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Gamete Biology |
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, 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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Newly synthesized proteins translated in the endoplasmic reticulum (ER) receive posttranslational modification (e.g., the removal of signal sequence, the formation of disulfide bonds, glycosylation, and proteolytic cleavage) before maturation. Among such modifications, it was found that proteolytic cleavage of the precursor protein was achieved by the specific endoprotease, generally referred to as proprotein convertase (PC), which is homologous to yeast subtilisin/kexin [6, 7]. Seven mammalian subtilisin/kexin-like PCs responsible for intracellular cleavage have been identified, including furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/8 (the latter is also referred to as LPC) [6, 8]. Among them, furin is ubiquitously expressed in all tissues and cell lines examined so far [7, 9]. Furin is mainly localized in the trans-Golgi network [10]. Because of its widespread expression, it has been suggested that furin might be responsible for the proteolytic processing of a broad range of proteins.
Individual PCs exhibit overlapping but distinct substrate specificities [11, 12]. It has been reported that the substrate for furin possesses a conserved consensus amino acid sequence, R-X-K/R-R [7, 13], and that this cleavage site is present near the C terminus of the deduced amino acid sequence of the cDNA of quail ZPC (R359-F-R-R362). Thus furin is a good candidate involved in the processing of the newly synthesized ZPC (proZPC) in quail. Previously, we reported that proZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion [14].
In the present study, we examined the role of the proteolytic cleavage of the proZPC on the secretion of the mature ZPC. To achieve this goal, we established an appropriate expression system for quail ZPC, using a mammalian cell line, and produced the site-directed mutant of ZPC, which encodes the mutated consensus furin cleavage site with the noncleavable form. Results of the present investigation strongly suggest that the C-terminal proteolytic processing of proZPC is a prerequisite event for quail ZPC secretion, and the absence of this cleavage results in the accumulation of proZPC in the ER.
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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 were 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 (PL) and the basal laminae, as previously described [15]. 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 of 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 the culture, the medium was recovered, and the granulosa cells were isolated by the procedure of 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).
ZPC Expression Constructs
Total RNA was extracted from the granulosa cells with a commercial kit, ISOGEN (Nippon Gene, Tokyo, Japan), according to the manufacturer's instructions. The first-strand cDNA was synthesized from 0.5 µg of total RNA using the Super Script First-Strand Synthesis System for the RT-PCR kit (Gibco BRL, Rockville, MD) with oligo(dT)-primed reverse transcription. Quail ZPC cDNA was amplified by PCR (cycling conditions: 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min for 30 cycles) in order to introduce HindIII and XbaI sites upstream of the initiator methionine and downstream of the chain termination codons, respectively. The sense and antisense primers used were 5'-AAAAAAGCTTAGGATGCAAGGCAGCTGCG-3' and 5'-AAAATCTAGATCACGCCGCAACCGAGGTTC-3', respectively. The PCR product containing the full-length quail ZPC cDNA 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, qZPC, was transformed into competent Escherichia coli, strain DH5
(Takara, Osaka, Japan), and the ampicillin-resistant clone containing qZPC was selected after the nucleotide sequence analysis was performed.
The site-directed mutant at the consensus furin cleavage site was generated with a Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) on qZPC using a sense primer (5'-CACGGCCGCCGCTTCGCCGCGGATGCCGGGAAAGAA-3') and an antisense primer (5'-TTCTTTCCCGGCATCCGCGGCGAAGCGGCGGCCGTG-3'), as suggested by the manufacturer's protocol. The resulting construct, qZPC-RFAA, which encoded the mutated consensus furin cleavage site (R359-F-R-R362) with the noncleavable form (R359-F-A-A362), was transformed into DH5. The mutation was verified by DNA sequencing.
The location of the signal peptide and its putative cleavage site in the ZPC amino acid sequence were presumed to be those suggested by the procedure of von Heijne [16] using PSORT Prediction (available on the World Wide Web at http://psort.nibb.ac.jp/form.html). The zona pellucida domain (ZP domain) and the putative transmembrane domain were detected by using the protein families database of alignments and HMMs (available at http://www.sanger.ac.uk/Software/Pfam/search.shtml).
