Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex Saeko Kanai1, Naoto Yonezawa1, Yuichiro Ishii1, Masaru Tanokura2 and Minoru Nakano1
1 Graduate School of Science and Technology, Chiba University, Japan 2 Graduate School of Agriculture and Life Science, The University of Tokyo, Japan
Keywords baculovirus-Sf9; fertilization; glycoprotein; zona pellucida; ZP domain
Correspondence M. Nakano, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Fax: +81 43 290 2874 Tel: +81 43 290 2794 E-mail: mnakano@faculty.chiba-u.jp
(Received 18 April 2007, revised 27 July 2007, accepted 24 August 2007)
doi:10.1111/j.1742-4658.2007.06065.x
The zona pellucida (ZP) is a transparent envelope that surrounds the mam- malian oocyte and mediates species-selective sperm–egg interactions. Por- cine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glyco- proteins (ZPGs) using baculovirus in insect Sf9 cells. Here we established a similar method for expression of bovine ZPGs. The recombinant ZPGs were secreted into the medium and purified by metal-chelating column chromatography. A mixture of bovine recombinant ZP3 (rZP3) and rZP4 coexpressed in Sf9 cells exhibited inhibitory activity for bovine sperm–ZP binding similar to that of a native bovine ZPG mixture, whereas neither bovine rZP3 nor rZP4 inhibited binding. An immunoprecipitation assay revealed that the coexpressed rZP3 ⁄ rZP4 formed a hetero-complex. We examined the functional domain structure of bovine rZP4 by constructing ZP4 mutants lacking the N-terminal domain or lacking both the N-termi- nal and trefoil domains. When either of these mutant proteins was coexpressed with bovine rZP3, the resulting mixtures exhibited inhibitory activity comparable to that of the bovine rZP3 ⁄ rZP4 complex. Hetero-com- plexes of bovine rZP3 and porcine rZP4, or porcine rZP3 and bovine rZP4, also inhibited bovine sperm–ZP binding. Our results demonstrate that the N-terminal and trefoil domains of bovine rZP4 are dispensable for formation of the sperm-binding active bovine rZP3 ⁄ rZP4 complex and, furthermore, that the molecular interactions between rZP3 and rZP4 are conserved in the bovine and porcine systems.
Abbreviations ACA, Amaranthus candatus agglutinin; BO, Brackett and Oliphant; FITC, fuorescein isothiocyanate; Fuc, fucose; GNA, Galanthus nivalis agglutinin; LC, liquid chromatography; LCA, Lens culinaris agglutinin; Man, mannose; MOI, multiplicity of infection; PA, pyridylamino; PHA, Phaseolus vulgaris agglutinin; PSA, Pisum sativum agglutinin; RCA120, Ricinus communis agglutinin; rZP2, recombinant ZP2; rZP3, recombinant ZP3; rZP3FLAG, FLAG-tagged rZP3; rZP4, recombinant ZP4; rZP4FLAG, FLAG-tagged rZP4; rZPG, recombinant ZPG; ZP, zona pellucida; ZPG, zona pellucida glycoprotein.
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Mammalian oocytes are surrounded by the zona pellu- cida (ZP), a transparent envelope that mediates several critical aspects of fertilization, including species-selective sperm recognition, blocking of polyspermy, and protec- tion of the oocyte and embryo until implantation [1–3]. The ZP consists of three or four kinds of glycoproteins (ZPGs). Human and rat ZPs consist of four ZPGs (ZP1, ZP2, ZP3, and ZP4) [4,5], whereas porcine and bovine ZPs comprise three ZPGs (ZP2, ZP3, and ZP4) that cor- respond to ZPA, ZPC, and ZPB, respectively, in other nomenclature [6]. Murine ZP also consists of three ZPGs (ZP1, ZP2, and ZP3) [7]. Porcine, bovine and murine ZPs have ZP2 and ZP3 in common, whereas ZP1 and ZP4 are products of distinct genes [8]. All ZPGs contain a domain that consists of (cid:2) 260 amino acids and contains eight conserved Cys residues [9].
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is consistent with a suggested model
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rZP2 (lane 1),
analyses of
Fig. 1. Recombinant bovine ZP proteins. (A) Schematic representa- tion of the rZP2, rZP4, rZP4136)464, rZP4182)464 and rZP3 polypep- tides. These recombinant polypeptides were expressed with His- and S-tags at their N-termini. Open square, region specific to ZP2, ZP4, or ZP3; dotted square, trefoil domain; filled square, ZP domain. Arrows indicate the putative furin cleavage sites that con- stitute the C-termini of the expressed polypeptides. The calculated molecular masses of the polypeptide moieties of the recombinants, excluding extra peptides derived from the transfer vector, are shown in kDa to the right of each polypeptide. (B, C) SDS ⁄ PAGE and immunoblot rZP4 (lane 2), rZP4136)464 (lane 3), rZP4182)464 (lane 4), and rZP3 (lane 5). The pro- teins were expressed in Sf9 cells, secreted into the culture med- ium, isolated using metal-chelation column chromatography, and detected by SDS ⁄ PAGE (B) or by immunoblot analysis using anti- bodies specific for each of the ZPGs (C). Arrowheads indicate the recombinant protein bands. Molecular mass markers are indicated in kDa on the left of each panel.
In mice, ZP3 is thought to be involved in gamete recognition [1–3]. ZP assembly is controlled by short, hydrophobic sequences in the C-terminal propeptides of ZPG precursors, and requires the ZP domains of ZP2 and ZP3 [10,11]. The molar ratio of murine tran- scripts is estimated at ZP1 ⁄ ZP2 ⁄ ZP3 ¼ 1 : 4 : 4 [12], a ratio that in which a ZP2 ⁄ ZP3 heterodimer forms filaments that are crosslinked by a ZP1 dimer [13]. However, the molar ratio of ZPGs in the murine ZP does not seem to correspond to the molar ratio of their transcripts [7]. In pigs, the estimated protein molar ratio of ZP2 ⁄ ZP3 ⁄ ZP4 is 1 : 6 : 6 [14]. Although neither ZP3 nor ZP4 exhibits porcine sperm-binding activity by itself, a high molecular mass ZP3 ⁄ ZP4 hetero-complex does exhibit this activity [15,16].
When subjected to nonreducing SDS ⁄ PAGE, bovine ZPGs form a band at an average apparent molecular mass of 74 kDa, which is broad owing to heterogeneity in glycosylation [17]. After endo-b-galactosidase-cata- lyzed removal of N-acetyl-lactosamine repeats at the nonreducing ends of carbohydrate chains, bovine ZP2, ZP3 and ZP4 migrate as three distinct bands of appar- ent molecular masses of 72, 45 and 58 kDa, respec- tively, under nonreducing conditions [17]. Under reducing conditions, the apparent molecular masses of the endo-b-galactosidase-digested components shift to 76, 63 and 21 kDa for ZP2, to 47 kDa for ZP3, and to 68 kDa for ZP4 [17]. Processing of bovine ZP2 occurs at a specific site upon fertilization, and yields disulfide- bonded polypeptides of 63 and 21 kDa [17,18]. A large fraction of ZP2 obtained from unfertilized eggs is already processed, probably as an artefact of the prep- aration, but the 76 kDa band of ZP2 completely dis- appears upon fertilization [17,18].
activity [21]. The components were not completely resolved by HPLC, indicating cross-contamination; thus, whether each bovine ZPG has sperm-binding activity by itself is not yet clear. A previous report that bovine sperm–egg binding is inhibited in the presence of anti-porcine ZP3 or ZP4 suggests that both ZP3 and ZP4 are involved in sperm–ZP binding [23].
The amino acid sequences of porcine and bovine ZP2, ZP3, and ZP4, which were previously determined by cDNA cloning and sequencing [6,19–21], are 77%, 85% and 75% identical, respectively. The mature por- cine and bovine ZP4 polypeptides differ in that an N-terminal region corresponding to residues 1–135 of bovine ZP4 (with the translation initiation Met num- bered 1) is lacking in the porcine protein [19,21,22] (Fig. 1A). The estimated protein molar ratio of bovine ZP2 ⁄ ZP3 ⁄ ZP4 is 1 : 2 : 1 [21], which differs signifi- cantly from the porcine molar ratio, suggesting that the structures of the bovine and porcine ZPs are differ- ent.
