Báo cáo Y học: Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells doc

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells Tomohiro Sasanami 1 , Jianzhi Pan 1 , Yukio Doi 2 , Miki Hisada 3 , Tetsuya Kohsaka 1 , Masaru Toriyama 1 and Makoto Mori 1 1 Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Japan; 2 Department of Food Science, Kyoto Women’s University, Higashiyama, Kyoto, Japan; 3 Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, Japan In avian species, an egg envelope homologous to the mam- malian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthe- sized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granu- losa cells. Western blot analysis of the cell lysates demon- strated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the pres- ence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-ter- minal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC 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. Keywords: zona pellucida; ZPC; granulosa cell; quail; post- translational modification. The plasma membrane of oocytes of all vertebrates is overlaid with extracellular matrix generally called the egg envelope, although different names have been adopted for different classes: zona pellucida for mammals, perivitelline layer (PL) for birds, vitelline envelope for amphibians, and chorion for fish. Mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2, and ZP3 [1], also known as ZPB, ZPA and ZPC, respectively [2]. For most mammalian species and other vertebrates, the homologous proteins are identified in the egg envelope [2–6]. The egg envelope plays a significant role in species- specific sperm–egg interaction. In mice, sperm binds to O-linked oligosaccharides of ZPC, and undergoes the acrosome reaction [7]. In humans and hamsters, ZPC participates in sperm–egg binding, whereas ZPB is the primary sperm binding protein in pigs and rabbits [6,8,9]. All of the zona pellucida glycoproteins in the mouse are synthesized coordinately by the oocytes [10], whereas the granulosa cells also participate in the formation of the zona pellucida proteins in the rabbit [11]. The amphibian vitelline envelope is synthesized by the oocytes [12] while a glyco- protein of fish chorion is produced in the liver and transported to the ovary by blood circulation [13]. Two major glycoproteins have been identified as compo- nents of the inner layer of the vitelline membrane in the avian oviposited eggs, a similar investment 1 to the PL of follicular oocytes before ovulation: 33 kDa and 175 kDa in quail [14] and 32 kDa and 183 kDa in hen [15]. The cDNAs encoding the 33-kDa protein in quail (GenBank Accession Number; AB012606) and the 32-kDa protein in the chicken (GenBank Accession Number; D89097) were cloned, and these proteins were designated as ZPC from the comparison of deduced amino-acid sequences of the known ZPC. Avian ZPC was found to be synthesized in the granulosa cells of the preovulatory follicles [16,17]. Because granulosa cells are arranged on the surface of the oocyte as a single layer of cells, their ZPC production provides a beneficial model for study of the vectorial 2 secretion of the protein. Nascent proteins translated in the rough endoplasmic reticulum (RER) receive post-translational modifications including removal of signal sequence, formation of disulfide bonds, glycosylation, and proteolytic cleavage. The proteo- lytic cleavage of the precursor protein is achieved by digestion by a proprotein convertase, homologous to yeast subtilisin/kexin that cleaves specific basic amino acid residues in the substrates [18,19]. So far, seven mammalian subtilisin/kexin-like proprotein convertases responsible for intracellular cleavages have been described, including furin, PC1 (also called PC3), PC2, PC4, PACE4, PC5 (also called PC6) and PC7 (also called LPC or PC8) [18,20]. PC1 and PC2 are found in the endocrine and neuroendocrine tissues, Correspondence to: M. Mori, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. Fax: + 81 54 2384866; E-mail: acmmori@agr.shizuoka.ac.jp Abbreviations: PL, perivitelline layer; BFA, brefeldin A; RER, rough endoplasmic reticulum; BL, basal laminae; PVDF, poly(vinylidene difluoride). Enzyme: lysylendopeptidase (EC 3.4.21.50). Note: the GenBank accession number of proteins mentioned in the text are: 33-kDa protein in quail (quail ZPC), AB012606; 32-kDa protein in chicken (chicken ZPC), D89097. (Received 23 October 2001, revised 26 February 2002, accepted 12 March 2002) Eur. J. Biochem. 