Expression of Recombinant ZPC in the Mammalian Cell Line
Chinese hamster ovary cells (CHO-K1 cells; generously provided by Dr. Kazuhiko Imakawa, Department of Animal Breeding, University of Tokyo, Tokyo, Japan) were cultured in Ham's 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. Cells were passaged with trypsin every 3–4 days and revived periodically from frozen stocks. Before transfection, cells were plated onto 6-well culture plates (Falcon Plastics, Los Angeles, CA) and were cultured for 48 h until the cells had grown to approximately 75% of confluence. DNA (2 µg) was introduced into CHO-K1 cells by using Lipofectamine Plus Reagent (Gibco BRL) as suggested by the manufacturer, and the cells were cultured in fresh medium (3 ml/well) for an additional 48 h for expressing recombinant ZPC. After the culture period, the conditioned medium was collected and centrifuged at 10 000 x g for 10 min to remove the cellular debris. Cells were washed twice with PBS (pH 7.4) and were scraped from the plates into SDS-Tris (500 µl/well) and dissolved 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.
Electrophoresis and Western Blot Analysis
SDS/PAGE under nonreducing conditions was carried out as described previously [17] using 12% and 5% polyacrylamide gels for resolving and stacking, respectively.
For Western blot analysis, proteins separated on SDS/PAGE were transferred to PVDF membranes (Immobilon-P, Millipore, Bedford, MA) [18]. The membranes were reacted with rabbit anti-ZPC antiserum (1:2000) [19] or rabbit anti-proZPC-derived peptide antiserum (1:1000) [14] and were visualized by means of a chemiluminescent technique (Amersham Pharmacia Biotech, Piscataway, NJ) using horseradish peroxidase-conjugated anti-rabbit IgG (Cappel, Durham, NC) as a secondary antibody.
Immunofluorescence Microscopy
The CHO-K1 cells transfected with qZPC or qZPC-RFAA were cultured for 48 h on 0.1% gelatin-coated coverslips and were fixed in 3.7% formaldehyde in PBS at room temperature for 30 min; they were 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 were incubated with rabbit anti-proZPC-derived peptide antiserum (1:100); mouse anti-KDEL antibody raised against the ER (1:100; StressGen Biotech, San Diego, CA) [20]; mouse anti-syntaxin 6 antibody, which specifically recognizes the Golgi apparatus (1:100, StressGen Biotech) [21]; or normal rabbit serum (1:200) for 2 h at room temperature. After several washings with PBS, the specimens were reacted with FITC-conjugated goat anti-rabbit IgG (1:200, Cappel) in the presence or absence of rhodamine-conjugated goat anti-mouse IgG (1:200, Cappel) for 1 h. After several washings with PBS, the samples were embedded in glycerol and were examined under a laser scanning microscope with an HeNe laser (543 nm) and an argon laser (488 nm) for multicolor fluorescence (LSM410, Carl Zeiss, Oberkochen, Germany). Granulosa layers cultured with RVKR (0 or 30 µM) for 6 h were also fixed and reacted with the antibodies, as in the procedure with the CHO-K1 cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of DNA sequencing of qZPC and its deduced amino acid sequence are shown in Figure 1. The qZPC cDNA (1314 bp) encoded a polypeptide of 437 amino acids, including a signal sequence (1–20), a ZP domain (57–316), a consensus furin cleavage site (359–362), and a putative transmembrane domain (407–423). A single consensus site for N-glycosylation was also identified (N at position 159). The nucleotide sequence of qZPC revealed 15 variations in sequence that differed from the previously deposited sequence for quail ZPC (GenBank accession AB012606). These changes in sequence led to the substitution of an amino acid sequence (L to V at position 102), and we deposited this sequence in the database (GenBank accession AB081506).
