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BOR - Papers in Press, published online ahead of print September 27, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.056267
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BIOLOGY OF REPRODUCTION 76, 9–18 (2007)
DOI: 10.1095/biolreprod.106.056267
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Heterocomplex Formation and Cell-Surface Accumulation of Hen's Serum Zona Pellucida B1 (ZPB1)with ZPC Expressed by a Mammalian Cell Line (COS-7): A Possible Initiating Step of Egg-Envelope Matrix Construction1

Hiroki Okumura 3, Naohito Aoki 6, Chihiro Sato 4 5, Daita Nadano 3, and Tsukasa Matsuda 2 3

Departments of Applied Molecular Biosciences3 and Bioengineering Sciences,4 Graduate School of Bioagricultural Sciences, Bioscience and Biotechnology Center,5 Nagoya University, Nagoya 464-8601, Japan Faculty of Bioresources,6 Mie University, Tsu 514-8507, Japan

ABSTRACT

The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.

gamete biology, granulosa cells, oocyte development

INTRODUCTION

The egg envelope in vertebrates is a multifunctional extracellular matrix surrounding oocytes that plays important roles in gamete recognition, sperm activation, polyspermy blocking, and provides protection for the early embryo [1]. Although the egg envelopes of vertebrates share such common biological functions, there are large differences in the matrix histology. For example, mouse egg-envelope, referred to as zona pellucida (ZP), is 7 µm in thickness, consists of a fine meshwork of thin filaments, and surrounds a relatively small oocyte (~85 µm in diameter) [2]. In contrast, chicken has a thinner envelope (2–4 µm thickness), which consists of a relatively rough meshwork of thick filaments in spite of the huge oocyte (~40 mm in diameter) [3, 4]. Unlike eggs of eutherian mammals, birds oocytes markedly increase their size with the egg-yolk deposition during the follicular development and, especially during the final 8–9 days before ovulation, the oocyte diameter quickly increases from about 7 to 40 mm [3]. The bird egg-envelope must, therefore, stretch and extend quickly throughout this rapid growth of the oocyte.

The egg-envelope matrix is composed of at least three ZP glycoproteins that share a common C-terminal domain (named ZP domain) consisting of ~260 amino acid residues with 8 or 10 conserved cysteine residues [5]. In early studies of mammalian egg-envelope, these ZP glycoproteins were named ZP1, ZP2, and ZP3, according to their apparent mobility in electrophoresis [6], or ZPA, ZPB, and ZPC, according to their protein sequence length [7], although ZP1, ZP2, and ZP3 do not necessarily correspond to ZPA, B, and C, respectively. Subsequent studies identifies nonmammalian ZPAX (Xenopus) [8] and ZPD (Xenopus and chicken) [9, 10], which show lower sequence identities with the ZP1–3 or ZPA-C. More recently, these ZP glycoproteins have been classified into four subfamilies: ZPA, ZPB (ZPB1, ZPB2, and others), ZPC, and ZPX (ZPX1 and ZPX2), according to their phylogenetic analyses [11]. According to this nomenclature, mouse ZP1, ZP2, and ZP3 correspond to ZPB1, ZPA, and ZPC, respectively [11], while chicken ZP1, ZPC, and ZPD correspond to ZPB1, ZPC, and ZPX2, respectively [10–12]. We use this nomenclature based on phylogenetic relationships described below. The egg envelopes surrounding chicken and mouse oocytes share two common components, ZPB1 and ZPC, and have individually specific components, ZPX2 and ZPA, respectively. Although ZPB1 is common in both mouse and chicken egg-envelopes, only chicken ZPB1 has a unique repeat structure between the N-terminal domain and trefoil domain. Furthermore, chicken ZPX2 has an epidermal growth factor-like domain [10], which has not been found in mammalian egg-envelopes so far. As for biosynthesis of ZP proteins, mouse ZPA, ZPB1, and ZPC are all synthesized in the growing oocyte [13–15], whereas chicken ZPC and ZPX2 are in the granulosa cells surrounding the preovulately growing oocyte [10, 16]. Furthermore, chicken ZPB1 is synthesized in and secreted from laying hen's liver and transported through the bloodstream to the ovary [17].

In addition to these egg-envelope components (ZP glycoproteins), several extracellular glycoproteins sharing the ZP domain have been identified (ZP domain proteins), and some of them are thought to be important in human pathologies [18]. Interestingly, all of these ZP domain proteins are present in filaments or matrices like the egg-envelope ZP glycoproteins. They have been believed to associate via ZP domains [19], and a model for the molecular mechanism for ZP-ZP interaction has been proposed [20].

Earlier biochemical and electron microscopic studies [2], and recent studies using ZP-knockout mice [21–23], suggest that ZPC associates with ZPB1 and/or ZPA to form filaments, ZPB1 and ZPA do not associate with each other, and ZPB1 cross-links the filaments by forming a disulfide-linked homodimer [24, 25]. This mouse model of egg-envelope structure might not necessarily be applied to all mammals or vertebrates because of the diversity in their ZP-glycoprotein composition, structure, and synthesis, as described above. Actually, a recent investigation on fish egg-envelope ZP glycoproteins suggested some diversity in the matrix construction, including disulfide-linked heterodimer formation [26], and differential C-terminal processing [27]. Concerning bird egg-envelope, our previous study revealed that ZPX2 was easily released from the chicken egg-envelope matrix by ultrasonication, and the remaining insoluble precipitate composed of ZPB1 and ZPC was solubilized in urea solution without reducing agent [10]. These results suggested that ZPX2 associated loosely with noncovalently formed ZPB1-ZPC complex. Interestingly, ZPB1 was present as both monomeric and disulfide-linked homodimeric forms either in the egg envelope or the ZPB1-ZPC complex preparation [10, 28]. The interaction of ZPC and ZPB1 has also been suggested in Japanese quail egg-envelope [29].