269, 2223–2231 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02880.x and recognize the paired basic amino acids (Arg–Arg or Lys–Arg) in the substrates [18,21–23]. Furin is ubiquitously expressed in all tissues and cell lines examined so far [19,24], and is localized in the trans-Golgi network [25]. The substrates for furin possesses a conserved consensus amino-acid sequence, Arg–X–Lys/Arg–Arg [19,26]. In the present study, we examined the proteolytic cleavage of the newly synthesized ZPC (proZPC) during post-translational modification and the secretion of the mature ZPC from quail granulosa cells. To achieve this, we used two inhibitors that affect the secretory process in the cell: monensin, an inhibitor of intracellular transport of protein at the level of Golgi apparatus [27], and brefeldin A (BFA), a specific inhibitor of membrane transport [28,29]. MATERIALS AND METHODS Animals and tissue preparation Female Japanese quail, Coturnix japonica, 15–30 weeks of age (Tokai-Yuki, Toyohashi, Japan), were main- tained individually under a photoperiod of 14 h light/10 h dark with light-on at 0500, and provided with water and a commercial diet (Tokai-Kigyo, Toyohashi, Japan) ad libitum. Animals were decapitated and the largest preovu- latory follicles were dissected and transferred to a physio- logical saline. The granulosa layer was isolated as a sheet of granulosa cells sandwiched between the PL and the basal laminae (BL) as described previously [30]. Culture of granulosa cells The granulosa layer was cut into 10 pieces, each approxi- mately 8 mm · 8 mm in size. Each piece was placed into one well of a 24-well culture plate (Falcon Plastics) and covered with 1 mL RPMI-1640 medium (Gibco BRL). A stock solution of monensin (10 m M ; Wako Pure Chemicals) and BFA (5 mgÆmL )1 ; Wako Pure Chemicals) was pre- pared in methanol and stored at )80 °C until use. When monensin or BFA was added to the medium, the methanol concentration never exceeded 0.1%. Granulosa layer was cultured at 41 °C in a humidified atmosphere of 5% CO 2 and 95% air. After culture, medium was collected and stored at )20 °C. To separate the granulosa cells and the PL, the granulosa layer was placed into a drop of distilled water (400 lL per piece) and washed with a flush of water from a Pasteur pipette under a dissecting microscope. Isolated PL was confirmed to be free from the granulosa cells by phase contrast microscopy. After removal of the PL and the BL, the residual solution, a mixture of intact granulosa cells and cell debris, was confirmed not to contain the PL and the BL by examination under a dissecting microscope. Electrophoresis and Western blot analysis The PL and the suspension of granulosa cells and cell debris was solubilized in SDS/Tris (1% SDS buffered at pH 6.8 with 70 m M Tris/HCl). Insoluble materials were removed by centrifugation at 14 500 g for 15 min and clear super- natants served as PL lysates and total cell lysates. The protein concentration in each sample was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). SDS/PAGE under nonreducing conditions was carried out as described previously [31], using 12 and 5% polyacrylamide for resolving and stacking gels, respec- tively. For separation of low molecular mass proteins, tricine/SDS/PAGE was performed [32] with 16.5, 10, and 5% polyacrylamide for resolving, spacer, and stacking gels, respectively. The gels were stained with Coomassie brilliant blue R 250 or a silver staining kit (Wako Pure Chemicals). For Western blotting, proteins separated on SDS/PAGE were transferred to a poly(vinylidene difluoride) (PVDF) membrane (Immobilon-P, Millipore) [33]. After reacting with antiserum, bands were visualized by a chemilumines- cent technique (Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated anti-rabbit 3 IgG (Cappel, Durham, NC, USA) as a secondary antibody. Determination of the C-terminus of ZPC ZPC was purified as described previously from the PL of preovulatory follicles [34]. Aliquots (2 mg protein) separ- ated by SDS/PAGE were transferred to PVDF