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In mice, in vitro studies have proposed that sperm ligands consist of O-linked carbohydrate chains linked to Ser332 and Ser334 of ZP3 [24,25]. Nevertheless, a recent structural analysis using MS did not show evi- dence for glycosylation [26]. The in vivo studies per- formed to date using transgenic mice lacking each glycosyltransferase gene do not support the involve- ment of carbohydrate chains of mouse ZP in sperm binding [27–29]. In pigs, neutral tri-antennary and tetra-antennary complex-type chains have the strongest sperm-binding activity of the N-linked chains of ZP [30], and O-linked chains also have sperm-binding activity [31]. The nonreducing terminal b-galactosyl In a previous study, we partially separated an endo- b-galactosidase-digested bovine ZPG mixture into the three three components by RP-HPLC [21]. Of components, ZP4 exhibited the strongest sperm-bind- ing activity. ZP2 and ZP3 exhibited much weaker
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that porcine, murine and human ZPGs are processed at consensus sites for furin or furin-like processing enzymes [35–38]. In at least three murine ZPGs and in porcine ZP3 and ZP4, this processing is followed by removal of the basic amino acid residues in the consen- sus sites by a carboxypeptidase [26,35]. We presume that bovine ZPGs are processed similarly.
Two N-terminal deletion mutants of bovine rZP4 were also expressed in this study. The rZP4136)464 mutant lacks residues Lys25 to Pro135 and consists of the trefoil and ZP domains of rZP4. The rZP4182)464 mutant lacks residues Lys25 to Tyr181 and thus con- sists only of the ZP domain (Fig. 1A).
the complex-type N-linked chains are residues of involved in sperm binding [32]. In cows, the major neutral N-linked chain of ZP consists of only one structure, a high-mannose-type chain containing five mannose residues [33]. Thus, the structures of the por- cine and bovine neutral chains are quite different. a-Mannosyl residues at nonreducing termini are essen- tial for the sperm-binding activity of bovine ZP [34], although the participation of O-linked chains in sperm binding has not yet been investigated. Recently, we reported that porcine recombinant ZPGs (rZPGs) expressed in insect Sf9 cells have pauci-mannose and high-mannose-type chains and bind to bovine sperm but not to porcine sperm [16]. This result supports a significant in bovine role for a-mannosyl residues sperm recognition and also demonstrates the utility of rZPG expression in Sf9 cells.
The apparent molecular masses of the recombinant proteins, as determined by SDS ⁄ PAGE, agreed with the molecular masses predicted from their encoded amino acid sequences, and immunoblots with specific antibodies to ZPG confirmed the presence of the pro- teins (Fig. 1B,C). The absorbance at 280 nm of the eluted fractions was used to estimate the yield of the recombinant proteins; about 15 lg of each rZPG was obtained from 200 mL of culture medium.
Sperm-binding activity of bovine rZPGs In this study, we used the Sf9 expression system to obtain each of the bovine rZPGs without the possibil- ity of contamination by the other rZPGs and examined the sperm-binding activity and complex formation of these rZPGs. We also created deletion mutants of recombinant (r)ZP4 to examine whether its N-terminal region and trefoil domain are necessary for sperm–ZP binding activity.
Results
Expression of bovine rZP2, rZP3, rZP4 and rZP4 mutants in Sf9 cells infected with recombinant baculoviruses
synthesized as the bovine We examined the inhibitory activity of rZPGs towards binding of bovine sperm to plastic wells coated with solubilized bovine ZP (Method 1; Fig. 2). In the presence of 2 lgÆmL)1 of solubilized bovine ZP, sperm binding to solubilized ZP-coated wells was reduced to its plateau level, which was about 10% of the level observed in the absence of solubilized ZP. In contrast, none of the bovine rZPGs significantly inhibited binding.
Native ZPGs are transmembrane proteins, processed at a site N-terminal to their trans- membrane regions, and then secreted as mature poly- peptides without their transmembrane regions. Here, His- and S-tagged recombinant polypeptides corre- sponding to bovine ZP2 (Ile36 to Arg637), ZP3 (Arg32 to Arg348) and ZP4 (Lys25 to Arg464) were expressed in Sf9 cells (Fig. 1A). The N-termini of these rZPGs correspond to those previously reported for mature native bovine ZPGs [17]. We presume that the N-ter- the native ZP3 and ZP4 polypeptides are mini of blocked and that the N-termini reported previously might have been a result of degradation [17]. Thus, the N-termini of rZP3 and rZP4 expressed here are likely to closely correspond to the N-termini of their native counterparts. Sf9 cells were coinfected with the appropriate re- combinant viruses to form rZP3 ⁄ rZP4, rZP2 ⁄ rZP4, rZP2 ⁄ rZP3 and rZP2 ⁄ rZP3 ⁄ rZP4 mixtures. Expression of the mixtures was confirmed by SDS ⁄ PAGE (Fig. 3A) and immunoblot analysis (data not shown). Bovine sperm binding to solubilized bovine ZP-coated wells was not significantly inhibited by rZP2, rZP3, or rZP4 (Fig. 3B; see also Fig. 2), but it was inhibited by the rZP3 ⁄ rZP4 mixture. The mixture reduced binding to a level similar to that observed with solubilized bovine ZP (Fig. 3B). The rZP2 ⁄ rZP4 and rZP2 ⁄ rZP3 mixtures did not significantly inhibit binding (Fig. 3B). When rZP3 and rZP4 were expressed separately in Sf9 cells and then mixed, the mixture did not inhibit binding (Fig. 3B).
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The C-termini of the mature bovine ZP2, ZP3 and ZP4 polypeptides have not yet been determined. The immature proteins have putative furin-processing sites at Arg634 to Arg637, Arg345 to Arg348, and Arg461 to Arg464, respectively. Recent studies have revealed To assess the effect of rZP2 on the inhibitory activ- ity of the rZP3 ⁄ rZP4 mixture, we compared the inhibi- tory activity of the rZP3 ⁄ rZP4 mixture to that of the rZP2 ⁄ rZP3 ⁄ rZP4 mixture. The total amount of rZP3 and rZP4 in the mixtures was the same and was equal
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Fig. 2. Inhibitory effects of rZP2, rZP3, rZP4 and solubilized bovine ZP on bovine sperm-solubilized ZP binding. Solubilized native bovine ZP was adsorbed to each well of a 96-well plate (0.2 lg per well; Method 1). Bovine sperm (4 · 105) were incubated with 0.2, 0.4 or 0.6 lg of solubilized ZP (·), rZP2 (r), rZP3 (m), or rZP4 (j) for 30 min, and then transferred to the coated wells. After incubation for 2 h, the wells were washed and 50 lL of glycerol ⁄ NaCl ⁄ Pi was added to each well. The sperm that bound to the ZP were recov- ered from the wells by vigorous pipetting, and the number of sperm in 0.1 lL of the suspension was determined. The number of sperm binding to the ZP in the absence of inhibitors is designated 100%. Assays were repeated at least three times, and the data shown represent means ± SD.
In a previous study, we examined the inhibitory activity of each bovine ZPG for the binding of sperm to ZP-encased eggs using an in vitro competition assay (Method 2 [21]). Recently, we established a competi- tion assay using solubilized ZP-coated plastic wells (Method 1 [16]). In Method 1, washing to remove sperm loosely attached to ZP does not require mouth pipetting; therefore, Method 1 is technically much eas- ier and more reproducible than Method 2. The inhibi- tory activity of a larger number of ZPGs can be examined at one time in Method 1 than in Method 2. However, Method 2 is an accepted assay system that has been used to evaluate the inhibitory activity of materials sperm–ZP binding in many species, including mouse, cow, and pig. Thus, we determined whether Method 2 yields parallel results to Method 1. In Method 2, bovine sperm binding to bovine eggs was not inhibited by rZP3 or rZP4, whereas binding was reduced by the rZP3 ⁄ rZP4 mixture to a level simi- lar to that observed with solubilized native bovine ZP (Fig. 4). Thus, the two competition assay systems gave similar results.
We examined whether the incubation of bovine sperm with solubilized bovine ZP or rZP3 ⁄ rZP4 induced the acrosome reaction of the sperm by using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglu- tinin (PSA) (FITC-PSA). This lectin binds to the acro- somal area of acrosome-intact, acrosome-damaged and to 0.2 or 0.4 lg (Fig. 3C). The inhibitory activity of rZP2 ⁄ rZP3 ⁄ rZP4 was not significantly different from that of rZP3 ⁄ rZP4.