In almost all vertebrates, especially in birds and reptiles with large eggs, physical strength is required for the egg envelope to hold and protect a large oocyte. At the same time, on fertilization, such a hard and solid envelope has to be proteolytically degraded and solubilized rapidly to make sperm penetrate into the egg envelope and interact with the egg. In our earlier study, some anti-ZPB1 antibodies blocked the proteolytic degradation of not only ZPB1, but also ZPC, during the egg-sperm interaction [28]. Interaction and association among the three components, ZPB1, ZPC, and ZPX2, would play an important role, not only in egg-envelope construction during the oocyte development, but also in the matrix deconstruction on egg-sperm interaction at the initial stage of fertilization.

The main goal of this study was to examine the possibility of reconstruction of egg-envelope matrix in vitro and to clarify the roles of each ZP glycoprotein in the matrix architecture of chicken egg-envelope. We examined whether hen's serum ZPB1 can bind specifically to the other components, ZPC and/or ZPX2, of egg envelope in vitro, and whether it accumulates around the cells secreting the other ZP protein in a cell culture system. Consequently, ZPB1 was found to associate with ZPC, and ZPB1-ZPC complexes were shown to form a matrix-like structure around ZPC-secreting cells of kidney cell line derived from African green monkey (COS-7) cells. A possible contribution of the ZP domain of ZPB1 to the specific association with ZPC is also discussed.

MATERIALS AND METHODS

All animal procedures were performed according to the guidelines for the care and use of experimental animals of Nagoya University. The animal protocols were approved by an institutional animal care and use committee (Nos. 2002041914 and 2003070901).

Antibodies

Mouse monoclonal antibodies (mAB) against chicken ZPB1 (5G9 and 8E1) were as described in [28], and mouse polyclonal antisera against recombinant proteins of chicken ZPB1 and ZPC were prepared in this study. Mice (6-wk-old female ddY; Japan SLC, Inc., Hamamatsu, Japan) were immunized by intraperitoneal injection of the purified fusion proteins described below (20–40 µg/mouse) emulsified with Freund's complete adjuvant (Difco Laboratories, Detroit, MI). At 2 wk after the first injection, mice were boosted by injection with each fusion protein (10–20 µg/mouse) emulsified with Freund's incomplete adjuvant (Difco Laboratories), which was repeated after an additional 3 wk. At 7 days after the last injection, blood was collected from individual mice. The serum was separated and stored at –20°C before use. Rabbit polyclonal antibody against hemagglutinin (HA) epitope tag was purchased from Covance Research Products (Denver, PA).

Preparation of Chicken Serum

Blood was collected from carotid artery of laying White Leghorn hens, coagulated overnight at 4°C, and centrifuged at 2000 x g for 10 min to remove a clot. After further centrifugation at 13 000 x g for 10 min, the supernatant was collected as the laying hen's serum (LHS) and stored at –20°C before use. When subjected to the following experiments, thawed LHS was filtered through a 0.2-µm-pore-size cellulose acetate filter (DISMIC-13CP; Advantec, Tokyo, Japan) for sterilization.

Production of Recombinant Chicken ZP Proteins

Complimentary DNA of chicken ZPB1 was amplified by RT-PCR. The first-strand cDNA was synthesized from the poly(A)+RNA isolated from laying White Leghorn hen's liver using Superscript II RNase H– reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamer primers. The PCR was performed using the first-strand cDNA as a template with gene-specific primers synthesized based on the cDNA sequence of chicken ZPB1 (GenBank accession no. AJ289697) [17]. Amplified cDNA was cloned into a pTargeT vector (Promega, Madison, WI) according to the manufacturer's instructions, and the DNA sequence was verified using an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA).

A ZPB1 cDNA fragment, coding for approximately three fourths of the repeat region (nucleotides 594-1475), was amplified by PCR using the ZPB1 cDNA as a template with a sense primer, 5'-GGATCCGCACACACTCAATCCATCCTG-3', and an antisense primer, 5'-GGATCCGACAAACCCTGCTTGTGT-3' (BamHI sites introduced in the primers are underlined). The resultant DNA fragment was digested with BamHI and inserted via the same restriction sites into the pET-32a(+) vector (Novagen, Darmstadt, Germany). The ZPB1/thioredoxin fusion protein was expressed in Escherichia coli Origami B(DE3)pLysS (Novagen) and purified by a stepwise elution with varied imidazole concentrations, according to the manufacturer's instructions. The purified protein was dialyzed against PBS and stored at –20°C until use for immunization.

Similarly, a cDNA fragment coding mature ZPC (nucleotides 282-1047 of the ZPC cDNA), without the N-terminal signal sequence and the C-terminal putative transmembrane region, was amplified by PCR using the full-length cDNA [16] as a template, cloned, and inserted into pET-32a(+) vector (Novagen). The thioredoxin fusion protein was produced and processed as described above.

