cell lysates leads to the detection of two proZPC bands: the 52-kDa Endo H-resistant- and the 45-kDa Endo H-sensitive forms (Fig. 4A, panel 3, lane +). The presence of the former species of proZPC suggests that the furin-resistant-ZPC mutant that has the intact cytoplasmic tail apparently was transported from the ER to the Golgi apparatus. On the other hand, in the case of the qZPC--CT–expressing cells, we failed to detect the Endo H resistant-form in the cell lysate (Fig. 4A, panel 2, lane +). Taken together, these results suggest that the deletion of the cytoplasmic tail of proZPC, but not the failure of the proteolytic processing, interferes with the intracellular trafficking of proZPC from the ER to the Golgi apparatus in CHO-K1 cells. Similar results were obtained from Western blot analyses using anti-proZPC-derived peptide antiserum (Fig. 4B).
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Intracellular Localization of the ZPC Variant Lacking the Cytoplasmic Tail
To examine the intracellular location of the ZPC variant in the cultured cells, the qZPC--CT-transfected cells were stained with anti-proZPC-derived peptide antiserum in combination with antibody directed against the ER (anti-KDEL antibody). As reported previously [18], when the qZPC-expressing cells were detected using anti-proZPC-derived peptide antiserum, a loose-reticular staining pattern extending from the nucleus was observed and was colocalized with anti-KDEL antibody-derived signals (data not shown). As shown in Figure 5, immunoreactive materials with anti-KDEL antibody were localized to the region that surrounded the nucleus in the qZPC--CT-transfected cells (Fig. 5A). When the same cells were detected with anti-proZPC-derived peptide antiserum, a similar reticular staining pattern was seen in the qZPC--CT-positive cells (Fig. 5B, arrowhead) and was coincident with the fluorescence from the anti-KDEL antibody (Fig. 5C, arrowhead). Taken collectively with the data in Figure 4, these results indicated that the truncated form of ZPC was retained in the ER, and this phenomenon leads to inhibit the following events in the cells, including the sugar moiety modifications, proteolytic processing, and secretion of ZPC. No positive immunostaining was seen when the mock-transfected cells were stained with anti-proZPC-derived peptide antiserum (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data showed that the recombinant quail ZPC expressed in CHO-K1 cells evidently received similar sugar moiety modification as in the case of the native ZPC because the qZPC-expressing cells contained both the Endo H-sensitive proZPC and the Endo H-resistant ZPC. Therefore, our ZPC expression system using CHO-K1 cells is an appropriate tool for further analysis of ZPC biosynthesis.
Protein exit from the ER does not occur through a constitutive "bulk flow" process but is accomplished via a "signal-mediated" event to sort and concentrate into vesicular carriers that are formed by specific machinery, referred to as coat protein (COP) [26]. It is currently thought that the interaction between the specific localization signal located in the cytoplasmic or transmembrane domain and its receptor molecule is important in controlling the export of protein from the ER [27]. Actually, Nakamura et al. [28] reported that the C-terminal leucine and valine of Emp24p, the protein enriched in COP II-coated vesicles, were necessary and sufficient to accelerate the transport from ER to the Golgi apparatus. In other words, C-terminal leucine and valine might act as the "sorting signal" responsible for the Emp24p transport from the ER to the Golgi apparatus. In the case of the yeast Sec12p, a type II transmembrane protein in the ER, the transmembrane domain acts mainly as the retrieval signal from the Golgi apparatus, and the cytoplasmic domain contains the retention signal in the ER [29]. On the other hand, the protein exit from the ER is also retarded if sufficient folding, the oligomerization of the protein, or the core N-glycosylation of the protein in the ER is impeded. These retained proteins tend to be degraded by the complex of events generally termed as the unfolded protein response (UPR) [30]. In light of these facts, the reason the truncated ZPC showed ER retention could be explained by the following two possibilities. First, the determinant responsible for the transportation of proZPC into the Golgi apparatus exists in the cytoplasmic tail, so the qZPC--CT proZPC was not able to be recognized with the putative sorting receptor. Second, the detection of the unfolded-truncated ZPC with a sensor of the UPR caused the retention in the ER lumen.