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Fig. 3. Inhibitory effects of various bovine rZPG mixtures on bovine sperm-solubilized ZP binding. (A) rZP2 ⁄ rZP4 (lane 1), rZP2 ⁄ rZP3 (lane 2), rZP3 ⁄ rZP4 (lane 3), rZP3 ⁄ rZP4136)464 (lane 4), rZP3 ⁄ rZP4182)464 (lane 5) and rZP2 ⁄ rZP3 ⁄ rZP4 (lane 6) mixtures were expressed by simulta- neous infection of Sf9 cells with the two or three corresponding recombinant viruses. The rZPGs were collected from the culture superna- tant using metal-chelation column chromatography and detected by SDS ⁄ PAGE with silver staining. Arrowheads indicate the recombinant protein bands. Molecular mass markers are indicated in kDa. (B) Bovine sperm were incubated with 0.2 lg of solubilized native ZP, 0.4 lg of each rZPG, 0.27 lg of each bi-component rZPG coexpressed mixture, or a mixture of 0.4 lg of rZP3 and 0.4 lg of rZP4 that were sepa- rately expressed, purified and mixed (rZP3 + rZP4) for 30 min, and the inhibitory effect of the proteins was determined by Method 1 as described in the legend to Fig. 2. The number of sperm binding to the ZP in the absence of inhibitors is designated 100%. Assays were per- formed at least three times, and the data shown represent means ± SD. (C) Bovine sperm were incubated for 30 min with a coexpressed mixture of rZP3 and rZP4 or a coexpressed mixture of rZP2, rZP3, and rZP4. The total amount of rZP3 and rZP4 was 0.2 or 0.4 lg, and the inhibitory effect of the rZPG mixtures was determined by Method 1 as described in the legend to Fig. 2.
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Fig. 4. Inhibitory effects of various bovine rZPGs on bovine sperm– egg binding. Bovine sperm were incubated with 0.7 lg of solubilized native ZP, rZP3, rZP4 or rZP3 ⁄ rZP4 mixture for 30 min and then incubated with bovine eggs. The inhibitory effects of the proteins were determined by Method 2. The number of sperm binding to eggs in the absence of inhibitors is designated 100%. Assays were performed six times, and the data shown represent means ± SD.
sperm were observed for each incubation condition. The percentages of sperm positively stained with FITC-PSA were 97.8 ± 0.9% for the sperm before incubation with the zona proteins, 93.8 ± 2.2% for the sperm after 3 h of incubation in the absence of the zona proteins, 94.2 ± 3.6% for the sperm after 3 h of incubation with solubilized bovine ZP, and 92.8 ± 1.3% for the sperm after 3 h of incubation with rZP3 ⁄ rZP4. This indicates that the percentages of acrosome-reacted sperm, which were not stained with FITC-PSA, increased significantly but only slightly after 3 h of incubation in the absence and also in the presence of zona proteins, and therefore neither solubilized bovine ZP nor rZP3 ⁄ rZP4 induced the acrosome reaction of bovine sperm under the experi- mental conditions used in this study. Neither solubilized bovine ZP nor rZP3 ⁄ rZP4 affected sperm motility as compared to the sperm incubated without the zona proteins (data not shown).
Fig. 5. Indirect immunofluorescence staining of sperm-bound bovine rZPGs. Suspensions of bovine sperm (50 lL at 2 · 106 mL)1) were incubated with 0.2 lg of rZP2, rZP3, rZP4, rZP3 ⁄ rZP4, rZP3 ⁄ rZP4136)464, rZP3 ⁄ rZP4182)464 or solubilized native ZP for 30 min. The proteins that bound to sperm were detected using a mixture of anti-porcine ZP2, ZP3, and ZP4 as the primary antibodies, and Alexa Fluor 546-conju- gated goat anti-(rabbit IgG) as the secondary antibody. The sperm were observed using fluorescence microscopy. As a control, the sperm were incubated without solubilized native ZP or rZPGs and then treated with the antibodies. Insets, magnified fluorescence images of the sperm head. Phase, phase-contrast image; fluorescence, fluorescence image.
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partially acrosome-reacted bovine sperm but not to acrosome-reacted bovine sperm [39]. We performed this experiment four times, and in each experiment, 100 The binding of sperm to rZPGs and to solubi- lized ZP was compared by indirect immunofluores- cence detection of rZPG-bound sperm. Solubilized, native bovine ZP and the rZP3 ⁄ rZP4 mixture bound to the acrosomal region of bovine sperm, as shown by fluorescent staining, but rZP2, rZP3 and rZP4 did not bind to sperm (Fig. 5). These results suggest that the inhibition of sperm–ZP binding by the rZP3 ⁄ rZP4 mixture is due to specific binding of rZP3 ⁄ rZP4 to to the acrosomal area of sperm, but not due
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the acrosome reaction of sperm by induction of rZP3 ⁄ rZP4.
inhibitory slightly
Effect of N-terminal deletions of rZP4 on the sperm-binding activity of rZP3 ⁄ rZP4 Neither rZP4136)464 nor rZP4182)464 significantly inhib- ited bovine sperm-solubilized ZP binding (data not shown). Mixtures of rZP3 with each of these N-termi- nal deletion mutants were prepared by coinfection of Sf9 cells with the corresponding baculoviruses, and protein expression was confirmed by SDS ⁄ PAGE (Fig. 3A). The rZP3 ⁄ rZP4136)464 mixture exhibited inhibitory activity similar to that of solubilized native ZP and rZP3 ⁄ rZP4 (Fig. 3B), indicating that residues 25–135 of rZP4 are not necessary for the sperm-bind- rZP3 ⁄ rZP4182)464 rZP3 ⁄ rZP4. The ing activity of mixture was the than less rZP3 ⁄ rZP4136)464 mixture. Although statistically signif- icant, this difference was very small, indicating that the trefoil domain of rZP4 is not essential for the sperm- binding activity of rZP3 ⁄ rZP4.
The rZP3 ⁄ rZP4136)464 and rZP3 ⁄ rZP4182)464 mix- tures exhibited significant binding to the acrosomal region, as shown by fluorescent staining (Fig. 5), in a manner similar to the rZP3 ⁄ rZP4 mixture, suggesting that the inhibition of sperm-solubilized ZP binding by the mixtures is due to specific binding of the mixtures to the acrosomal area of sperm. immunoprecipitation, neither rZP3 nor rZP4 was pre- cipitated by the anti-FLAG gels (Fig. 6A, lane 4 in the left panel), but they were precipitated using S-protein agarose from the supernatant of the immunoprecipita- lane 5 in the tion with anti-FLAG gels (Fig. 6A, left panel). Antibody to FLAG detected rZP3FLAG lanes 6 and 7 in the right panel) but not (Fig. 6A, rZP3 or rZP4 (Fig. 6A, lanes 3 and 5 in the right panel). When the rZP3FLAG ⁄ rZP4 mixture coexpressed in Sf9 cells was subjected to immunoprecipitation, rZP4 and rZP3FLAG were coprecipitated and detected by immunoblots with antibody to His (Fig. 6A, lane 7 in the left panel) and antibody to FLAG (Fig. 6A, lane 7 in the right panel), respectively. These results indicate that there was no nonspecific binding of rZP4 or rZP3 ⁄ rZP4 mixture to the anti-FLAG gels and that rZP4 was pulled down by the anti-FLAG gels through the FLAG-tag of rZP3FLAG. Thus, we found that the immunoprecipitation assay using FLAG-tag is useful for examining complex formation between rZPGs. When rZP3FLAG and rZP4 were expressed separately in Sf9 cells and the culture supernatants were mixed, incubated overnight, and subjected to immunoprecipi- tation using anti-FLAG gels, rZP3FLAG was pulled down, as revealed by the detection with antibody to FLAG (Fig. 6B, lane 4 in the right panel), but rZP4 was not coprecipitated with rZP3FLAG (Fig. 6B, lane 4 in the left panel). This result indicates that the sepa- rately expressed rZP3 and rZP4 did not form a com- plex. When the rZP3FLAG ⁄ rZP4182)464 mixture
Complex formation of FLAG-tagged rZP3 (rZP3FLAG) with rZP4
left panel), indicating that
the FLAG-tag pulled down coex- pressed in Sf9 cells was subjected to immunoprecipi- tation, rZP4182)464 and rZP3FLAG were coprecipitated by anti-FLAG gels and detected by antibody to His lane 4 in the left panel) and antibody to (Fig. 6C, FLAG (Fig. 6C, lane 4 in the right panel), respec- tively. When the coexpressed rZP3 ⁄ rZP4182)464 mix- ture was subjected to immunoprecipitation, neither rZP4182)464 was detected in the pellet rZP3 nor (Fig. 6C, lane 1 in the left panel) but both were pulled down by S-protein agarose from the superna- tant of the immunoprecipitation (Fig. 6C, lane 2 in rZP3FLAG and the rZP4182)464 formed a complex and that the complex was of through rZP3FLAG.