Electrophoresis, Immunoblotting, and Ligand Blotting

SDS-PAGE and Tricine SDS-PAGE were performed according to the method of Laemmli [30], and Schägger and Von Jagow [31], respectively. Protein samples were boiled for 3 min in SDS-PAGE sample buffer in the presence or absence of 2-mercaptoethanol. To detect protein bands, gels were stained with Coomassie brilliant blue R-250 (CBB). For immunoblotting, proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA). After blocking with 2.5% gelatin, the membrane was incubated with primary antibody and then with horse radish peroxidase-labeled anti-mouse immunoglobulin (Ig) G or anti-rabbit IgG antibody (Cappel, Costa Mesa, CA), as previously described [32]. The signal was detected by the ECL (enhanced chemiluminescence) Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). For a ligand blotting analysis, electroblotting, blocking, incubation with antibodies, and detection were performed in the same manner as described above, except that, after blocking, the membrane was incubated with ZPB1 solution (1% LHS in the incubation buffer of 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0, 0.05% Triton X-100, and 0.25% geratin) or 1% fetal bovine serum (FBS) in the incubation buffer, at 4°C for 24 h. The mouse antiserum to the recombinant ZPB1 fragment was used for the primary antibodies specific for ZPB1 (anti-ZPB1), unless otherwise noted.

Transient Expression of ZPC-HA In COS-7 Cells

Expression plasmid of HA-tagged chicken ZPC (ZPC-HA) was constructed through a PCR-based strategy using the overlapping PCR method [33]. Two primers for HA-tag insertion, C-HA-S, 5'-TACGACGTGCCCGACTACGCGCGCTTCCGTCGTGATGCC-3' and C-HA-AS, 5'-GTAGTCGGGCACGTCGTACGGGTAGCGGCTCTGCCATCTCTC-3', including parts of the HA sequence (underlined nucleotides), of which 18 nucleotides from each 5' terminal were complement, and two gene-specific primers LFSI, 5'-TGGAAGCAGGCGGGATGCAA-3' and LFAS, 5'-TGCACTCACACCGCAGCTGAG-3' were used for PCR. The first PCR was performed at an annealing temperature of 50°C for 25 cycles using the ZPC cDNA [16] as a template with two primer sets: LFSI and C-HA-AS; and C-HA-S and LFAS. The PCR products were annealed to form hybrids in those complementary sequences, and primer extension reaction was performed. The resultant mutant product was amplified by the second PCR at an annealing temperature of 55°C for 25 cycles with the primer pair LFSI and LFAS. Amplification and primer extension reaction were both carried out with Pyrobest DNA polymerase (Takara Bio Inc., Otsu, Japan). The final PCR product was cloned into pTargeT vector and sequenced as described above (ZPC-HA/pTargeT [pZPC-HA]). HA-tag (-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-) was placed upstream of a conserved furin cleavage site, between amino acids Arg367 and Arg368.

African green monkey kidney epithelial cells, COS-7 were obtained from American Type Culture Collection and cultured in Dulbecco modified Eagle medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS or 10% LHS, and 100 U/ml penicillin, 0.1 mg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2. The plasmid DNA (pZPC-HA or empty pTargeT, 4 µg/well) was introduced into the cells according to Chen and Okayama's calcium phosphate method [34], as described in [32]. The cells were washed three times with ice-cold PBS and cultured in 1 ml/well of fresh medium containing 10% FBS or 10% LHS for an additional 48 h. The cells were then washed three times with ice-cold PBS and lysed on ice with 100 µl/well of lysis buffer (1% Triton X-100, 5 mM EDTA, 10% glycerol, 150 mM NaCl, and 50 mM HEPES, pH 7.5) supplemented with 100 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were then centrifuged at 13 000 x g for 10 min, and the resultant Triton X-100-insoluble precipitates and soluble supernatants were subjected to subsequent analyses. The culture supernatants collected were centrifuged at 1200 x g for 15 min to remove detached cells, and immediately subjected to immunoprecipitation experiments.

For the experiments on the binding efficiency of two mAbs (5G9 and 8E1), the culture supernatant of transfected COS-7 cells was pooled, mixed with 1/10 volume of LHS or FBS as a control, and incubated at 37°C for 60 min, followed by further incubation at 4°C for 22 h. Each solution of the culture supernatant/LHS mixture was divided in two and then subjected to immunoprecipitation using two distinct monoclonal anti-ZPB1 antibodies, 5G9 and 8E1, respectively.

Immunoprecipitation

Protein G magnetic beads (25 µl of 50% slurry; New England Biolabs, Beverly, MA) was prepared according to the manufacturer's instructions and rotated for 24–48 h at 4°C in 80 µl binding buffer (0.1 M sodium phosphate, pH 8.0) with each antibody described above to couple IgG to the beads, pulled by magnetic field, and washed three times with 500 µl binding buffer. A total of 800 µl of the culture supernatant of COS-7 (described above) was precleared with 25 µl of the beads, without the antibody coupling by rotating for 24–48 h at 4°C. The beads were pulled by magnetic field, and the supernatants were incubated with the antibody-bound beads for 24–48 h at 4°C. The immunoprecipitate was washed twice, as described above, dissolved in 25 µl of SDS-PAGE sample buffer in the absence of 2-mercaptoethanol (nonreducing conditions), and subjected to electrophoresis.

Identification of the ZPB1 Peptide Fragment Recognized by 8E1 and Mass Spectroscopic Analysis of the Antigenic Peptide

The egg envelope was collected mechanically with forceps from preovulatory mature follicles of laying White Leghorn hens as described previously [10, 16], and stored at –20°C until use.