In our results, the Western blot analysis using either anti-ZPC antiserum or anti-proZPC-derived peptide antiserum demonstrated that the qZPC--CT-expressing cells were shown to contain two immunoreactive proZPC bands of 41 and 90 kDa (Fig. 2, B and C, lane 3). Furthermore, the intracellular 41-kDa and 90-kDa proZPC bands were shifted to migrate to the position at 45 kDa in the molecular mass on SDS-PAGE gel under reducing condition (Fig. 4, A and B, panel 2, lane -). Such analogy between the native and wild-type proZPC with the qZPC--CT proZPC indicates that the cytoplasmic tail-deficient proZPC not only could form the oligomer but also could have a folded conformation similar to that of the native and wild-type proZPC. We think, therefore, that the cytoplasmic tail of proZPC contains the putative "sorting signal" responsible for the transport of proZPC from the ER to the Golgi apparatus, and it seems unlikely that the ER retention of this mutant is due to the action of a UPR mechanism in the ER. This idea is also supported by the present result showing that the qZPC--CT proZPC received the core N-glycosylation because the Endo H digestion apparently increases the mobility of the qZPC--CT proZPC on SDS-PAGE gel (Fig. 4, A and B, panel 2, lane +). Consistent with our results, Linstedt et al. [31] reported that the mutant of the Golgi-resident protein, giantin, lacking a portion of its cytoplasmic domain adjacent to the membrane anchor (transmembrane domain), fails to reach the Golgi apparatus and that even this mutant has a stable folded structure. Further studies are needed to identify such sorting signals in the proZPC molecule using specific mutation analysis of the cytoplasmic domains of quail ZPC.
The proZPC from the cells that were transfected with the site-directed mutant of ZPC (qZPC-RFAA), which encodes the mutated consensus furin cleavage site with the noncleavable form, acquired Endo H resistance (Fig. 4, A and B, panel 3, lane +). In addition, the granulosa cells treated with RVKR were also shown to contain the Endo H-resistant proZPC (Fig. 3, A and B, panel 3, lane +). These results clearly demonstrated that the noncleavable species of proZPC at a consensus furin cleavage site could exit from the ER. However, we found by means of immunocytochemical observations using anti-proZPC-derived peptide antiserum that both the furin-resistant proZPC mutant and RVKR-treated proZPC accumulated in the ER [18]. Superimposed with these results, although the unprocessed proZPC indeed was transported to the Golgi apparatus, the inhibition of the proteolytic processing at a consensus furin cleavage site causes the retrograde transport of proZPC from the Golgi apparatus back to the ER. The reason the noncleavable proZPCs are retrieved from the Golgi apparatus is currently unknown; however, it is reported that the interaction between COP and the specific amino acid sequence (e.g., dilysine motif) of type I transmembrane proteins is important for such selective retrograde transport of the protein from the post-ER compartment back to the ER [23]. The question of whether such retrograde transport is a regulatory mechanism in ZPC biosynthesis remains to be resolved because we could not find a dilysine motif in quail ZPC.
In contrast to our results, Jovine et al. [32] demonstrated that the C-terminal transmembrane domain and the following short cytoplasmic tail of mouse ZP2 and ZP3 are not required for secretion but are essential for polymerization into the zona pellucida. Although the reason for this difference has not yet been elucidated, it is interesting to note that the egg envelope subunit proteins in medaka (Oryzias latipes) and ZP1 protein in quail liver have not been shown to contain such C-terminal regions [33, 34]. Thus, the ZPC biosynthesis in the cells could appear to be regulated differently in different species, although a common ancestor might be suggested for the ZPC homologs of various species [35, 36].
In conclusion, our findings suggest that either the C-terminal proteolytic processing of proZPC or the secretion of ZPC is severely inhibited when the cytoplasmic tail-deficient ZPC mutant was introduced into CHO-K1 cells. This is due to the mutant's inability to exit from the ER, as revealed by the immunofluorescence analysis and the acquisition of resistance to Endo H digestion. Therefore, it is thought that the cytoplasmic tail of quail ZPC might possess the determinant responsible for the efficient transport of proZPC from the ER to the Golgi apparatus. Additional study is needed to identify the targeting signals in the ZPC molecules and characterize the machinery that mediates the intracellular sorting of proZPC from the ER to the Golgi apparatus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Makoto Mori, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. FAX: 81 54 238 4866; acmmori{at}agr.shizuoka.ac.jp
Received: 14 April 2003.
First decision: 1 May 2003.
Accepted: 4 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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