correlated with the inhibitory activity of
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These results of the immunoprecipitation assay indi- cate that complex formation between rZP3 and rZP4 is the rZP3 ⁄ rZP4 mixture for sperm–ZP binding. In addition, these results indicate that the N-terminal and trefoil domains of rZP4 are dispensable for complex forma- tion of rZP4 with rZP3. To examine whether rZP3 associates with rZP4, we prepared rZP3 whose N-terminal His-tag was changed to FLAG-tag (rZP3FLAG) and investigated whether rZP4 (without FLAG-tag) was coimmunoprecipitated with rZP3FLAG using anti-FLAG M2 gels. rZP3FLAG expressed alone in Sf9 cells was precipitated with anti- FLAG gels and detected by antibody to FLAG (Fig. 6A, lane 6 in the right panel) but not by antibody to His (Fig. 6A, lane 6 in the left panel). The bands indicated by closed circles in Fig. 6 were detected in the culture supernatants both in the absence and in the presence of baculovirus infection, and therefore were unrelated to rZPGs. rZP4 expressed alone was not pre- cipitated by the anti-FLAG gels, as rZP4 was not detected by antibody to His in the pellet (Fig. 6A, lane 2 in the left panel), although the rZP4 was precip- itated using S-protein agarose from the supernatant of the immunoprecipitation from the anti-FLAG gels lane 3 in the left panel). When the coex- (Fig. 6A, subjected to the pressed rZP3 ⁄ rZP4 mixture was
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(C)
Fig. 6. Complex formation between rZP3FLAG and rZP4. (A) Immu- noprecipitation of the coexpressed mixture of rZP3FLAG ⁄ rZP4. Cul- ture supernatants without rZPGs (lane 1 in each panel), containing rZP4 expressed alone (lanes 2 and 3 in each panel), containing coexpressed rZP3 ⁄ rZP4 mixture (lanes 4 and 5 in each panel), con- taining rZP3FLAG expressed alone (lane 6 in each panel), or contain- ing coexpressed rZP3FLAG ⁄ rZP4 mixture (lane 7 in each panel), as indicated above each panel, were subjected to anti-FLAG immuno- precipitation. The rZPGs pulled down by the anti-FLAG gels (F) were detected by immunoblotting with antibody to His (left panel) and with antibody to FLAG (right panel). The rZP3 and rZP4 remain- ing in the supernatant after the immunoprecipitation were sub- jected to pull-down by S-protein agarose (S) to examine the expression of the rZPGs. (B) Immunoprecipitation of rZP3FLAG ⁄ rZP4 mixture individually expressed and then combined. Culture superna- tants containing rZP4 expressed alone (lanes 1 and 2 in each panel), rZP3FLAG expressed alone (lane 3 in each panel), or a mix- ture of rZP3FLAG and rZP4 individually expressed, mixed, and incu- bated overnight (lane 4 in each panel), as indicated above each panel, were subjected to anti-FLAG immunoprecipitation. The rZPGs pulled down by anti-FLAG gels (F) were detected by immu- noblotting with antibody to His (left panel) and with anti-FLAG M2 (right panel). rZP4 remaining in the supernatant after the immuno- precipitation was subjected to pull-down by S-protein agarose (S) Immunoprecipitation of to examine the expression of rZP4. rZP3FLAG ⁄ rZP4182)464 mixture coexpressed in Sf9 cells. Culture supernatants containing coexpressed rZP3 ⁄ rZP4182)464 (lanes 1 and 2 in each panel), rZP3FLAG expressed alone (lane 3 in each panel), or coexpressed rZP3FLAG ⁄ rZP4182)464 (lane 4 in each panel), as indi- cated above each panel, were subjected to anti-FLAG immunopre- cipitation. The rZPGs pulled down by anti-FLAG gels (F) were detected by immunoblotting with antibody to His (left panel) and with anti-FLAG M2 (right panel). rZP3 and rZP4182)464 remaining in the supernatant after the immunoprecipitation were subjected to pull-down by S-protein agarose (S) to examine the expression of the rZPGs (lane 2 in each panel). The rZP3 and rZP3FLAG bands are indicated by arrowheads in (A), (B), and (C). The rZP4 band is indi- cated by an arrow in (A) and (B). The ZP4182)464 band is indicated by an asterisk in (C). Bands detected by the antibodies but unre- lated to rZPGs are indicated by closed circles in (A), (B), and (C). Molecular mass markers are indicated in kDa on the left of each panel in (A), (B), and (C). IB, immunoblot.
Glycosylation of rZPGs
The carbohydrate moieties of the rZPGs were analyzed by digestion with glycopeptidase F. The mobility of rZP3 on SDS ⁄ PAGE increased as digestion progressed (Fig. 7A), and three bands with higher mobilities appeared, indicating that rZP3 has three N-linked chains. Although the mobilities of rZP2 and rZP4 also increased after digestion with glycopeptidase F, indi- cating that rZP2 and rZP4 have N-linked chains (Fig. 7A), the resulting bands were not sufficiently resolved to deduce the number of N-linked chains in these proteins. Native bovine ZP2 has three N-linked chains [40], but the numbers of N-linked chains in native bovine ZP3 and ZP4 have not been reported. Therefore, whether the N-linked glycosylation charac- teristics of the recombinant proteins are similar to those of their native counterparts cannot be deter- mined at present. ([M + H]+) [41–43]. Two minor peaks were also observed by LC, and were assigned as Man2-GlcNAc- (Fuc-)GlcNAc-PA and Man3-GlcNAc-GlcNAc-PA from m ⁄ z ¼ 973.3 ([M + H]+) and 989.4 ([M + H]+), respectively [41–43]. The calculated m ⁄ z ([M + H]+) values of these structures were 1135.4, 973.4, and 989.4, respectively. We carbohydrate examined the
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fucose) structures of rZP4136)464 by liquid chromatography (LC) ⁄ MS analy- sis of its pyridylaminated chains. This protein was cho- sen for MS analysis because its yield was the highest among the bovine rZPGs described here. Only one major peak was observed by LC, and was assigned as Man3-GlcNAc-(Fuc-)GlcNAc-pyridylamino (PA) from m ⁄ z ¼ 1135.5 (Man, mannose; Fuc, We also compared the carbohydrate structures of the recombinant and native ZPGs using five different lectins. The two ZP4 deletion mutants and all three rZPGs were recognized by Galanthus nivalis agglutinin (GNA) and Lens culinaris agglutinin (LCA) (Fig. 7B), but not by Ricinus communis agglutinin (RCA120), Phaseolus vulgaris agglutinin (PHA-L4), or Amaranthus
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A
B
Fig. 7. N-glycans of bovine rZPGs. (A) The rZP2, rZP3 and rZP4 proteins were digested with glycopeptidase F for 0 min or 24 h (for rZP2 and rZP4), or for 0, 1 or 5 min or 24 h (for rZP3), and the mobility shifts of the rZPGs on SDS ⁄ PAGE (8% separating gel) were examined. After 1 min of digestion, the rZP3 sample yielded three bands (indicated by bars) of higher mobility than undigested rZP3 (0 min), indicating that rZP3 contains three N-linked chains. After 24 h of digestion, rZP2 and rZP4 also migrated faster than undigested rZP2 and rZP4 (0 min), indicating that rZP2 and rZP4 contain N-linked chain(s) as well. The bands were not sufficiently resolved, however, to allow determination of the number of N-linked chains. Molecular mass markers are indicated in kDa on the left of each panel. (B) GNA and LCA recognized the endo-b-galactosidase-digested native bovine ZPGs (lane 1 in each panel), as expected from the reported structures of the major N-linked chains [33]. rZP2 (lane 2), rZP4 (lane 3), rZP4136)464 (lane 4), rZP4182)464 (lane 5) and rZP3 (lane 6) were also recognized by GNA and LCA. Molecular mass markers are indicated in kDa on the left of each panel.
candatus agglutinin (ACA) (data not shown). In con- trast, all tested lectins recognized native bovine ZP2, ZP3, and ZP4. This latter result is consistent with the the bovine ZP [33]; a native known structures of bovine ZPG mixture has a high-mannose-type chain and acidic di-antennary, tri-antennary, and tetra-anten- nary complex-type chains. The lectin staining results for rZP4136)464 are consistent with the above MS assignments. N-linked chains of similar structure to those of rZP4136)464; i.e. pauci-mannose-type chains with or without fucose, may be abundant in rZPGs, and these chains were recognized by GNA and LCA. Since rZPGs were not recognized by RCA or PHA-L4, complex-type chains may not be abundant in rZPGs.
The lectin-staining results for rZPGs and the MS results for rZP4136)464 are consistent with the major structures of N-linked chains found in recombinant glycoproteins expressed in Sf9 cells, i.e. pauci-man- nose-type chains with or without fucose residues linked to the innermost GlcNAc residue [41–43].