The egg envelope (3.6 mg wet weight) was solubilized in 0.1% SDS without 2-mercaptoethanol and digested with trypsin for 6 h, as described previously [10]. The digest was reduced with 2-mercaptoethanol followed by acrylamide treatment for blocking [35] and subjected to Tricine SDS-PAGE (16.5% T, 6% C) under reducing conditions. The peptide bands were detected by CBB staining or immunoblotting using the anti-ZPB1 antibody (8E1) described above. The 8E1-positive band was excised and cut into small pieces for the following in-gel digestion. After being destained with a 50% acetonitrile/25 mM NH4HCO3 mixture, the gel pieces were washed with 100 µl of acetonitrile, dried by vacuum centrifugation, and then rehydrated in buffer containing 25 mM NH4HCO3 and 1.1 U trypsin (Trypsin Gold, mass spectrometry [MS] Grade; Promega). Following the digestion for 17 h at 37°C, the peptides were extracted from the gel stepwise with 0.1% trifluoroacetic acid, 0.1% trifluoroacetic acid/50% acetonitrile mixture, and 100% acetonitrile. The pooled extract was concentrated by vacuum centrifugation.

The extracted peptides were subjected to matrix-assisted laser-desorption ionization (MALDI) MS, as described previously [10]. The MS/MS data were analysed using DeNovo Explorer and Data Explorer software (Applied Biosystems).

Immunofluorostaining and Confocal Laser Microscopy

COS-7 cells were plated onto poly-L-lysine-coated coverslips in a six-well culture plate, transiently transfected, and cultured under the same conditions as described above. The cells on coverslips were washed with PBS and fixed with 3% paraformaldehyde in PBS on ice for 30 min. The cells for permeabilization were washed again with PBS and incubated with 0.1 % Triton X-100 in PBS on ice for 30 min. The permeabilized and nonpermeabilized cells were washed again with PBS and blocked with 2% BSA in PBS (2% BSA/PBS) for 30 min at room temperature. The cells were incubated with 2% BSA/PBS containing mouse anti-ZPC (polyclonal anti-recombinant ZPC), rabbit anti-HA, or mouse anti-ZPB1 (polyclonal anti-recombinant ZPB1) overnight at 4°C, washed with PBS, and finally incubated with 2% BSA/PBS containing Alexa Fluor 488 goat anti-mouse IgG (6.7 µg/ml; Molecular Probes, Eugene, OR) and Alexa Fluor 568 goat anti-rabbit IgG (6.7 µg/ml; Molecular Probes) for 30 min at room temperature in darkness. After washing with PBS, the cells on coverslips were mounted onto glass slides containing a drop of 90% glycerol in PBS. Imaging was performed on a Zeiss Axioplan2 microscope equipped with LSM5 PASCAL laser scanning confocal optics (Carl Zeiss, Thornwood, NY) in the multitrack mode. A 488-nm excitation filter and a 505–530 nm band-pass emission filter was used for imaging Alexa Fluor 488; a 543-nm excitation filter and a 560-nm long-pass filter was used for imaging Alexa Fluor 568. Differential interference contrast (DIC) microscopy images were taken on the same system. Three-dimensional image reconstruction and orthogonal sectioning were carried out using the Zeiss LSM Image Browser, Version 3.5 (Carl Zeiss).

RESULTS

Specific Binding of Serum ZPB1 to Egg-Envelope ZPC as Analyzed by Ligand Blotting

It was expected that the ZPB1 in LHS would bind to the other ZP glycoproteins when it joins the formation of egg-envelope matrix in the ovary. To identify egg-envelope components that interact with ZPB1, a type of ligand blotting analysis was performed (Fig. 1). Egg-envelope proteins were solubilized with the SDS sample buffer under nonreducing conditions and subjected to SDS-PAGE followed by electroblotting onto a PVDF membrane. The membrane was incubated in the presence or absence of ZPB1 as a ligand, and the bound ZPB1 was detected immunologically with anti-ZPB1 antibody. A 35-kDa band corresponding to ZPC, but not to a 40-kDa ZPX2, was clearly detected with anti-ZPB1 antibody only on the membrane incubated with LHS, indicating the specific binding of ZPB1 to ZPC. Except for this 35-kDa band, there were no significant differences in the anti-ZPB1 immunostaining pattern between the two membranes incubated with and without LHS containing ZPB1.


Figure 76010301
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  		FIG. 1. A ligand-blotting analysis showing the specific binding of serum ZPB1 to electrophoretically separated egg-envelope ZPC. Aliquots of egg-envelope (670 µg wet weight for lane 1, and 11 µg each for lanes 2–4) were solubilized by SDS sample buffer and separated by SDS-PAGE under nonreducing conditions. One lane of the gel was stained with CBB (lane 1) and the other lanes (lanes 2–4) were used for electroblotting. After incubation with (+; lane 2) or without (–; lanes 3 and 4) ZPB1-containing serum, the blotted membranes were immunostained with anti-ZPB1 (lanes 2 and 3) or anti-ZPC (lane 4) antibody. The migration positions of the protein markers are shown on the right. The bands of monomeric (mono-ZPB1) and dimeric ZPB1 (di-ZPB1), ZPC, and ZPX2 are indicated on the left.

Expression, Processing and Secretion of HA-Tagged ZPC by Transfected COS-7 Cells

For the in vitro analyses of ZPB1-ZPC interaction, an epitope (HA)-tagged ZPC was expressed transiently in a mammalian cell line, COS-7. To confirm whether the recombinant ZPC-HA was processed correctly and secreted into the culture medium, the cell lysate and the culture supernatant were subjected to immunoblotting and immunoprecipitation analyses, respectively (Fig. 2). A 42-kDa band, which was expected to be a proform of ZPC-HA, was clearly detected in the cell lysate, either by anti-ZPC or anti-HA antibody. In contrast with cell lysate, a 35-kDa band corresponding to the mature form of ZPC (see Fig. 1, lane 1) was detected in the culture supernatant by the specific antibodies. The mature form was below the detectable level in the cell lysates. These results suggest that HA-tagged ZPC expressed as a proform with its carboxyterminal hydrophobic region in transfected COS-7 cells was processed, at least in part, to an expected mature form and secreted to the culture medium.