membranes. The band containing approximately 40 nmol ZPC was digested at 37 °C for 16 h with 400 pmol lysylendopepti- dase (EC 3.4.21.50, Wako Pure Chemicals) dissolved in 10% acetonitrile buffered at pH 9.0 with 50 m M Tris/HCl. The ZPC digests were fractionated by RP-HPLC (Model 600, Waters) using a 40–60% acetonitrile gradients in 0.1% trifluoroacetic acid at a flow rate of 1.0 mLÆmin )1 . A peak at 6.1 min (48% acetonitrile) was collected, and the N-terminal amino-acid sequence was confirmed as Ala318- Arg-Asn-Thr-Trp-Val-Pro-Val-Glu-Gly327 by an auto- mated gas-phase protein sequencer (Model 492, Applied Biosystems). Theexactmolecularmassofthisfragmentwasdeter- mined using MALDI-TOF MS by means of a Voyager-DE mass spectrometer (PE Biosystems) with a-cyano-4- hydroxycinnamic acid (Aldrich Chemical) as a matrix. In order to identify the C-terminal amino acid of ZPC, 18.5 lg of the purified ZPC as described above was applied directly to an automated C-terminal protein sequencer (Procise 494-C, Applied Biosystems). Production of antiserum against proZPC-derived peptide A peptide (Pro-Val-Leu-Leu-Ser-Ala-Asp-Pro-Gly-Ala- Val-Gly-Gln-Gln) corresponding to the sequence 376–389 of quail ZPC coupled with an extra Cys residue at the N terminus was synthesized using multiple peptide synthesizer (SYRO II, MultiSynTech GmbH). A mature female rabbit was immunized with the hemocyanin-coupled peptide (200 l 4 g of peptide) as described previously [35]. N-Terminal sequence analysis of proZPC-derived peptide Granulosa layers were cultured for 6 h in the presence of 200 ngÆmL )1 monensin (1 mL medium per granulosa layer). After culturing, the granulosa layer was extracted with ice- cold RIPA buffer (300 m M NaCl, 2% Nonidet P-40, 1% deoxycholate, 0.2% SDS, 50 m M Tris/HCl pH 7.5) at 4 °C for 16 h. Insoluble constituents were removed by centrifu- gation at 14 500 g at 4 °C for 20 min and the supernatant served as granulosa cell extracts. 2224 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002 To prepare the affinity gel, the IgG fractionated from anti-(proZPC-derived peptide) serum using a HiTrap Pro- tein A FF affinity column (Amersham Pharmacia Biotech) was covalently coupled to Protein A Sepharose FF (Amer- sham Pharmacia Biotech) with dimethylpimelimidate [36]. The granulosa cell extracts were incubated with the affinity gel for 16 h at 4 °C. After extensive washing, the gel was eluted with 1% SDS and the effluent containing proZPC- derived peptide was dried under a stream of nitrogen gas. The sample was dissolved in Laemmli’s sample buffer [31], separated by tricine/SDS/PAGE, and the band of proZPC- derived peptide transferred to PVDF membrane was applied directly to an automated gas-phase protein sequencer (Model 492, Applied Biosystems). Immunohistochemical observation For localization of proZPC and ZPC, granulosa layers cultured with monensin or BFA were fixed in Bouin’s fixative solution and embedded in Paraplast (Wako Pure Chemicals). Immunohistochemical techniques were as described previously [37] using anti-ZPC serum (1 : 300), anti-(proZPC-derived peptide) serum (1 : 200), or normal rabbit serum (1 : 200). The immunolabelled sections were examined under an interference-contrast photomicroscope (BX 50, Olympus Optics). RESULTS ZPC secretion by granulosa cells Western blotting with anti-ZPC serum of the SDS-solubi- lized granulosa cells, the PL, and the culture medium is shown in Fig. 1. The lysates of the granulosa cells before culture were shown to contain three immunoreactive bands of 35, 43, and 94 kDa (lane 1). The SDS-solubilized PL contained only the 35-kDa protein (lane 2). After 6 h of culture, a 35-kDa band was detected in the culture medium (lane 5). The intensity of the 43-kDa band in the cell lysates appeared to decrease during culture, whereas that of the 94-kDa band tended to increase (lane 3). From the comparison of the intensity of the band as a proportion to the total ZPC in the culture well, the amount of secreted ZPC is larger than that of cellular ZPC after culture for 6 h. To refute the possibility of the release of ZPC from the PL in the medium during culture, the isolated PL alone was incubated for 6 h. Because the culture medium of the isolated PL did not contain any immunoreactive bands (lane 