Sperm-binding activity of interspecific mixtures of porcine and bovine rZP3 and rZP4
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porcine ZP4, or porcine ZP3 and bovine ZP4. We examined these mixtures for inhibitory activity towards bovine sperm-solubilized ZP binding after confirming expression by immunoblotting (Fig. 8A). Both of the interspecific rZP3 ⁄ rZP4 mixtures inhibited binding to an extent similar to that observed for the bovine rZP3 ⁄ rZP4 mixture (Fig. 8B). None of the interspecific rZP3 ⁄ rZP4 mixtures coexpressed in Sf9 cells was immunoprecipitated by anti-FLAG gels (Fig. 8C,D, lane 1 in the left panels), whereas both interspecific rZP3 ⁄ rZP4 mixtures were precipitated by S-protein agarose from the supernatants of the immunoprecipita- tion assays (Fig. 8C,D, lane 2 in the left panels). When bovine rZP4 whose N-terminal His-tag was changed to FLAG-tag (rZP4FLAG) and porcine rZP3 were coex- pressed and subjected to the immunoprecipitation using anti-FLAG gels, porcine rZP3 and bovine rZP4FLAG were coprecipitated and detected by anti- body to His (Fig. 8C, lane 3 in the left panel) and anti- body to FLAG (Fig. 8C, lane 3 in the right panel), respectively. When bovine rZP3FLAG and porcine rZP4 were coexpressed and subjected to immunoprecipita- tion, bovine rZP3FLAG and porcine rZP4 were copre- cipitated and detected by antibody to FLAG (Fig. 8D, lane 3 in the right panel) and antibody to His (Fig. 8D, lane 3 in the left panel), respectively. These results indicate that porcine rZP3 ⁄ bovine rZP4FLAG and bovine rZP3FLAG ⁄ porcine rZP4 complexes were formed and immunoprecipitated through FLAG-tag. In the interspecific rZP3 ⁄ rZP4 mixtures, complex for- mation was parallel to sperm-binding activity. Recently, we reported that a porcine rZP3 ⁄ rZP4 mix- ture coexpressed in Sf9 cells binds bovine, but not porcine, sperm, owing to the presence of pauci- mannose-type and high-mannose-type chains on por- cine rZP3 ⁄ rZP4 [16]. In this study, we obtained inter- specific rZP3 ⁄ rZP4 mixtures by coinfection of Sf9 cells with baculoviruses encoding either bovine ZP3 and
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A
B
C
D
Fig. 8. Inhibitory effect of heterospecific porcine ⁄ bovine rZP3 ⁄ rZP4 mixtures on bovine sperm-solubilized ZP binding. (A) Mixtures of porcine rZP3 and bovine rZP4 (rpZP3 ⁄ rbZP4, lane 1 in each panel) or of bovine rZP3 and porcine rZP4 (rbZP3 ⁄ rpZP4, lane 2 in each panel) were expressed by simultaneous infection of Sf9 cells with the two corresponding recombinant viruses. The rZPGs were collected from the cul- ture supernatant using metal-chelation column chromatography and detected by SDS ⁄ PAGE (left panel) and immunoblotting (right panel) using a mixture of antibodies specific for each ZPG. Arrowheads indicate the rZPG bands. Molecular mass markers are indicated in kDa on the left of each panel. (B) Bovine sperm were incubated with 0.4 lg of the rbZP3 ⁄ rbZP4, rpZP3 ⁄ rbZP4 or rbZP3 ⁄ rpZP4 mixtures for 30 min. The assay (Method 1) was performed as described in the legend to Fig. 2. The number of sperm binding to the solubilized ZP in the absence of inhibitors was designated 100% (without inhibitors). Assays were performed at least three times, and the data shown represent means ± SD. (C) Immunoprecipitation of rpZP3 ⁄ rbZP4FLAG mixture coexpressed in Sf9 cells. Culture supernatants containing coexpressed rpZP3 ⁄ rbZP4 (lanes 1 and 2 in each panel) or coexpressed rpZP3 ⁄ rbZP4FLAG (lane 3 in each panel), as indicated above each panel, were sub- jected to anti-FLAG immunoprecipitation. rZPGs pulled down by anti-FLAG gels (F) were detected by immunoblotting with antibody to His (left panel) and with anti-FLAG M2 (right panel). The rpZP3 and rbZP4 remaining in the supernatant after the immunoprecipitation were sub- jected to pull-down by S-protein agarose (S) to examine the expression of the rZPGs (lane 2 in each panel). (D) Immunoprecipitation of rbZP3FLAG ⁄ rpZP4 mixture coexpressed in Sf9 cells. Culture supernatants containing coexpressed rbZP3 ⁄ rpZP4 (lanes 1 and 2 in each panel) or coexpressed rbZP3FLAG ⁄ rpZP4 (lane 3 in each panel), as indicated above each panel, were subjected to anti-FLAG immunoprecipitation. The rZPGs pulled down by anti-FLAG gels (F) were detected by immunoblotting with antibody to His (left panel) and with anti-FLAG M2 (right panel). The rbZP3 and rpZP4 remaining in the supernatant after the immunoprecipitation were subjected to pull-down by S-protein aga- rose (S) to examine the expression of the rZPGs (lane 2 in each panel). The rpZP3, rbZP3FLAG and rbZP3 bands are indicated by arrowheads in (C) and (D). The rbZP4, rbZP4FLAG and rpZP4 bands are indicated by arrows in (C) and (D). The bands detected by the antibodies but unre- lated to rZPGs are indicated by closed circles in (C) and (D). Molecular mass markers are indicated in kDa on the left of each panel in (C) and (D). IB, immunoblot.
Discussion
this activity is significant
We previously reported that native bovine ZP3 and ZP4 partially purified by RP-HPLC each has sperm- binding activity, although the activity of ZP3 is much weaker [21]. Native ZP2 also has weak sperm-binding is activity, but whether unknown. We also reported that a mixture of native ZP3 and native ZP4 proteins has sperm-binding activ- ity that is slightly stronger than that of ZP4 alone, sug- gesting that ZP3 promotes binding of ZP4 to sperm [21].
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rZPGs, only the rZP3 ⁄ rZP4 mixture bound to sperm. rZP3 and rZP4 coexpressed in Sf9 cells formed a het- ero-complex. When rZP3 and rZP4 were expressed separately in Sf9 cells and then mixed, the mixture did not inhibit sperm–ZP binding, and an interaction between rZP3 and rZP4 was not detected. As complex formation between rZP3 and rZP4 was parallel to the sperm-binding activity of the rZP3 ⁄ rZP4 mixture, sperm binding to the bovine ZP in vitro is mediated by a hetero-complex of rZP3 and rZP4. This conclusion obtained using the rZPGs further suggests that the pre- viously reported sperm-binding activity of partially purified native ZP4 [21] was due to contamination with ZP3. The weak sperm-binding activities that we reported for native ZP2 and ZP3 [21] may be also ascribed to contamination with both ZP3 and ZP4 In pigs, native ZP4 or with ZP4, respectively. In this study, we found that none of the bovine rZPGs bound to sperm when assayed alone, as revealed by two kinds of in vitro competitive inhibition assays and indirect immunofluorescence staining. Of the three the three possible dual combinations of
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uncontaminated with ZP3 exhibits no sperm-binding activity, and only the ZP3 ⁄ ZP4 hetero-complex has sperm-binding activity [15]. Recently, we reported a parallel result for porcine rZPGs; neither rZP3 nor sperm-binding rZP4 has physiologically significant activity, but rZP3 ⁄ rZP4 coexpressed in Sf9 cells does have activity [16]. Thus, in both the porcine and bovine systems, sperm binding to the ZP is mediated by a ZP3 ⁄ ZP4 hetero-complex. Furthermore, all three ZPGs are shared in the porcine and bovine systems. The molecular mechanisms by which sperm interact with the ZP appear to be similar for pigs and cows.