Figure 76010302
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  		FIG. 2. Expression, processing, and secretion of HA-tagged ZPC by transfected COS-7 cells. COS-7 cells were transfected with an expression plasmid for the epitope-tagged ZPC (pZPC-HA) or an empty vector (mock), and cultured. The culture supernatants were collected, while the cells were lysed with Triton X-100 containing lysis buffer and harvested. The cell lysates were separated into two fractions: soluble supernatant and insoluble pellet. The Triton X-100-insoluble (I; 1/5 of the total cell lysates) and -soluble (S; 1/6 of the total cell lysates) fractions were subjected to SDS-PAGE under nonreducing conditions (two left panels), followed by immunoblotting with anti-ZPC (top) or anti-HA (bottom) antibodies. The secreted ZPC-HA was immunoprecipitated (IP) from the culture supernatants (800 µl; 4/5 of the culture supernatant) using anti-ZPC, and subjected to SDS-PAGE/immunoblotting analysis (two right panels). The migration positions of protein markers are shown on the right. Protein bands estimated to be proZPC-HA (arrows) and mature ZPC-HA (open arrowheads) are also indicated on the left and the right, respectively. Asterisks represent nonspecific staining.

Specific Binding of Serum ZPB1 to ZPC Secreted by the Transfected COS-7 Cell

The culture supernatants of the COS-7 cells, which had been transfected and then cultured in the presence of serum ZPB1, were subjected to reciprocal coimmunoprecipitation analysis using anti-ZPB1 mAB (5G9) and anti-HA antibody, and the cell lysates were directly subjected to immunoblotting with anti-ZPB1 and anti-HA antibodies (Fig. 3). The probable mature ZPC-HA secreted into the medium (Fig. 3, open arrowheads) was coprecipitated with ZPB1 (Fig. 3, closed arrowheads) by immunoprecipitation using the anti-ZPB1 (Fig. 3, A and B), and, conversely, ZPB1 was coprecipitated with the ZPC-HA by immunoprecipitation using the anti-HA (Fig. 3, C–E), indicating the presence of ZPB1-ZPC complexes in the culture supernatant of ZPC-expressing cells cultured in the ZPB1-containing medium. A 180-kDa band, corresponding to dimeric ZPB1 (Fig. 3, C and D, striped arrowheads), was also detected, particularly in the immunoprecipitate by the anti-HA, which was expected to be richer in ZPB1-ZPC complexes as compared with the immunoprecipitates by anti-ZPB1 because of an excess of free ZPB1 was in the culture medium. This 180-kDa band was confirmed to migrate on SDS-PAGE under reducing conditions (data not shown) to the same position of 97-kDa monomeric ZPB1 as the dimeric ZPB1 of egg envelope [28]. These results indicate that ZPB1 interacted specifically with ZPC-HA secreted into the medium and formed ZPB1-ZPC heterocomplexes. Both ZPB1 and mature ZPC-HA were also detected in the Triton X-100 insoluble fraction of the cell lysate (Fig. 3, F and G), implying accumulation of the ZPB1-ZPC complex around the transfectants cultured with ZPB1, although the dimeric ZPB1 was below the detectable level in the cell lysate. Furthermore, the coprecipitated ZPB1 (monomer and dimer) was also detected by another anti-ZPB1 mAB, 8E1 (Fig. 3D), indicating the presence of 8E1-epitope in the ZPB1 coprecipitated with ZPC-HA.


Figure 76010303
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  		FIG. 3. Immunoprecipitation of ZPB1-ZPC heterocomplexes from the culture supernatant and detection of ZPB1 in the cell lysates of the transfected COS-7 cells cultured in the presence of ZPB1. COS-7 cells transfected with an expression plasmid for ZPC-HA (pZPC-HA) or a control plasmid (mock) were cultured in the presence (+) or absence (–) of ZPB1. The culture supernatants were collected and subjected to immunoprecipitation (IP) using anti-ZPB1 mAB, 5G9 (A and B) or anti-HA (C–E). The immunoprecipitates (1/5 [A, C, and D] and 1/2 [B and E] of the precipitates), Triton X-100-insoluble (I; 1/5 of the confluent culture) and -soluble (S; 1/6 of the confluent culture) fractions of the cell lysates (F and G), egg-envelope (EE), and LHS were subjected to SDS-PAGE under nonreducing conditions, followed by immunoblotting with anti-ZPB1 (polyclonal anti-recombinant ZPB1) (A, C, and F), monoclonal anti-ZPB1, 8E1 (D), or anti-HA (B, E, and G) antibody. The migration positions of the protein markers are shown on the right. Protein bands corresponding to dimeric ZPB1 (di-ZPB1; striped arrowheads), monomeric ZPB1 (mono-ZPB1; closed arrowheads), mature ZPC-HA (open arrowheads), and proZPC-HA (arrow) are indicated on the left. Asterisks represent nonspecific staining.

Contribution of ZPB1 C-Terminal Region to the Association with ZPC

To compare more quantitatively the binding efficiency of the two anti-ZPB1 mABs (5G9 and 8E1) [28] to the ZPB1-ZPC heterocomplexes, the culture supernatants were pooled from the ZPC-HA-expressing COS-7 cells, mixed with ZPB1-containing serum, and incubated to induce the complex formation. The ZPC-HA-ZPB1 mixture was subjected to immunoprecipitation by each of the mABs, 5G9 or 8E1 (Fig. 4). Interestingly, one mAB, 5G9, effectively immunoprecipitated the ZPB1-ZPC heterocomplex, whereas 8E1 did not. The mAB 8E1, of course, immunoprecipitated free ZPB1, particularly from the culture supernatant of the mock transfectants.