7), the 35-kDa immunoreactive protein in the medium must be secreted from the granulosa cells during culture. Next, we cultured granulosa layers for 8 h to assess the time-related changes of ZPC contents in the medium and in the cell lysates. As shown in Fig. 2A, the intensity of the 35-kDa band increased during culture. The content of immunoreactive 43-kDa protein in the cell lysates decreased in a time-related manner (Fig. 2B). These results suggest that the 43-kDa protein is the precursor (proZPC) of 35-kDa ZPC. Effect of monensin on ZPC secretion Granulosa layers were cultured with increasing concentra- tions of monensin, 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 inhibitor, a decreased intensity was detected in the medium supplemented with monensin in a dose-dependent manner (Fig. 3A). The addition of 400 ngÆmL )1 monensin com- pletely abolished ZPC secretion. In contrast, an increase in the intensity of all of the bands in the cell lysates was observed with the addition of 160 ngÆmL )1 of monensin (Fig. 3B). Thus, monensin inhibits the secretion of ZPC without interfering with the conversion of proZPC to 35-kDa ZPC. Effect of BFA on ZPC secretion We next investigated the effects of BFA on ZPC secretion. The addition of 50 ngÆmL )1 BFA caused a decrease in ZPC contents in the medium, and 100 ngÆmL )1 BFA completely abolished ZPC secretion (Fig. 4A). Although 25 ngÆmL )1 BFA failed to affect the contents of ZPC in the cell lysates, the addition of 50 ngÆmL )1 BFA caused a distinct accumu- lation of 43-kDa and 94-kDa proteins (Fig. 4B). The addition of 100 ngÆmL )1 BFA caused a decrease in the 35-kDa ZPC content of the cell lysates (Fig. 4B). These results indicate that BFA inhibits the secretion of ZPC by inhibiting the conversion of proZPC to 35-kDa ZPC. C-terminal sequence of 35-kDa ZPC In order to determine the C-terminal amino acid of 35-kDa ZPC, the electroblotted ZPC on PVDF membrane was digested with lysylendopeptidase. As shown in Fig. 5A, seven major bands were detected in the ZPC digests (lane 2). From the amino-acid sequencing, we purified a 5.4-kDa fragment by RP-HPLC. MALDI-TOF MS analysis demonstrated that the molecular mass of this fragment is Fig. 1. Western blot analysis of ZPC in medium, cells and PL. Gran- ulosa layers were cultured for 0 (lanes 1 and 2) or 6 h (lane 3–5), and the ZPC protein in the granulosa cells (lanes 1 and 3; 0.5 lgproteinper lane; approximately 1/50 of the cell in one well), in the PL (lanes 2 and 4; 0.3 lg protein per lane; approximately 1/90 of the PL in one well) and in the medium (lane 5, 8 lL culture medium per lane; 8/1000 of the total volume in one well) were detected by using anti-ZPC serum (1 : 2000 dilution). Isolated PL alone was also incubated for 6 h, and theZPCproteininthePL(lane6;0.3lg protein per lane) and in the medium (lane 7; 8 lL culture medium per lane) was analyzed. Immunoblots shown are representative of at least three experiments. Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2225 Fig. 2. Time course of ZPC content in the medium and the cell lysate during 8 h of culture. Granulosa layers were cultured for 0, 2, 4, 6, or 8 h, and ZPC protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. The intensities of bands were quantified and plotted as arbitrary units. Values are means ± SEM of three independent experiments with triplicate wells. Fig. 3. Effects of monensin on ZPC secretion. Granulosa layers were cultured with 0, 80, 160, 240, 320, or 400 ngÆmL )1 monensin for 6 h. The ZPC protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. Values are means ± SEM of three independent experiments with triplicate wells. Fig. 4. Effects of BFA on ZPC secretion. Granulosa layers were cultured with 0, 12.5, 25, 50, or 100 ngÆmL )1 BFA for 6 h. The ZPC protein in the medium (A) and in the cell lysate (B) were detected by using anti-ZPC serum. Values are means ± SEM of three independent experiments with triplicate wells. 