Neither rZP2 ⁄ rZP4 nor rZP2 ⁄ rZP3 coexpressed in Sf9 cells exhibited sperm-binding activity. Thus, we found no evidence for involvement of ZP2 in sperm–ZP bind- ing. In mice, a ZP consisting of mouse ZP1, human ZP2 and mouse ZP3 was made using transgenic mice [49]. Human ZP2 in the chimeric ZP remained unc- leaved after fertilization, and mouse sperm continued to bind to the ZP. On the basis of these observations, a model was proposed in which mouse sperm recognize the supramolecular structure of the ZP but not the car- bohydrate structure of the ZP [3,49]. Additionally, sperm cannot recognize the supramolecular structure modulated by ZP2 processing. Considering this model, it remains to be clarified whether processed ZP2 inhib- its the sperm-binding activity of the ZP3 ⁄ ZP4 complex in cows.
in mouse,
The mature bovine ZP4 polypeptide consists of a unique N-terminal region, a trefoil domain, and a ZP domain. Although porcine and bovine ZP4 are homol- ogous, the mature porcine ZP4 polypeptide lacks the N-terminal region found in the bovine protein [21,22]. The trefoil domain was first discovered in proteolysis- resistant trefoil factor peptides that play roles in muco- sal defense and healing [50]. As trefoil factor peptides are expressed in association with mucins, they are likely to interact with mucins through carbohydrate or polypeptide moieties [50]. The roles of the N-terminal region and trefoil and ZP domains of bovine ZP4 have not yet been clarified; however, the ZP domain is essential for the assembly of ZP2 and ZP3 [10]. In this study, both coexpressed rZP3 ⁄ rZP4136)464 and coexpressed rZP3 ⁄ rZP4182)464 mixtures showed sperm-binding activity similar to that of the rZP3 ⁄ rZP4 mixture, as revealed by a competitive inhibition assay (Method 1) and indirect immunofluorescence staining. Moreover, rZP3 and rZP4182)464 formed het- ero-complexes. These data indicate that the N-terminal region and trefoil domain of rZP4 are not necessary for the sperm-binding activity and hetero-complex for- mation of rZP3 ⁄ rZP4. Neither solubilized bovine ZP nor rZP3 ⁄ rZP4 signif- icantly induced the acrosome reaction of bovine sperm in this study. However, this does not mean that solubi- lized bovine ZP does not have acrosome reaction- inducing activity. Previous reports have shown that 30–35% of bovine sperm complete the acrosome reac- tion after incubation with 50 ngÆlL)1 of solubilized bovine ZP as compared to about 10% after incubation with unrelated glycoproteins [44,45]. The induction of the acrosome reaction is only 3–4% in those reports at 9 ngÆlL)1 of solubilized bovine ZP, however, which is the concentration examined in the present study. As the concentrations of the zona proteins examined in the competitive inhibition assays in the present study were lower than 9 ngÆlL)1, it could be concluded that the acrosome reaction of bovine sperm was not significantly induced under the experimental conditions used in this study. Because in mice a recent report sug- gested that an intact porous structure of ZP is neces- sary for mechanical induction of the acrosome reaction of mouse sperm [46], it remains to be clarified whether solubilization of bovine ZP reduces its acrosome reac- tion-inducing activity for sperm. According to previous reports, 4 h of incubation is necessary for complete capacitation of bovine sperm [44,45]. Then, it is also possible that the bovine sperm used in this study were not completely capacitated after 30 min of incubation, and therefore the acrosome reaction was not induced significantly by incubation with the zona proteins.
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a-Mannosyl residues at the nonreducing termini of high-mannose-type chains of the bovine ZP are essen- tial for sperm binding, as previously shown by the fact that a-mannosidase treatment greatly reduces the inhibitory activity of native ZP against sperm–egg binding [34]. Porcine rZPGs expressed in Sf9 cells have chains pauci-mannose-type and high-mannose-type with or without fucose at the innermost GlcNAc, and do not have detectable amounts of complex-type chains [16]. Porcine rZP3 ⁄ rZP4, which binds to bovine loses most of its sperm but not to porcine sperm, inhibitory activity towards bovine sperm–ZP binding upon a-mannosidase treatment [16]. Here, MS and sperm-binding activity of the Native bovine, porcine and murine ZP2 are pro- cessed at a specific site by an unidentified enzyme upon fertilization [17,47,48]. This processing plays a role in blocking polyspermy by the ZP [49]. Specific proteo- lysis of bovine ZP2, together with formation of intra- is molecular and intermolecular disulfide linkages, involved in ZP hardening [18], but the role of ZP2 in sperm binding is not yet clear. Because, here, a bovine rZP2 ⁄ rZP3 ⁄ rZP4 mixture coexpressed in Sf9 cells inhibited bovine sperm–ZP binding at a level similar to that of rZP3 ⁄ rZP4, we conclude that rZP2 does not rZP3 ⁄ rZP4. affect
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template. The ZP4 mutants bovine ZP4 cDNA as rZP4136)464 and rZP4182)464 correspond to regions Asp136 to Arg464 and Gly182 to Arg464, respectively. The 5¢-sense primers contained SmaI sites, and the 3¢-antisense primer used for rZP4 was also used for both ZP4 mutants.
residues that are essential
The PCR products were electrophoresed on 1% agarose gels, bands of expected sizes were excised from the gels, and the recovered DNA was ligated to pGEM-T Easy vec- tor (Promega, Madison, WI, USA). The DNA sequences of the PCR products were confirmed by DNA sequencing and then subcloned into the baculovirus transfer vector pBAC- gus-6 (Novagen, Madison, WI, USA). The resulting recom- binant proteins had N-terminal His- and S-tags and were secreted into the medium.
lectin blot analyses indicated that the major N-linked chains of bovine and porcine rZPGs are similar. Thus, bovine and porcine rZPGs have nonreducing terminal a-mannosyl for bovine sperm binding. This study further suggests that the presence of nonreducing terminal a-mannosyl moieties is insufficient for bovine sperm binding, which addi- tionally requires a specific three-dimensional inter- action between bovine rZP3 and rZP4.
Construction of recombinant baculovirus transfer plasmids for bovine rZP3FLAG and rZP4FLAG
The baculovirus transfer vector pBACgus-6 was digested with NcoI and SacII to remove the region encoding His- tag. Two synthetic DNA oligomers, sense oligomer 5¢-CAT GGATTACAAGGACGACGATGACAAGTCCGC-3¢ and antisense oligomer 5¢-GGACTTGTCATCGTCGTCCTTG TAATC-3¢, were annealed and ligated to the digested plas- mid to insert the sequence encoding FLAG-tag in place of His-tag. The cDNAs encoding bovine ZP3 and ZP4 were inserted into the plasmid as described above. The DNA sequences including the region encoding FLAG-tag of the plasmid and the 5¢- and 3¢-terminal restriction sites of ZP3 and ZP4 cDNAs ligated to the plasmid were confirmed by DNA sequencing. The resulting rZP3FLAG and rZP4FLAG had N-terminal FLAG- and S-tags, but did not have His- tag, and were secreted into the medium.
Studies using transgenic mice have demonstrated that coexpression of mouse ZP1, human ZP3 and either mouse or human ZP2 leads to successful forma- tion of the ZP matrix [49,51]. The interactions between ZP2 and ZP3, and between ZP1 and the ZP2 ⁄ ZP3 complex, are conserved in humans and mice. As por- cine rZPGs expressed in Sf9 cells bind to bovine sperm, we examined the bovine sperm-binding activity of heterospecific bovine ⁄ porcine rZP3 ⁄ rZP4 mixtures. Both porcine rZP3 ⁄ bovine rZP4 and bovine rZP3 ⁄ por- cine rZP4 inhibited bovine sperm–ZP binding at a level similar to that of the bovine ⁄ bovine mixture. The het- erospecific mixtures formed complexes. These results suggest that the polypeptide regions involved in the interaction between ZP3 and ZP4 are highly conserved in cows and pigs.
The porcine and bovine rZPGs may be useful for obtaining new insights into structure–function relation- ships of ZPGs, although at present it is uncertain how closely the rZPGs resemble their native counterparts. Further experimental work is necessary to determine whether the rZPGs can represent the native ZPGs in an in vitro functional analysis of ZPGs.
Experimental procedures
Expression and purification of rZPGs, mutant rZP4s, and rZPGFLAGs
Plasmid DNA preparations containing individual cDNAs (0.25 lg) were transfected along with 0.1 lg of BacVector- 2000 Virus DNA (Novagen) into Sf9 cells using Eufectin (Novagen), according to the manufacturer’s protocol. Recombinant plaques were identified and purified by the plaque assay protocol supplied with the BacVector-2000 kit. Sf9 cells were routinely propagated in Sf-900II serum- free medium (Invitrogen, Groningen, the Netherlands). Several purified plaques were examined for expression and secretion of the recombinant proteins.