Figure 76010304
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  		FIG. 4. Differential accessibility of two distinct mABs to ZPB1 of the ZPB1-ZPC heterocomplexes. The culture supernatants of COS-7 cells transfected with an expression plasmid for ZPC-HA (pZPC-HA) or a control plasmid (mock) were collected, mixed, and incubated with (+) or without (–) ZPB1, and subjected to immunoprecipitation (IP) using the anti-ZPB1 mAB, 5G9 or 8E1. The immunoprecipitates were subjected to SDS-PAGE under nonreducing conditions, followed by immunoblotting with anti-ZPB1 (polyclonal anti-recombinant ZPB1; upper panels) or anti-HA (lower panels) antibody.

The lack of binding of 8E1 to ZPB1 associated with ZPC suggested that ZPC bound to the region nearby an 8E1-binding site (epitope) in ZPB1. To map the 8E1 epitope, 8E1-positive ZPB1 fragments were screened among the tryptic peptides of whole-egg envelope by Tricine-SDS-PAGE, followed by immunoblotting (Fig. 5A). A 5.5-kDa band was clearly stained with 8E1. The MS analyses on the in-gel digest of this band revealed that this 8E1-positive fragment contained two ZPB1-derived sequences (Fig. 5B, peaks a and b), and that the two sequences were identical except for one residue at position 897 (Val or Leu, probably due to ZPB1 isoforms). The proprotein of ZPB1 has a consensus sequence, RARR (from 900 to 903), for the furin cleavage. Accordingly, the 5.5-kDa fragment was found to contain a decapeptide (Pro891–Arg900) corresponding to the putative C terminus of mature ZPB1 (Fig. 5, B and C), suggesting that the 5.5-kDa fragment was Phe852–Arg900, with theoretical molecular masses of 5258.82 and 5272.84 (containing the peak a and b sequences, respectively). The peaks c, d, and f were derived from ZPC, while the peak e was from ZPX2 (data not shown). Thus, the 8E1 epitope was mapped in a C-terminal peptide within ZP domain of a ZPB1 molecule.


Figure 76010305
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  		FIG. 5. Epitope mapping for an anti-ZPB1 mAB, 8E1, by mass spectrometry analysis. A) The tryptic digest of egg envelope (0.81 mg wet weight) was reduced with the SDS sample buffer containing 1% 2-mercaptoethanol, and resultant free sulfhydryl groups were blocked with acrylamide. Peptide fragments were separated by Tricine SDS-PAGE under reducing conditions. Polypeptide bands were detected by CBB (left lane) or immunoblotting with an mAB, 8E1 (right lane). The band in the CBB-stained gel corresponding to the 8E1-positive band on the immunostained membrane (arrowhead) was excised. The migration positions of the protein markers are shown on the left. B) Polypeptides in the excised band were further digested in gel with trypsin and analyzed by MALDI-time-of-flight/time-of-flight mass spectrometer. The six major peaks (arrowheads a–f) were detected. C) Each peak shown in (B) was subjected to further MS/MS analysis to obtain sequence data, and typical collision-induced dissociation spectra and sequence data of peaks a and b are shown. The second Val of peak a was substituted by a Leu in peak b. All Cys were modified with acrylamide (C*). The molecular mass of each peak is shown in brackets.

Colocalization of ZPB1 and ZPC Around the ZPC-Transfected COS-7 Cells Cultured in the ZPB1-Containing Medium

To determine whether ZPB1 is accumulated with ZPC on the surface of ZPC-expressing cell, the transfected COS-7 cells were cultured in the presence of serum ZPB1. After being cultured for 2 days, the cells were fixed with or without the permeabilization treatment. The fixed cells were stained with anti-ZPB1 and anti-HA antibodies, followed by incubation with fluorescence-conjugated secondary antibodies, and observed under confocal laser scanning microscopy (Fig. 6, A and B). Both ZPB1 (green) and ZPC-HA (red) were detected on the surface of the ZPC-HA-expressing cells treated without permeabilization. A strong ZPC-HA signal was observed in the cytoplasm of permeabilized cells. Three-dimensional graphic images constructed from the multiple confocal images demonstrated that not only ZPC, but also ZPB1, were accumulated and distributed widely on the cell surface, and that localization of the two proteins was identical, indicating that ZPB1-ZPC complexes were formed and accumulated on the cell surface. As expected, no ZPB1 accumulation was observed in the case of mock transfectants.