2226 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002 4970 Da (Fig. 5B), which coincides with the calculated molecular mass of the fragment ending at Phe360 (4972.6 Da). This was also supported by the fact that the C-terminal amino acid was determined as Phe by an automated C-terminal protein sequencer. Proteolytic processing of proZPC in granulosa cells In order to investigate the proteolytic processing of proZPC in the granulosa cells, we raised antiserum against the tetradeca peptide located on the C-terminal side of Phe360 (Pro376 to Gln389). Anti-(proZPC-derived peptide) serum reacted with 43-kDa and 94-kDa but not with 35-kDa ZPC in the cell lysates and in the PL (Fig. 6, panel 3). In comparison with that of anti-ZPC serum, anti-(proZPC- derived peptide) serum tended to react well with the 94-kDa ZPC but had only poor reactivity with 43-kDa ZPC (panels 1 and 3). This immunostaining was diminished by the addition of antigen (Fig. 6, panel 4). These results indicate that proZPC is cleaved between Phe360 and Pro376 during proteolytic processing. In addition, anti-(proZPC-derived peptide) serum detects the 12-kDa band (Fig. 6, panel 3), which could not be detected by the anti-ZPC serum (Fig. 6, panel 1). This suggests that the 12-kDa protein is the cleaved peptide derived from the processing of proZPC. Effects of monensin and BFA on proteolytic processing of proZPC Next, we evaluated the effects of monensin and BFA on the proteolytic processing of proZPC. After the culture of the granulosa layer with monensin or BFA, the cell lysates were subjected to Western blot analysis using anti-(proZPC- derived peptide) serum. As shown in Fig. 7A, the addition of monensin caused an increase in the intensity of the 12-kDa band. This is consistent with the result shown in Fig. 3B in which monensin inhibits the secretion of 35-kDa ZPC but does not disturb the conversion of proZPC to ZPC. On the other hand, > 80 ngÆmL )1 BFA, which inhibits the conversion of proZPC to ZPC (see Fig. 4B) decreased the intensity of the 12-kDa band (Fig. 7B). Detection of proZPC in granulosa cells To determine the localization of proZPC, the sections of the granulosa layers were analysed by immunohistochem- istry. As shown in Fig. 8A, the immunoreactive material recognized by anti-ZPC serum was concentrated in the PL. No positive immunostaining was seen when the granulosa layer was stained with normal rabbit serum (Fig. 8B). In contrast, anti-(proZPC-derived peptide) serum showed the localization of proZPC in the peri- nuclear region of the cells, but not in the PL (Fig. 8C). This staining displayed a highly polarized pattern, that is, the staining was restricted at the apical side of the Fig. 5. C-Terminal sequence analysis of 35-kDa ZPC. (A) Represen- tative silver staining pattern of ZPC digested with lysylendopeptidase. Each PVDF membrane electroblotted with 0 (lane 1) or  40 nmol ZPC (lane 2) was digested by 400 pmol lysylendopeptidase. The supernatant of each digest was separated by tricine/SDS/PAGE, and silver stained. (B) MALDI-TOF MS analysis of purified C-terminal fragment. Fig. 6. Representative Western blot analysis of proZPC and ZPC in the granulosa cells and PL. Granulosa cell lysate (Cell) and PL were detected with anti-ZPC serum (panel 1, 1 : 2000 dilution), anti-ZPC serum preincubated with vitelline membrane of oviposited eggs (panel 2), anti-(proZPC-derived peptide) serum (panel 3, 1 : 1000 dilution), or anti-(proZPC-derived peptide) serum preincubated with antigen pep- tide (panel 4). Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2227 perinuclear region corresponding to the PL side, but not basal side apposed to the BL. When the granulosa layer was cultured without inhibitors, a similar staining pattern was obtained by anti-(proZPC-derived peptide) serum, but the amount of immunoreactive material tended to decrease (Fig. 8D). The granulosa layer cultured with monensin was shown to swell, and the entire cytoplasm was stained (Fig. 8E). The addition of BFA caused a strong staining of the entire cytoplasm without swelling (Fig. 8F). This staining pattern might reflect the accumu- lation of proZPC in the granulosa cells. N-Terminal sequence of the 12-kDa fragment derived from proZPC The 12-kDa fragment cleaved from proZPC was analysed for the N-terminal amino-acid sequence. The first eight residues are Asp-Ala-Gly-Lys-Glu-Val-Ala-Ala, which cor- responds to the sequence Asp363–Ala370 deduced from the cDNA. This result indicated that the proteolytic cleavage of proZPC occurs at the consensus furin cleavage site, Arg359- Phe360-Arg361-Arg362. DISCUSSION