N-Glycosylation Site in the Middle Region Is Involved in the Sperm-Binding Activity of Bovine Zona Pellucida Glycoproteins ZP3 and ZP4

Mammalian fertilization is a species-selective event that involves a series of interactions between sperm proteins and the oocyte’s zona pellucida (ZP) glycoproteins. Bovine ZP consists of three glycoproteins: bZP2, bZP3, and bZP4. In our previous study, we demonstrated that bovine sperm binds to plastic wells coated with recombinant bZP4 and identified that the N-terminal domain and the middle region of bZP4 are critical for sperm-binding activity. Here, we investigated the sperm-binding site in the middle region (residues 290 to 340) of bZP4, which includes the hinge region. We showed that bovine sperm binds to bZP4’s middle region in a species-selective manner. We mapped the function of bZP4’s middle region to its N-glycosylation site at Asn-314 using several recombinant mutated proteins. Moreover, we showed that mutations of the N-glycosylation sites at Asn-314 close to the hinge region and Asn-146 of the hinge region of bZP4 and bZP3, respectively, reduced the sperm-binding activity of the complex of the bZP3 (from 32 to 178) and bZP4 (from 136 to 464) fragments. Together, these results suggest that ZP’s middle regions of bZP3 and bZP4 form one of the sperm-binding sites of bovine ZP.

Studies have shown the role of ZP proteins in sperm binding in several mammalian species.In mice and humans, sperm binds to the N-terminal domain of ZP2 in a speciesselective manner [10].Its polypeptide moiety, instead of the carbohydrate moiety, is responsible for this binding [11].Though only in vitro studies have been reported, evidence suggests that different ZP proteins are involved in the sperm-binding processes in pigs and bovines [3,12,13].Nevertheless, ZP4 predominantly binds to the sperm in both pigs and bovines [12,13], indicating that the mechanisms of sperm-ZP interaction are somewhat  .This is a top-ranked model made using AlphaFold2.The previously proposed three sites involved in sperm binding on bZP4  are shown in red in the ZP-N1 domain.The previously identified sperm-binding region from 290 to 340 on bZP4 is shown in orange extending from ZP-N domain to ZP-C domain.This orange region corresponds to the two-headed orange arrow shown in (A).(C) Domain architecture of mature bZP3 (32-348) and schematic representation of truncated bZP3 (32-178).The inverted tripods mark potential N-glycosylation sites.Oglycosylation sites are not shown.
Notably, the N-terminal fragment of bZP3 (Arg-32 to Glu-178), which includes the hinge region (Figure 1C), interacts with bZP4.Competitive inhibition assay results Competitive inhibition assays have shown that solutions containing bovine bZP3/bZP4 heterocomplex can inhibit sperm-ZP binding, whereas neither bZP3 nor bZP4 alone could inhibit this binding [17].This suggests that the bZP3/bZP4 heterocomplex formation is necessary for in vitro sperm-binding activity.Recently, using ZP proteins fixed to plastic wells, we demonstrated that bovine sperm binds to bZP4 but not to bZP3, and that two regions of bZP4 are involved in sperm binding [13].The first region, ranging from Lys-25 to Asp-136, nearly corresponds to the N-terminal ZP-N1 domain (Figure 1A,B).The second region, extending from Ser-290 to Lys-340, comprises the flexible hinge region and the Nterminus of the ZP-C domain (Figure 1A,B) [3,18].Bovine sperm binds to bZP4 (25-136) in a species-selective manner without requiring N-glycosylation [19].Additionally, we identified three sites involved in sperm binding on bZP4 (25-136) using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins (Figure 1B) [19].However, the role of the bZP4 (290-340) region in sperm-ZP binding remains to be determined.
Notably, the N-terminal fragment of bZP3 (Arg-32 to Glu-178), which includes the hinge region (Figure 1C), interacts with bZP4.Competitive inhibition assay results showed that this complex inhibits bovine sperm-ZP binding [20].Furthermore, when the Nglycosylated Asn-146 from the bZP3 fragment's hinge region was mutated to Asp, the inhibitory activity was reduced, even though this mutation did not diminish the interaction between the bZP3 fragment and bZP4 [20].This result suggested the significance of the Nglycosylation site on bZP3's hinge region in sperm recognition by the bZP3/bZP4 complex.
To understand the role of the bZP4 middle region (290-340) in sperm-binding activity, we conducted an in vitro sperm-ZP protein-binding assay using recombinant bZP4 mutated proteins.
The DNA sequences of the constructed plasmids were confirmed using a commercial DNA sequencing service (Eurofins Genomics, Tokyo, Japan).