Sf9 cells (1.8 · 106) were attached to flasks, infected with recombinant virus from each purified plaque at a multiplic- ity of infection (MOI) of 5–10, and cultured in 2.5 mL of Sf-900II serum-free medium for 48 h at 27 (cid:2)C. The culture supernatant fraction was recovered, and 500 lL was mixed with 10 lL of S-protein agarose suspension (Novagen) that had been prewashed with NaCl ⁄ Pi (pH 7.4). The mixture
RT-PCR was used to obtain cDNA encoding the secreted, mature bovine ZP3 polypeptide. Bovine ovary poly(A)+ RNA, isolated according to the methods of Chomczynski & Sacchi [52], was used as the template. The cDNAs encod- ing the secreted, mature polypeptides for bovine ZP2 and ZP4 were obtained by PCR using their previously described cDNAs [21] as templates. The resulting constructs encoded regions Ile36 to Arg637 of ZP2, Arg32 to Arg348 of ZP3, and Lys25 to Arg464 of ZP4, with the translation initiation Met residues numbered 1. The 5¢-sense primers for ZP2, ZP3 and ZP4 contained EcoRI, BamHI and SmaI sites, contained XhoI, respectively. The 3¢-antisense primers BamHI and HindIII sites, respectively, in addition to a stop codon. Preparation of cDNAs encoding N-terminal deletion mutants of bovine ZP4 was performed by PCR using
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Construction of recombinant baculovirus transfer plasmids for bovine rZPGs and rZP4 mutants
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was shaken gently at room temperature for 30 min to allow binding of the recombinant proteins to the S-protein agarose through their N-terminal S-tags. The agarose beads were washed three times with NaCl ⁄ Pi, followed each time by centrifugation at 2000 g using a Sprout minicentrifuge (Heathrow Scientific, Vernon Hills, IL, USA). The pellet that contained the agarose beads was prepared for SDS ⁄ PAGE.
For large-scale protein production, Sf9 cells (200 mL at 1.0 · 106 cellsÆmL)1) were infected with recombinant virus at an MOI of 10. When two or three proteins were coex- pressed, each virus was used at an MOI of 5. After 48 h of culture in suspension, the medium was centrifuged at 800 g for 10 min using an SCT5B centrifuge (Hitachi Koki Co. Ltd., Ibaraki, Japan) with RT5S2 swing-type rotor, and the supernatant fraction was filtered through a 0.45 lm filter. The filtrate was sonicated and then stored at 4 (cid:2)C.
For lectin blots, membranes were blocked with T-NaCl ⁄ Tris for 1 h and then incubated for 2 h with 1 lgÆmL)1 of either horseradish peroxidase-conjugated or biotin-conju- gated lectin in T-NaCl ⁄ Tris containing 1 mm each MgCl2 and CaCl2. The horseradish peroxidase-conjugated lectins were LCA and RCA120. The biotin-conjugated lectins were PHA-L4, ACA, and GNA. ACA and GNA were purchased from EY Laboratories (San Mateo, CA, USA), and the remaining lectins were from Seikagaku Kogyo. For the per- oxidase-conjugated lectins, membranes were washed three times for 15 min each with T-NaCl ⁄ Tris containing 1 mm each MgCl2 and CaCl2, and developed as described above. For the biotin-conjugated lectins, membranes were incu- bated for an additional hour with 0.5 lgÆmL)1 of horserad- ish peroxidase-conjugated streptavidin (Sigma, St Louis, MO, USA) in T-NaCl ⁄ Tris containing 1 mm each MgCl2 and CaCl2 before washing and color development as described above.
For purification of His-tagged proteins, the filtered and sonicated supernatants were subjected to metal-chelation column chromatography using His-Bind resin (Novagen) equilibrated with column buffer (20 mm Tris ⁄ HCl, pH 7.9, 0.5 m NaCl) containing 5 mm imidazole at a flow rate of 0.5 mLÆmin)1 at 4 (cid:2)C. The column was washed with 10 col- umn volumes of the equilibration buffer, and the bound protein was eluted with six column volumes of column buffer containing 60 mm imidazole followed by six column volumes of column buffer containing 1 m imidazole.
rZP3FLAG and rZP4FLAG were not purified, but the su- pernatants were directly used in a pull-down assay using anti-FLAG gels as described below or S-protein agarose as described above.
Glycopeptidase F was obtained from Takara (Kyoto, Japan). Each rZPG (0.4 lg) was mixed with 10 lL of 1% SDS solution and boiled for 2 min. The resulting solutions were then mixed with 40 lL of reaction buffer (50 mm sodium phosphate, pH 8.0, 12.5 mm EDTA, 5 mm sodium azide, 1.25% Nonidet P-40), boiled for 2 min, and cooled on ice. Aliquots corresponding to 0 min of digestion were taken from the solutions, and enzymatic digestion was initi- ated with 1 mU of glycopeptidase F. Additional aliquots were taken at 1 and 5 min and at 24 h, and digestion was terminated by boiling.
Glycopeptidase F digestion of rZPGs
SDS ⁄ PAGE was performed under reducing conditions according to the Laemmli protocol [53]. The proteins were separated on gels of various polyacrylamide concentrations (8–13%), and either visualized by silver staining or trans- ferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) according to the method of Towbin for immu- noblot and lectin blot analyses [54]. The membranes were blocked with 3% BSA in Tris-buffered saline (NaCl ⁄ Tris; 20 mm Tris ⁄ HCl, pH 7.5, 500 mm NaCl) for 1 h. The membranes were then incubated for 2 h with rabbit poly- clonal antibodies against porcine ZP2, ZP3 and ZP4 [17,30] that were diluted 1 : 200, 1 : 2000, and 1 : 2000, respec- tively, in NaCl ⁄ Tris containing 1% BSA. The membranes were washed three times for 15 min each with NaCl ⁄ Tris containing 0.05% Tween-20 (T-NaCl ⁄ Tris) and then incu- bated for 1.5 h with horseradish peroxidase-conjugated goat anti-(rabbit IgG) that was diluted to 1 lgÆmL)1 in NaCl ⁄ Tris containing 1% BSA. The membranes were again washed three times with T-NaCl ⁄ Tris, and the blots were developed using an Immunostain Kit (Seikagaku Kogyo, Tokyo, Japan).
Solubilized bovine ZP (0.2 lg in 50 lL of NaCl ⁄ Pi) was added to each well of a 96-well plate (Nalge Nunc, Roches- ter, NY, USA), which was then incubated at 4 (cid:2)C overnight. The wells were rinsed with NaCl ⁄ Pi and then blocked with NaCl ⁄ Tris containing 3% BSA at 37 (cid:2)C for 2 h. Frozen bovine sperm were thawed and then washed twice in pre- warmed (38.5 (cid:2)C) Brackett and Oliphant (BO) solution without BSA [21,55]. The bovine sperm were then capaci- tated by incubation in BO solution containing BSA for 30 min. Capacitation and subsequent incubations were car- ried out at 38.5 (cid:2)C under 2% CO2. Aliquots (50 lL) con- taining 4 · 105 capacitated sperm were mixed with 50 lL of BO solution containing solubilized bovine ZP or each rZPG and then incubated for 30 min. The amounts of protein used are indicated in the legends for Figs 2, 3 and 8. The wells were rinsed three times with NaCl ⁄ Pi, the preincubated sperm solutions were transferred into the wells, and the plates were incubated for 2 h. The wells were then washed three times with BO solution, 50 lL of 70% glycerol in NaCl ⁄ Pi was added to each well, and the sperm bound to
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Electrophoresis, immunoblot analysis and lectin blot analysis of rZPGs Competitive inhibition assay ) Method 1
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Recombinant bovine zona pellucida glycoproteins
the wells were recovered by 20 strokes of vigorous pipetting. The number of sperm in 0.1 lL of suspension was deter- mined using a hemocytometer. The average number of sperm in control experiments without inhibitors was 60.4.
without zona proteins, or BO solution containing 0.9 lg of solubilized bovine ZP or 1.0 lg of rZP3 ⁄ rZP4 at 38.5 (cid:2)C for 3 h under 2% CO2. The sperm were washed three times by centrifugation at 2000 g for 1 min using a Sprout mini- centrifuge (Heathrow Scientific), suspended in NaCl ⁄ Pi, transferred onto cover glasses, and fixed with 3.7% formal- dehyde in NaCl ⁄ Pi at 37 (cid:2)C for 30 min. The cover glasses were then rinsed with NaCl ⁄ Pi and blocked with 3% BSA in NaCl ⁄ Tris at 37 (cid:2)C for 1 h. Sperm were incubated in NaCl ⁄ Pi containing 10 lgÆmL)1 of 4¢,6-diamidino-2-phenyl- indole and 1 lgÆmL)1 of FITC-PSA (Sigma) at 37 (cid:2)C for 1 h. The cover glasses were rinsed with NaCl ⁄ Pi and mounted on slide glasses. Sperm heads stained with 4¢,6-diamidino-2-phenylindole (total sperm), and the sperm stained with FITC-PSA (acrosome-intact, acrosome- damaged or partially acrosome-reacted sperm [39]) were counted under a fluorescence microscope. This experiment was repeated four times, and 100 sperm were observed for each incubation condition of each experiment.