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Western blot analyses of the anti-ZPC antiserum of the culture medium and of the SDS-solubilized CHO-K1 cells were performed to compare the native ZPC from granulosa cells and the recombinant ZPC. The results are shown in Figure 2A. The conditioned medium from the CHO-K1 cells transfected with qZPC contained a 35-kDa immunoreactive ZPC band (lane 2) that migrated to the same position as that of the native ZPC secreted from the granulosa cells (lane 3). The lysates of the CHO-K1 cells were shown to contain three immunoreactive bands of 35-kDa ZPC and 43- and 94-kDa proZPC (lane 5). The SDS/PAGE mobility of these bands corresponded to that of the respective bands of native ZPC from granulosa cells (lane 6). Both the medium and the lysates from the cells into which the vector alone had been introduced did not contain any immunoreactive bands (lanes 1 and 4). These results demonstrate that quail ZPC is expressed in CHO-K1 cells and that it appears to receive similar posttranslational modifications as those of the native ZPC.
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The same samples in Figure 2A were probed with anti-proZPC-derived peptide antiserum raised against the tetradeca peptide located on the C terminus of ZPC (Fig. 1). As shown in Figure 2B, this antibody recognized the 43- and the 94-kDa proZPC band (lanes 5 and 6), whereas it reacted with neither the secreted (lanes 2 and 3) nor the cellular (lanes 5 and 6) 35-kDa ZPC from either the granulosa or the CHO-K1 cells. In addition, anti-proZPC-derived peptide antiserum revealed the presence of a 12-kDa cleaved peptide that was derived from the processing of proZPC [14] in CHO-K1 cells (lane 5). This band migrated to the same position on SDS/PAGE as that observed in the case of the granulosa cell lysates (lane 6). These results suggest that the C-terminal proteolytic processing of proZPC in CHO-K1 cells occurs prior to secretion, which is consistent with a previous finding in quail granulosa cells [14].
Effects of RVKR on ZPC Secretion and Proteolytic Processing
Next, we considered the effects of RVKR, a specific inhibitor of furin, in order to assess the possibility that this endoprotease plays a role in the processing and secretion of ZPC. Granulosa layers were cultured with increasing concentrations of RVKR (0, 0.3, 1, 3, 10, and 30 µM), and the media and the cell lysates were subjected to Western blot analysis. Although an intense band of 35-kDa ZPC was observed in the medium without an inhibitor, decreased intensity was detected in the medium supplemented with RVKR in a dose-dependent manner (Fig. 3A). The addition of 30 µM RVKR completely abolished ZPC secretion. In contrast, an increase in the intensity of the 43-kDa proZPC band in the cell lysates was observed by the addition of more than 1 µM RVKR (Fig. 3B). Furthermore, Western blot analysis of anti-proZPC-derived peptide antiserum showed that the intensity of the 12-kDa band decreased by the addition of 3 µM RVKR, whereas the intensity of the 43-kDa proZPC band increased (Fig. 3C). These results clearly demonstrated that this compound efficiently inhibits the proZPC conversion.
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Mutation in the Consensus Furin Cleavage Site Inhibits ZPC Secretion
We next investigated the potential roles of C-terminal proteolytic processing in the secretion of quail ZPC. To achieve this goal, we produced a site-directed mutant (qZPC-RFAA) that possessed a mutated consensus furin cleavage sequence with a noncleavable form (R359-F-A-A362); this mutant was then introduced into CHO-K1 cells. Although the cells expressing wild-type ZPC secreted significant levels of 35-kDa ZPC (Fig. 4A, lane 3), the trace amount of immunoreactive band in the medium were detected when the cells were transfected with the qZPC-RFAA construct (Fig. 4A, lane 5). ZPC secretion from the qZPC-expressing cells was inhibited by the addition of 30 µM RVKR (Fig. 4A, lane 4). These results indicate that the elimination of convertase cleavage attenuates, but does not completely inhibit, ZPC secretion. Although the cells carrying qZPC-RFAA contained a large amount of 43-kDa proZPC, we did not detect 35-kDa ZPC in the lysates (Fig. 4B, lane 5). This result demonstrated that the mutated form (i.e., lacking the consensus furin cleavage site) did not undergo C-terminal proteolytic processing in CHO-K1 cells.