Expression of Recombinant ZP Proteins
The recombinant baculoviruses for bZP4, bZP4 (136-464), pZP4 (137-466), bZP3 (32-178), and bZP3 (32-178) N146D, all of which are N-terminally His-and S-tagged, were reported previously [17,20,21].New recombinant baculoviruses were prepared based on a previously reported procedure [13].Sf9 cells were routinely propagated in Sf-900II serum-free medium (Invitrogen, Carlsbad, CA, USA) and transfected with each baculovirus transfer plasmid construct along with flashBAC DNA (Oxford Expression Technologies, Oxford, UK), following the manufacturer's protocol.The Sf9 cells were then infected with each recombinant virus.The expression and secretion of each recombinant protein into the culture supernatant were verified, as previously reported [17,19].All recombinant ZP proteins were expressed as secretory proteins using the signal peptide from pBACgus6.

Purification of Recombinant ZP Proteins from Culture Supernatants
The recombinant ZP proteins were purified as reported previously [17,19].Briefly, for large-scale protein expression, either an individual or a mixture of corresponding recombinant virus(es) was used to infect 200 mL of Sf9 cells (1 × 10 6 cells/mL).These suspensions were cultured for 48 h at 27 • C. Each N-terminal His-tagged recombinant protein or complex was purified using TALON metal affinity resin ® (Clontech, Mountain View, CA, USA).The protein concentrations were determined at an absorbance of 280 nm using the absorbance value of 1 mg/mL protein, which was calculated based on their amino acid compositions (https://web.expasy.org/protparam/(accessed on 18 April 2023)).

SDS-PAGE
SDS-PAGE was performed on 11 or 12.5% (w/v) separating gels under reducing conditions according to the Laemmli method [22].The gels were silver-stained, and the standard proteins with a broad molecular mass range (Takara) were used to estimate the apparent protein molecular masses.
2.6.Sperm Binding to Recombinant ZP Proteins Adsorbed to Plastic Wells [13,19] Specific amounts of the proteins were added to a 96-well plate (Nalge Nunc) and incubated overnight at 4 • C.Only 50 µL of TALON elution buffer was adsorbed as the negative control.After discarding the solution, the wells were washed with PBS and blocked with 3% BSA in TBS at 38.5 • C for 2 h.Frozen Holstein bull sperm straws, purchased from Animal Genetics Japan Co., Ltd.(Matsuzaka, Japan), were used for artificial insemination.Frozen bovine sperm was thawed, washed twice in pre-warmed (38.5 • C) Brackett and Oliphant (BO) solution without BSA [23], and then capacitated by incubating in BO solution containing BSA for 30 min.The capacitation and subsequent incubations were performed at 38.5 • C under 2% CO 2 .Aliquots (50 µL) containing 4 × 10 5 capacitated sperm were transferred into the wells, and the plates were incubated for 2 h.After incubation, the wells were washed thrice with BO solution, and 50 µL of 70% glycerol in PBS was added to each well.The sperm bound to the wells was recovered by 20 strokes of vigorous pipetting.Then, the number of sperm in 0.1 µL of suspension was determined using a hemocytometer.The number of sperm bound to the uncoated wells was subtracted from the number of sperm bound to the wells coated with recombinant ZP proteins.The average number of sperm in 0.1 µL of suspension in the 100% sperm-binding control of experiments is provided in the figure legends (Figures 2-4).

Statistical Analysis
We used Welch's t-test to determine whether there was a significant difference in sperm count between the two groups.Differences with p < 0.05 were considered statistically significant.

Modeling of bZP Proteins
Models of bZP4 (25-464) and heterodimer of bZP3(32-178) and bZP4 (25-464) were generated using the ColabFold v1.5.2-patch:AlphaFold2 website using MMseqs2 (https:// colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb(accessed on 11 October 2023)) with template mode: none [24].(C) Adsorption of each complex protein to plastic wells shown above.The amount of each complex protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with a mixture of anti-His tag and anti-FLAG tag antibodies.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each protein complex shown above.Plastic wells were coated with each protein complex (0.8 µg for each protein complex).The number of sperm bound to wells coated with bZP4 (25-464) varied from 21 to 39 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.(C) Adsorption of each complex protein to plastic wells shown above.The amount of each complex protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with a mixture of anti-His tag and anti-FLAG tag antibodies.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each protein complex shown above.Plastic wells were coated with each protein complex (0.8 µg for each protein complex).The number of sperm bound to wells coated with bZP4 (25-464) varied from 21 to 39 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.reduced compared to that of bZP4 (136-464) (Figure 2D).Meanwhile, the sperm-binding activity of bZP4 (136-464)/pZP4 (312-315) was not significantly different from that of bZP4 (136-464)/pZP4 (312-315, 299-302).These results suggest that the corresponding bovine residues at 311 and 314 are necessary for the sperm-binding activity of bZP4 (136-464).