Figure 76010306
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  		FIG. 6. Colocalized deposition of ZPB1 with ZPC around the ZPC-transfected COS-7 cells cultured in the presence of ZPB1. A) COS-7 cells transfected with an expression plasmid for ZPC-HA (pZPC-HA; panels 1, 2, 4, 5, 7, 8, 10, and 11) or a control plasmid (mock; panels 3, 6, 9, and 12) were cultured in the presence of ZPB1. The cells were fixed with (+; panels 1, 4, 7, and 10) or without (–; panels 2, 3, 5, 6, 8, 9, 11, and 12) the permeabilization treatment, treated with a combination of anti-ZPB1 (polyclonal anti-recombinant ZPB1; green; Alexa Fluor 488) and anti-HA (red; Alexa Fluor 568), and then observed under a laser-scanning confocal microscope equipped with a 488-nm (panels 1, 2, and 3) or 543-nm (panels 4, 5, and 6) excitation filter . Superimposed images constructed from the two color images are shown (Composite; panels 7, 8, and 9). Light images of differential interference contrast microscopy (Light-DIC) of the cells are also shown (panels 10, 11, and 12). Bar = 10 µm. B) COS-7 cells transfected with an expression plasmid for ZPC-HA were cultured in the presence of ZPB1. The cells were fixed with (+) or without (–) the permeabilization treatment, stained, and observed as described above. A series of 13–18 horizontal (XY) contiguous sections, 0.74–0.78 µm apart, were collected at each depth along the vertical (Z) axis of each cell from bottom to top (see right panel). Based on these data, the images of vertical sections (XZ and YZ) were constructed digitally using Zeiss LSM Image Browser Version 3.5 software. Typical XY sections are shown in blue frames. The constructed XZ and YZ sections, at a position shown by green and red lines on the XY sections (in blue frames), are indicated in the green and red frames, respectively. Depth of the XY sections is indicated as a blue line both in the XZ and YZ sections. Bar = 10 µm.

Release of ZPC from Transfected COS-7 Cells Cultured in the Absence of ZPB1

As shown in Figure 6, ZPC was required for ZPB1 in the medium to be accumulated on the cell surface. Another experiment was conducted to elucidate whether ZPC-HA alone accumulated on the surface of transfected cells. The transfected COS-7 cells were cultured for 2 days in the absence of serum ZPB1, fixed, and stained with anti-ZPC antibody as well as anti-HA antibody. As shown in Figure 7, when serum ZPB1 was not present in the medium, no ZPC accumulation on the surface of nonpermeabilized cells was observed by using anti-ZPC in addition to anti-HA. The intracellular signal of ZPC-HA was detected in the permeabilized cells, and the stained image with anti-ZPC (green) was identical to that with anti-HA (red).


Figure 76010307
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  		FIG. 7. Contribution of ZPB1 for ZPC accumulation on the surface of ZPC-expressing COS-7 cells. COS-7 cells, transfected with expression plasmid for ZPC-HA (pZPC-HA), were cultured in the absence of ZPB1. The cells were fixed with (+; panels 1, 3, 5, and 7) or without (–; panels 2, 4, 6, and 8) the permeabilization treatment, treated with a combination of anti-ZPC (green; Alexa Fluor 488) and anti-HA (red; Alexa Fluor 568), and then observed under a laser-scanning confocal microscope equipped with a 488-nm (panels 1 and 2) or a 543-nm (panels 3 and 4) excitation filter. Superimposed images constructed from the two color images are shown (Composite; panels 5 and 6). Light images of differential interference contrast microscopy (Light-DIC) of the cells are also shown (panels 7 and 8). Bar = 10 µm.

DISCUSSION

In our previous study, ZPB1 was solubilized from isolated egg-envelope with SDS or a urea solution without reducing agents, and was eluted as monomer and dimer into two separate fractions from a gel-filtration column in the presence of urea [10]. In contrast to the egg-envelope ZPB1, the ZPB1 secreted from liver cells was present stably as a soluble protein in blood, even in the absence of such denaturing agents or strong detergents. This indicates that noncovalent association of ZPB1 with the other ZP components is important for the egg-envelope matrix formation. Interestingly, the serum ZPB1 specifically bound to ZPC, but to neither ZPX2 nor ZPB1 itself blotted on the membrane (Fig. 1), and not only ZPB1 monomer, but also ZPB1 dimer, were coprecipitated with ZPC in a proportion similar to that of the egg envelope (see Fig. 3, C–E). These results, in addition to the fact that serum ZPB1 is not cross-linked to each other through disulfide bridges [17], suggest that serum ZPB1 interacts with ZPC secreted from granulosa cells, and then ZPB1 dimerization by intermolecular disulfide bonding is induced during the formation of ZPB1-ZPC heterocomplexes and further polymerization.

The disulfide-linked dimer of ZPB1 present in part in the isolated egg-envelope is believed to play important roles in not only the sperm activation during fertilization [10], but also the construction of fibrous matrix of egg envelope. Presumably, at least some conformational changes in ZPB1 molecules are required for a free sulfhydryl group to be exposed for interaction with a sulfhydryl or disulfide group of another ZPB1. The monoclonal anti-ZPB1, 8E1, could precipitate free ZPB1, but not ZPB1-ZPC complexes, in the culture medium (see Fig. 4). A simple explanation for this result is that the C-terminal region of the ZP domain in a ZPB1 molecule was masked by the associated ZPC, resulting in the prevention of the antibody, 8E1, from binding. Such ZP domain-dependent interaction between ZPB1 and ZPC could be reasonably expected, because the ZP domain proteins, including the egg-envelope ZP glycoproteins, are suggested to associate via ZP domains [19]. An alternative speculation is that a certain level of conformational changes in the C-terminal region of a ZPB1 molecule, including the 8E1 epitope, was induced through ZPB1-ZPC interaction or complex formation. We have ruled out the possibility that the epitope for 8E1, but not that for 5G9, of ZPB1 in the ZPB1-ZPC complexes in the COS-7 culture supernatant had already been removed by partial proteolytic degradation before secretion, because ZPB1 coprecipitated with ZPC-HA by anti-HA was immunostained with 8E1 as a protein band identical to that of egg-envelope ZPB1 (Fig. 3D). ZPB1 detected in the cell lysate of the ZPC transfectants cultured with ZPB1 was mainly the monomeric form, and the dimeric form was below the detectable level (Fig. 3F), whereas both the dimer and monomer of ZPB1 were detected in the ZPB1-ZPC heterocomplexes in the culture supernatant of the ZPC transfectants cultured with ZPB1 (Fig. 3C). The manner of ZPB1-ZPB1 interaction, including intermolecular disulfide-bonding formation, through the association with ZPC might differ between the cell surface and the culture medium. Further studies are needed to determine how ZPB1-ZPC polymerization and inter-ZPB1 disulfide cross-linking are induced during the egg-envelope construction in the laying hen's ovary. It would be of special interest to examine a possible C-terminal processing of serum ZPB1 after its transport to the oocyte surface, because such processing has recently been reported in fish ZP glycoproteins [27], which are also synthesized in liver.