Competitive inhibition assay: Method 2
Method 2 was performed according to our previous report [21]. Frozen bovine sperm were thawed and capacitated as described in Method 1. Several drops (50 lL each) of BO solution containing the rZPGs to be examined were pre- pared under paraffin oil. The amounts of protein used are indicated in the legend for Fig. 4. A 10 lL aliquot of the capacitated sperm was added to the drop to give a sperm concentration of 1.0 · 106 cellsÆmL)1. The suspension was incubated at 38.5 (cid:2)C for 30 min in 2% CO2. Then, 10–13 bovine eggs prepared from ovaries were added to each drop, and the mixture was incubated at 38.5 (cid:2)C for another 3 h. After the eggs were washed 10 times to remove the loosely attached sperm by transfer to fresh BO solution using a pipette with a bore size of around 200 lm, the eggs were fixed with 3% glutaraldehyde in NaCl ⁄ Pi. The sperm heads binding to ZP were counted after staining with 4¢,6-diamidino-2-phenylindole. The average number of sperm bound to one egg in control experiments without inhibitors was 70.0.
Detection of complex formation between rZP3 and rZP4
1% BSA in NaCl ⁄ Tris
Frozen bovine sperm were washed and capacitated as described above, and 50 lL aliquots (2 · 106 spermÆmL)1) were incubated with 0.2 lg of solubilized bovine ZP or rZPGs in BO solution at 38.5 (cid:2)C for 30 min under 2% CO2. The sperm were washed three times by centrifugation at 2000 g for 1 min using a Sprout minicentrifuge (Heathrow Scientific), suspended in NaCl ⁄ Pi, transferred onto cover glasses, and fixed with 3.7% formaldehyde in NaCl ⁄ Pi at 37 (cid:2)C for 30 min. The cover glasses were then rinsed with NaCl ⁄ Pi and blocked with 3% BSA in NaCl ⁄ Tris at 37 (cid:2)C for 1 h. The proteins that bound to sperm were detected using a mixture of anti-porcine ZP2, ZP3, and ZP4 (diluted 1 : 25, 1 : 100 and 1 : 1000 with 1% BSA in NaCl ⁄ Tris, respectively) as primary antibodies, and Alexa Fluor 546- conjugated goat anti-(rabbit IgG) antibody (diluted to 1 lgÆmL)1 with (Mole- cular Probes, Eugene, OR, USA) as the secondary antibody. The sperms were observed under a fluorescence microscope.
Indirect immunofluorescence staining of rZPGs bound to sperm
aliquots
above,
50 lL
and
Culture media containing rZP3FLAG or rZP4 expressed alone, and containing a coexpressed mixture described in the legends for Figs 6 and 8, were prepared by infection of Sf9 cells (40 mL at 1.0 · 106 cellsÆmL)1) with a correspond- ing baculovirus at an MOI of 10 or a mixture of corre- sponding baculoviruses each at an MOI of 5 and sub sequent culture for 48 h at 27 (cid:2)C. Culture medium without rZPGs was prepared by culturing Sf9 cells without infec- tion. Culture medium for Fig. 6B was prepared by combin- ing the culture medium (20 mL) containing rZP3FLAG and the culture medium (20 mL) containing rZP4 and incubat- ing the mixture overnight at 4 (cid:2)C. The culture media were centrifuged at 800 g for 10 min using an SCT5B centrifuge (Hitachi Koki Co. Ltd.) with RT5S2 swing-type rotor, and each supernatant was divided into two 20 mL aliquots. Each aliquot was mixed with 14 lL of a 50% suspension of anti-FLAG M2 gels (Sigma), and the mixture was gently shaken for 1 h at room temperature. The anti-FLAG gels were recovered as pellets by centrifugation at 800 g for 3 min using an SCT5B centrifuge (Hitachi Koki Co. Ltd.) with RT5S2 swing-type rotor, and the supernatants were used to check the expression of each rZPG using S-protein agarose as described above. The pellets were then washed three times with NaCl ⁄ Pi containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, followed each time by centrifugation at 2000 g for 1 min using a Sprout minicentrifuge (Heathrow Scientific). The pellet that con- tained the anti-FLAG gels was prepared for SDS ⁄ PAGE. Immunoblots were performed as described above. His- tagged rZPGs and FLAG-tagged rZPGs were detected with antibody to His (diluted 1 : 3000; GE Healthcare, Chalfont
Frozen bovine sperm were washed and capacitated as containing described 4 · 105 sperm were mixed with 50 lL of BO solution
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3 Hoodbhoy T & Dean J (2004) Insights into the molecu- lar basis of sperm–egg recognition in mammals. Repro- duction 127, 417–422.
St Giles, UK) and anti-FLAG M2 (diluted to 4.8 lgÆmL)1; Sigma), respectively, as primary antibodies, and horseradish peroxidase-conjugated anti-mouse IgG (diluted 1 : 2000; Sigma) as a secondary antibody.
4 Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ash- ton P, Pavlovic B, Lenton W, Afnan M, Brewis IA, Monk M et al. (2004) Four zona pellucida glycoproteins are expressed in the human. Hum Reprod 19, 1580– 1586.
5 Hoodbhoy T, Joshi S, Boja ES, Williams SA, Stanley P
& Dean J (2005) Human sperm do not bind to rat zonae pellucidae despite the presence of four homolo- gous glycoproteins. J Biol Chem 280, 12721–12731.
6 Harris JD, Hibler DW, Fontenot GK, Hsu KT,
Yurewicz EC & Sacco AG (1994) Cloning and charac- terization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Seq 4, 361–393.
7 Bleil JD & Wassarman PM (1980) Structure and func- tion of the zona pellucida: identification and character- ization of the proteins of the mouse oocyte’s zona pellucida. Dev Biol 76, 185–202.
8 Hughes DC & Barratt CLR (1999) Identification of the true human orthologue of the mouse Zp1 gene: evidence for greater complexity in the mammalian zona pellu- cida? Biochim Biophys Acta 1447, 303–306.
Forty micrograms of rZP4136)464 was purified from the Sf9 culture supernatant fraction as described above, desalted by dialysis against water, and lyophilized. N-linked carbohy- drate chains were released by hydrazinolysis [56], and the carbohydrate labeled with chains were fluorescently 2-aminopyridine, as described previously [56]. LC ⁄ MS anal- ysis of pyridylaminated chains was commercially performed by the Asahi KASEI Analysis & Simulation Center (Fuji, Japan). Pyridylaminated chains were separated using Asahi- pak NH2P-50 4E (Showa Denko K.K., Kawasaki, Japan). The eluents were 0.05% formic acid in water (eluent A) and acetonitrile (eluent B). The pyridylaminated chains were eluted at a flow rate of 0.5 mLÆmin)1 with a gradient of eluent B from 80% to 50% in 40 min at 40 (cid:2)C. Mass spectrometer spectra were recorded on an LCQ mass (Thermo Electron, Waltham, MA, USA). The mass spec- trometer was operated in positive ion mode. Ions in the range of m ⁄ z 500–2000 were acquired.
9 Jovine L, Darie CC, Litscher ES & Wassarman PM
Analysis of carbohydrate chains by MS
(2005) Zona pellucida domain proteins. Annu Rev Bio- chem 74, 83–114.
10 Jovine L, Qi H, Williams Z, Litscher E & Wassarman PM (2002) The ZP domain is a conserved module for polymerization of extracellular proteins. Nat Cell Biol 4, 457–461.
Welch’s t-test was used to determine whether rZPGs had inhibitory activity against sperm-solubilized ZP or sperm– egg binding, and whether the inhibitory activities of the solubilized bovine ZP and the rZPG mixtures were significantly different. Differences were considered to be significant at P < 0.05.
11 Jovine L, Qi H, Williams Z, Litscher ES & Wassarman PM (2004) A duplicated motif controls assembly of zona pellucida domain proteins. Proc Natl Acad Sci USA 101, 5922–5927.
Statistical analysis
Acknowledgements
12 Epifano O, Liang LF, Familari M, Moos MC Jr &
Dean J (1995) Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121, 1947–1956.
13 Greve JM & Wassarman PM (1985) Mouse egg extra-
cellular coat is a matrix of interconnected filaments pos- sessing a structural repeat. J Mol Biol 181, 253–264. 14 Nakano M, Yonezawa N, Hatanaka Y & Noguchi S
(1996) Structure and function of the N-linked carbohy- drate chains of pig zona pellucida glycoproteins. J Reprod Fertil Suppl 50, 25–34.
We thank Dr Atsushi Tanaka and Dr Kazunori Toma for the LC ⁄ MS analysis of sugar chains. We also thank Naoto Yoda and Ai Mariko for technical assis- tance. This work was supported in part by Grants-in- Aid for Scientific Research and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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