In the present study, we have shown that newly synthesized proZPC is accumulated by inhibiting protein transport from RER to the Golgi apparatus by BFA, and that ZPC and the 12-kDa fragment generated by the proteolytic processing of proZPC are accumulated by inhibiting protein transport from the Golgi apparatus. As proZPC is not secreted without proteolysis, this process might be a prerequisite to ZPC secretion and its incorporation into the PL. Fig. 7. Effects of monensin and BFA on pro- teolytic processing. Granulosa layers were cultured with monensin (0, 80, 160, 240, 320, or 400 ngÆmL )1 ) or BFA (0, 20, 40, 60, 80, or 100 ngÆmL )1 ) for 6 h. The proZPC protein in thecelllysate(0.5lgproteinperlane)was detected by using anti-(proZPC-derived pep- tide) serum. Representative of repeated experiments. Fig. 8. Immunohistochemical localization of proZPC and ZPC in granulosa layer. Sections of granulosa layer obtained from 0 (A–C) or 6 h of culture with control medium (D), with 200 ngÆmL )1 monensin (E) and with 100 ngÆmL )1 BFA (F) were processed for immunohistochemical observation using anti-ZPC serum (A), normal rabbit serum (B), or anti-(proZPC-derived peptide) serum (C–F). Representative of repeated experiments. 2228 T. Sasanami et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Uchida et al. [38] reported that monensin inhibits the secretion of procollagen and fibronectin from cultured human fibroblasts. They also showed that this inhibition is accompanied by the accumulation of procollagen and fibronectin in the Golgi apparatus [39,40]. Accumulation of laminin in the Golgi apparatus was also observed in the monensin-treated rat astrocytes [41]. In our results, monen- sin inhibits ZPC secretion without disturbing the conversion of proZPC to ZPC (Fig. 3). On the other hand, monensin impedes the proteolytic processing of pro-opiomelanocortin in rat pituitary cells [42]. This might be due to the fact that proteolytic processing of pro-opiomelanocortin occurs in the secretary granule [43]. BFA blocks albumin secretion in rat hepatocyte by inhibiting the protein transport from RER to the Golgi complex [44]. As an accumulation of proalbumin in the RER was observed when cells were cultured with BFA [45], the proteolytic conversion of proalbumin to mature albumin takes place in the Golgi apparatus [44]. Our findings regarding the accumulation of proZPC in the presence of BFA and conversion of proZPC to ZPC in the presence of monensin indicate that the proteolytic processing of ZPC could occur in the Golgi apparatus. Amino-acid sequence analysis showed that the C termi- nus of mature ZPC protein is Phe360 (Fig. 5). The N-terminal amino acid of the proZPC-derived 12-kDa fragment was determined to be Asp363, located just after the consensus furin cleavage site. These results indicate that the Arg361-Arg362 sequence might be missing. This may be accounted for the following two possibilities: (a) proZPC is initially digested at the consensus furin cleavage site and the resulting C-terminal dibasic residues are trimmed off to generate mature ZPC; and (b) proZPC directly receives the proteolytic cleavage between Phe360 and Arg361, and the N-terminal two residues of the proZPC-derived 12-kDa fragment are trimmed off. In the case of neuropeptides and peptide hormones, the C-terminal basic amino acid is removed by a carboxypeptidase H in secretory granules after initial digestion [46,47]. Although the N-terminal amino-acid sequence of the proZPC-derived peptide was not determined, Kubo et al. [48] reported that the two basic C-terminal residues of gp43, a protein homologous to ZPC in Xenopus laevis, is removed to produce the mature protein. We think, therefore, that the processing event of proZPC to ZPC in quail granulosa cells might take place initially by a furin-like protease and then by a carboxypeptidase H-like protease. On the other hand, mouse ZPC was reported to be cleaved at the consensus furin cleavage site without further processing of its C-terminal paired Arg residues [49]. Such differences in the process of proteolytic processing between quail and mouse might reflect the marked species differences in the properties of their ZPC biosynthesis. Our results demonstrated that ZPC is never secreted in a precursor