After the separation from ZPC, ZPB1 could remain soluble even after removal of urea by dialysis, at least in a low-concentration (µg/ml level) solution [10]. In addition to this previous observation, the binding specificity of ZPB1 to ZPC, as demonstrated by using two distinct mABs in the present study, suggests that the association between ZPB1 and ZPC is not simply due to hydrophobic aggregation, but to some specific association that includes hydrogen and electrostatic bonds. We have previously suggested that the chicken egg-envelope is constructed with ZPB1 and ZPC, and that ZPX2 associates loosely with the ZPB1-ZPC complex because ZPX2 is easily released from the complex by a mechanical treatment, such as sonication [10] or vigorous washing [16, 17, 28]. This suggestion of egg-envelope construction by ZP proteins would be consistent with the results of the present study, which demonstrated that ZPB1 associated with ZPC, not ZPX2, and that only the two components, ZPB1 and ZPC, could form a type of matrix on the surface of cultured cells (see Figs. 1 and 6).

HA-tagged ZPC was successfully expressed, processed, and secreted in part by the transfected COS-7 cells (see Fig. 2) in a manner similar to endogenous ZPC of cultured chicken granulosa cells [16]. Mature native ZPC in the egg-envelope was detected as a 35-kDa signal in nonreducing SDS-PAGE (Fig. 1), and the calculated molecular mass of C-terminal hydrophobic peptide derived from the processing of proZPC at the conserved furin cleavage site was approximately 7 kDa. The sum of the masses for mature ZPC and the C-terminal peptide agrees well with that of probable proZPC (42 kDa) (see Fig. 2). Such a transient expression of ZPC has also been reported on Chinese hamster ovary (CHO) cells by using Japanese quail ZPC [36–38]. Furthermore, some ZP domain proteins, including FLAG-tagged mouse ZPC, are properly processed in and secreted from transfected CHO cells [19]. These results, and those of our present study, suggest that the intracellular accumulation of proproteins, and the extracellular secretion of processed and probably matured proteins, are common in both of the two cell lines originated from the different organs (kidney and ovary). COS-7 cells are known to endogenously express a processing enzyme, furin, and can process some ectopically expressed prohormones, resulting in the secretion of bioactive hormones [39–42]. As such, it is expected that the 35-kDa ZPC-HA secreted into culture medium by COS-7 cells is matured and correctly folded ZPC, with HA tag at its C-terminus.

The confocal laser scanning microscopic analyses clearly demonstrated that ZPB1 was deposited and accumulated together with the secreted ZPC on the cell surface (Fig. 6). No accumulation of ZPB1 on the mock transfectants was as expected, but it should be noted that the ZPC signal was also below detectable levels on the surface of pZPC-HA transfectants cultured in the absence of ZPB1 (see Fig. 7), which actually expressed and secreted ZPC-HA (see Fig. 2). Thus, it would be unlikely that ZPB1 was accumulated around the cell by binding to ZPC, which had been localized or predeposited alone on the cell surface. The ZPB1 would more likely associate with the secreted ZPC near the cell, leading to the accumulation of ZPB1-ZPC matrix-like complexes around the cell.

Although biochemical and physicochemical differences between the ZPB1-ZPC complexes present in the medium, and those accumulated on the cell surface, are still unclear, we speculate that a part of the ZPB1-ZPC complexes was diffused into the medium as small complexes or particles before larger particles or matrices were formed on the cell surface. In fact, in preliminary experiments, we have found that the ZPB1-ZPC complexes were precipitated by a simple ultracentrifugation at 100 000 x g for 60 min (data not shown), indicating that ZPB1-ZPC complexes in the culture medium were present as small particles. In hen's ovary, the secreted ZPC would be accumulated in a narrow space between a granulosa cell layer and an oocyte, and, therefore, such small particles would also participate in the construction of the egg-envelope matrix.

In eukaryotes, aside from the egg-envelope components, several extracellular or membrane proteins sharing the ZP domain (ZP domain proteins) have been reported [18]. When these ZP domain proteins are in disorder, some of them, such as {alpha}- and ß-tectorins [43], Tamm-Horsfall protein [44], transforming growth factor ß receptor type III [45], and DMBT1 (deleted in malignant brain tumors 1) [46], are thought to be associated with human pathologies. ZP domain proteins, including the egg-envelope ZP glycoproteins, are often present in filaments and/or matrices of specific tissues and organs. The results on the formation of ZPB1-ZPC heterocomplexes obtained in the present study could provide new insight into the molecular mechanism for construction of noncollagenous extracellular matrices by ZP domain proteins.

FOOTNOTES

1Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology in Japan to N.A., D.N., and T.M. Back

Correspondence: 2 Tsukasa Matsuda, Department of Applied Molecular Biosciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. FAX: 81 52 789 4128; e-mail: tmatsuda{at}agr.nagoya-u.ac.jp

Received: 6 August 2006.

First decision: 23 August 2006.

Accepted: 12 September 2006.

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