N-Glycosylation Sites on Both bZP3 and bZP4 Are Involved in Sperm-Binding Activity
Recombinant bZP3 and bZP4 expressed in Sf9 cells form a heterocomplex that exhibits bovine sperm-binding activity [17].Moreover, the complex between the N-terminal fragment of bZP3 (32-178) (Figures 1C and 4A) and bZP4 inhibits sperm-ZP binding, as shown using competitive inhibition assays.The inhibitory activity requires N-glycosylation at Asn-146 of bZP3 (32-178) [20].In this study, we analyzed the involvement of the Nglycosylation site at Asn-314 of bZP4 (136-464) in sperm-binding activity in the context of bZP3 (32-178)/bZP4 (136-464) complex using the solid support assay.

Discussion
The sperm-binding activity of bZP4 depends on its N-terminal ZP-N1 domain and the region between residues 290 and 340, with the former showing higher sperm-binding activity than the latter [13].bZP4 has four potential N-glycosylation sites at Asn-71, -202, -219, and -314 [25].We found that the potential N-glycosylation site at Asn-71 of the N-terminal ZP-N1 domain was not involved in sperm-binding activity, as mutation of the site did not change the sperm-binding activity of this domain [19].In this study, we further examined the sperm-binding site of the residues 290-340 of bZP4.We found that recombinant pZP4 (137-466) showed very low binding activity toward bovine sperm (Figure 2D), even though only eight amino acid residues in the 291-341 region of pZP4 are different from those at corresponding sites in the 290-340 region of bZP4 (Figure 2A).Therefore, our strategy for identification of sperm-binding sites in the 290-340 region of bZP4 was to systematically replace the 290-340 region from the bovine amino acid sequence with the porcine amino acid sequence and to examine bovine sperm-binding activities of the bZP4 mutants.As a result, we found that the N-glycosylation site at Asn-314 of bZP4 is important for its sperm-binding activity.We do not know, when each of the eight amino acid residues is mutated to other amino acids, such as Ala, whether the mutation would affect the sperm-binding activity of the 290-340 region of bZP4.This remains to be clarified.
Our competitive inhibition assays showed that neither bZP3 nor bZP4 expressed in Sf9 cells inhibited the binding of bovine sperm to ZP [17], indicating that individually, either bZP3 or bZP4 in a solution does not have sperm-binding activity.However, bovine sperm binds to bZP4-coated plastic wells but not to bZP3-coated ones [13], suggesting that in a solid-supported multivalent form, bZP4 shows sperm-binding activity, but bZP3 does not.The competitive inhibition assays also showed that the complex between the N-terminal fragment of bZP3 ranging from 32 to 178 and bZP4 recombinantly expressed in Sf9 cells inhibited sperm binding to ZP [20].The mutation of the N-glycosylation site at Asn-146 of the bZP3 fragment to Asp significantly reduces the inhibitory activity of the complex between the bZP3 fragment and bZP4, but the mutation does not reduce the interaction between the bZP3 fragment and bZP4 [20].This indicated that when complexed with bZP4, the N-glycosylated Asn-146 of bZP3 is probably involved in sperm-binding activity.Here, we found that mutation of Asn-314 of bZP4 to Asp significantly reduced the sperm-binding activity of the complex between the bZP3 fragment and the bZP4 fragment lacking the N-terminal ZP-N1 domain.Taken together with the previous results, the presence of both N-glycosylated Asn-146 of bZP3 and N-glycosylated Asn-314of bZP4 is essential for the formation of an active sperm-binding complex.
When expressed in Sf9 cells, the recombinant bZP4 contains pauci-mannose type N-glycans with α-Man residues at the non-reducing termini [17].Bovine sperm exhibits a preference for α-Man residues, as shown by its binding to plastic wells coated with α-mannosylated glycolipid analog [26].The binding of bovine sperm to native ZP requires non-reducing terminal α-Man residues [16].Therefore, these residues of the recombinant bZP3/bZP4 complex might participate in the binding of bovine sperm to the complex.However, remains to be clarified whether the N-glycans directly bind to bovine sperm, or whether they have roles in correct folding of the polypeptide moiety of the spermbinding site.
Native pZP4 purified from ovaries lacks the N-terminal ZP-N1 domain [27,28], which could be due to post-translational processing, resulting in a mature pZP4 polypeptide that stretches from Asp-137 to Arg-466.pZP4 has three N-glycosylation sites at Asn-203, Asn-220, and Asn-333.Mutation of Asn-203 or Asn-220 to Asp significantly reduces the spermbinding activity of the pZP3/pZP4 complex, whereas that of Asn-333 to Asp does not [21].Based on the 3D model of pZP4, Asn-203 and Asn-220 are in close proximity [3,29,30].A 3D model of pig ZP filament was proposed based on the cryo-EM structure of human uromodulin filament [29,30].In this model, the N-glycosylation site at Asn-220 of the pZP4-ZP-N domain, O-glycosylation sites in the hinge region of pZP3, Thr-155, Thr-161, and Thr-162, and the N-glycosylation site at Asn-271 of pZP3 were located close to the interface of the pZP3-ZP-C and pZP4-ZP-N domains that form a sperm-binding surface.This region might also contain the N-glycosylated Asn-203, which is close to Asn-220 of the pZP4-ZP-N domain.However, the involvement of the N-glycosylation site at Asn-271 of pZP3 in the sperm-binding activity of the pZP3/pZP4 complex has not been clarified yet using in vitro sperm-binding assays.In chicken, O-glycosylation at Thr-168 in the hinge region of ZP3 homolog was shown to be important for the sperm-binding activity of ZP3 homolog [9], and this glycosylation site is located in the interfaces of domains, as proposed based on the cryo-EM structure of human uromodulin filament [29].
The 3D model of the bZP3 fragment/bZP4 complex, proposed by AlphaFold2/ ColabFold [24], showed that Asn-146 of bZP3 and Asn-314 of bZP4 are in close proximity, potentially forming a sperm recognition site at the interface of the bZP3-ZP-N and bZP4-ZP-C domains (Figure 5).Asn-314 is located at the N-terminal edge of the ZP-C domain within a consensus sequence called the internal hydrophobic patch (IHP) [8].In the IHP, the amino acid at this position is usually Pro, as shown in the pZP4 sequence (Figure 2A).The Asn at this position in ZP4 might be unique to bovines, making the N-glycosylation at this site a unique feature of bZP4.Therefore, the formation of a spermbinding site including N-glycosylated Asn residues at the ZP3-ZP-N-ZP4-ZP-C domain interface might also be unique to bovines.
In our previous study, incubation of bovine sperm with solubilized native bovine ZP for 3 h in BO solution, the same medium as used in the present study, induced only a 5% acrosome reaction [17].Therefore, we suppose that in the present study using bZP protein-coated plastic wells, the acrosome reaction of bovine sperm was not induced.