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Intracellular Localization of ProZPC
In order to examine the intracellular location of proZPC in the cultured cells, qZPC- or qZPC-RFAA-transfected CHO-K1 cells were stained with anti-proZPC-derived peptide antiserum in combination with an antibody directed against the ER (anti-KDEL antibody). These cells were then observed by laser scanning confocal microscopy. In our transient transfection procedure, as little as 10% of the total cells were stained with anti-proZPC-derived peptide antiserum after the culture (data not shown). As shown in Figure 5C, immunoreactive materials with anti-KDEL antibody and anti-proZPC-derived peptide antiserum colocalized in the region that surrounded the nucleus in the qZPC expressing cells (arrowhead). The fluorescence derived from anti-syntaxin 6 antibody raised against the Golgi apparatus was localized in the perinuclear region (Fig. 5B), in a pattern completely different from the staining pattern seen with the anti-KDEL antibody (Fig. 5A). These results indicated that the proZPC in these cells was mainly associated with the ER. Furthermore, in the case of qZPC-RFAA-transfected cells, the staining pattern did not change; that is, the mutated proZPC also accumulated in the ER of the qZPC-RFAA-positive cells (Fig. 5D, arrowhead). Taken together, these results indicated that the unprocessed form of ZPC accumulated in the ER, and that proteolytic processing was able to take place after the ZPC had been transported out of the ER.
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Next, we performed similar experiments using cultured granulosa cells in order to examine whether or not the native proZPC was also localized in the ER. As expected, fluorescence from both anti-KDEL and anti-proZPC-derived peptide antiserum corresponded to the perinuclear region (Fig. 6B), and the treatment of RVKR did not change the intracellular distribution of proZPC (Fig. 6C). These results indicated that proZPC in the granulosa cells was also localized in the ER. No positive immunostaining was seen when the cells were stained with normal rabbit serum (Fig. 6A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our data showed that the recombinant quail ZPC evidently received the C-terminal proteolytic processing in CHO-K1 cells, and the apparent molecular weight of the final product on SDS/PAGE corresponded to that of native ZPC from granulosa cells. These results indicate that the recombinant ZPC received similar posttranslational modification such as sugar moiety modification, disulfide bond formation, as well as the C-terminal proteolytic processing of native ZPC. Because of these appropriate intracellular modifications in the cells, our expression system provides a beneficial model for studying the mechanism and the role of posttranslational modification in ZPC biosynthesis.
Expression experiments with qZPC-RFAA suggest that proteolytic cleavage at a consensus furin cleavage site is required for quail ZPC secretion in CHO-K1 cells (Fig. 4). Furthermore, the inhibitor for furin, RVKR, efficiently blocked the proteolytic processing of proZPC and the accumulation of proZPC and the elimination of a proZPC-derived 12-kDa peptide in the cell lysates (Fig. 3). It is reported that RVKR binds covalently to the substrate binding site of furin and inhibits its activity [22]. Although the expression of furin in quail granulosa cells was not demonstrated, Hatsuzawa et al. [9] detected the presence of mRNA for furin in CHO-K1 cells by Northern blot analysis. Taken collectively, these results suggest the involvement of furin in the proteolytic processing and secretion of proZPC in quail granulosa cells.
Consistent with our results, it has recently been reported that the excision of the C-terminal region of mouse ZPC at a consensus furin cleavage site is required for the secretion and the subsequent assembly into zona pellucida [23, 24]. In contrast, Zhao et al. [25] reported that mutation of the furin site to a noncleavable form does not affect the intracellular trafficking or secretion of mouse ZPC fused with enhanced green fluorescent protein (GFP) in embryonic fibroblast cells. The reason for this discrepancy might be, at least in part, due to the different epitope-tags introduced to the recombinant protein (i.e., Myc and Flag vs. GFP) or of the different cell lines employed for the transfection studies (i.e., embryonic carcinoma cells vs. embryonic fibroblast cells).