form (see Figs 1–4). Williams and Wassarman [50] reported ) based on a site-directed point mutation study ) that secretion of mouse ZPC from transfected cells is dependent on the cleavage at the consensus furin cleavage site. The truncation of the C-terminal amino acid of choriogenin, the precursor protein of the component of chorion in Oryzias latipes, was also reported to be a prerequisite for formation of the mature protein and its assembly into chorion [51]. We suggest that the proteolytic processing of quail proZPC is considered to be, at least in part, required for ZPC secretion rather than ZPC biosyn- thesis. The consensus furin cleavage site was found within the hydrophobic domain near the C terminus in the ZPC of all mammalian and avian species studied, though the overall similarity in amino-acid sequence among the distal classes was relatively low [6,16]. This indicates that intracellular processing at the furin cleavage site might universally participate in the formation of mature ZPC from its precursor. The immunohistochemical study with anti-(proZPC- derived peptide) serum showed that immunoreactive material is present only on the apical side of the perinuclear region (Fig. 8C). Therefore ZPC might be transported selectively from the Golgi apparatus toward the apical surface of granulosa cells, which are apposed to the PL. In polarized Madin–Darby canine kidney cells, the O-glyco- sylated domain has critical role for apical secretion of neurotrophin receptors [52]. Fiedler et al. [53] reported that antibody for annexin XIIIb significantly inhibited the transport of influenza virus glycoprotein to the apical plasma membrane. Efforts are currently in progress to investigate the topology of ZPC secretion in which ZPC selectively secreted to the apical surface of the granulosa cells forms the PL. Our finding that the 12-kDa fragment cleaved from proZPC accumulated in the monensin-treated cells (Fig. 7A) and did not degrade immediately indicates the possibility of the physiological importance of this fragment, although its fate is currently unknown. The C-peptide, a by-product of proteolytic processing of proinsulin in the pancreas, is demonstrated to have important physiological effects on kidney and nerve functions, such that C-peptide binds to specific G protein-coupled receptors on human plasma membrane [54]. Nillni and Sevarino [23] also described that the seven peptides derived from thyrotro- pin-releasing hormone precursor are secreted from the hypothalamus, and have various biological functions. In the Western blot analysis, we found that the 94-kDa band reacted with both anti-ZPC serum and anti-(proZPC- derived peptide) serum in the cell lysates (Figs 1 and 6). This protein migrates at the same position on SDS/PAGE under reducing conditions as 43-kDa proZPC (data not shown). The high molecular mass immunoreactive band was also observed during insulin biosynthesis in pancreatic b cells [55], which is regarded as an intermediate of proinsulin to insulin conversion. Because the intensity of the 94-kDa band is always parallel to 43 kDa, we suggest that the 94- kDa protein is an oligomeric intermediate of the 43-kDa proZPC generated during post-translational modification. In conclusion, our study suggests that newly synthesized ZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the resulting two basic residues at the C-terminus are subsequently trimmed off with carboxypeptidase H-like protease to generate the mature 35-kDa ZPC prior to secretion. This process might be a prerequisite event for ZPC secretion and its incorporation into PL. ACKNOWLEDGEMENTS We are grateful to W. J. Schneider (Department of Molecular Genetics, Institute of Medical Biochemistry, University and Biocenter Vienna) for his helpful discussion. This work was supported in part by Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. 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Ó FEBS 2002 Proteolytic processing of ZPC in quail granulosa (Eur. J. Biochem. 269) 2231 . determined as Phe by an automated C-terminal protein sequencer. Proteolytic processing of proZPC in granulosa cells In order to investigate the proteolytic processing. Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells Tomohiro Sasanami 1 , Jianzhi Pan 1 , Yukio