Although the reason why the unprocessed quail ZPC was not secreted from the cells has not yet been investigated, immunocytochemical observations suggest that proZPC is present in the ER of both granulosa and CHO-K1 cells. In addition, the proZPC produced in the presence of RVKR or those produced by the furin-resistant mutant were accumulated in the ER. The association of proZPC in the ER may account for the following two possibilities: 1) the unprocessed proZPC was unable to exit the ER or 2) proZPC is able to exit the ER, enabling it to enter the Golgi apparatus, but there is a mechanism to retrieve the unprocessed form back to the ER. In mice, it has been reported that the predominant intracellular form of ZPC is localized in the ER, suggesting that the exit from the ER might be the rate-limiting step in secretion, and if the C-terminal proteolytic processing were inhibited, ZPC would be retained in the ER [23]. On the other hand, Kiefer and Saling [26] reported that although RVKR prevented the proteolytic processing of human ZPC, the ZPC received the Golgi-derived O-linked glycosylation. Our results from the granulosa cell culture demonstrated that RVKR prevented the processing of proZPC, whereas a slightly retarded mobility shift of this uncleaved species on SDS/PAGE was observed by close inspection of the gel (Fig. 3C, lanes 3–10 µM). This shift might have reflected that the unprocessed proZPC indeed was transported to the Golgi apparatus and received the Golgi-derived modification, as in the case with human ZPC. We think, therefore, that proZPC is able to exit the ER, but that it is unsuccessfully processed, which causes the retrograde transport of proZPC from the Golgi back to the ER. It is reported that both the cytoplasmic domain and the transmembrane domain of a protein play critical roles in the protein transport between the ER and the Golgi apparatus [27, 28]. The cytoplasmic and the putative transmembrane domains are also found downstream of the consensus furin cleavage site in the quail ZPC polypeptide (Fig. 1). Currently, searches for such signals of ZPC transportation are underway, using mutation analyses of the cytoplasmic and the transmembrane domains of quail ZPC.
Immunocytochemical observations further indicate the possibility that the proteolytic processing occurs after proZPC exits from the ER, because the unprocessed species have been observed as mainly localized in the ER. This conclusion gains support from our previous observations that newly synthesized proZPC accumulated in the ER by inhibition of protein transport from the ER to the Golgi apparatus by brefeldin A. Moreover, 35-kDa ZPC and the 12-kDa fragment generated by the proteolytic processing of proZPC both accumulated in the cells by inhibition of the protein transport from the Golgi apparatus by monensin [14].
Western blot analysis demonstrated that the 94-kDa band reacted with both anti-ZPC antiserum and anti-proZPC-derived peptide antiserum in either CHO-K1 and granulosa cell lysates. This protein migrated to the same position as that observed with 43-kDa proZPC on SDS/PAGE under reducing conditions (data not shown), which indicated the possibility that the 94-kDa protein was an oligomeric intermediate of the 43-kDa proZPC generated during posttranslational modification. In pancreatic ß cells, a high molecular weight immunoreactive band was observed during insulin biosynthesis [29]. This band is regarded as reflective of a proinsulin intermediate leading to insulin conversion. Moreover, a truncated mutant lacking the C terminus of rat intestinal mucin, which is normally cleaved at the consensus furin cleavage site, formed an incorrect dimer in the ER and was not secreted as efficiently as the wild-type mucin [30]. They suggested that the C-terminal peptide of mucin might play a role in ensuring that the dimer is correctly folded in the rat intestine. Although we do not know whether the 94-kDa protein plays a role in ZPC biosynthesis, it is of interest to identify whether the molecule that interacts with 43-kDa proZPC is itself (i.e., a homo-dimer), or if the 43-kDa proZPC interacts with another independent protein (i.e., a hetero-dimer). The identity of the molecule would help clarify the nature of the 94-kDa protein and its potential roles in ZPC biosynthesis.
In conclusion, our study suggested that newly synthesized ZPC is proteolytically cleaved at the consensus furin cleavage site with a furin-like protease, and the failure of this cleavage will result in the accumulation of proZPC in the ER. Therefore, it is thought that the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.
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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: 2 October 2002.
First decision: 25 October 2002.
Accepted: 20 November 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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