Figure 1 .
Figure 1.Domain architectures of bZP4 and bZP3 polypeptides.(A) Domain architecture of mature bZP4 (25-464) and schematic representation of truncated bZP4 (136-464) fragment.The orange twoheaded arrow shows the bZP4 (290-340) region examined in this study.The inverted tripods mark potential N-glycosylation sites.O-glycosylation sites are not shown.(B) A predicted structural model of bZP4 (25-464).This is a top-ranked model made using AlphaFold2.The previously proposed three sites involved in sperm binding on bZP4 (25-136) are shown in red in the ZP-N1 domain.The previously identified sperm-binding region from 290 to 340 on bZP4 is shown in orange extending from ZP-N domain to ZP-C domain.This orange region corresponds to the two-headed orange arrow shown in (A).(C) Domain architecture of mature bZP3 (32-348) and schematic representation of truncated bZP3 (32-178).The inverted tripods mark potential N-glycosylation sites.Oglycosylation sites are not shown.

Figure 1 .
Figure 1.Domain architectures of bZP4 and bZP3 polypeptides.(A) Domain architecture of mature bZP4 (25-464) and schematic representation of truncated bZP4 (136-464) fragment.The orange twoheaded arrow shows the bZP4 (290-340) region examined in this study.The inverted tripods mark potential N-glycosylation sites.O-glycosylation sites are not shown.(B) A predicted structural model of bZP4 (25-464).This is a top-ranked model made using AlphaFold2.The previously proposed three sites involved in sperm binding on bZP4 (25-136) are shown in red in the ZP-N1 domain.The previously identified sperm-binding region from 290 to 340 on bZP4 is shown in orange extending from ZP-N domain to ZP-C domain.This orange region corresponds to the two-headed orange arrow shown in (A).(C) Domain architecture of mature bZP3 (32-348) and schematic representation of truncated bZP3 (32-178).The inverted tripods mark potential N-glycosylation sites.O-glycosylation sites are not shown.

Figure 2 .
Figure 2. Sperm-binding sites in the middle region of bZP4.(A) Comparison of the amino acid sequences of bZP4 (290-340) and its corresponding pZP4 (291-341) counterpart and schematic diagrams of pZP4 (137-462), bZP4 (136-464), and bZP4 (136-464) mutant proteins.The non-identical amino acid residues between bovine and porcine sequences are highlighted in yellow and purple, respectively.Amino acids are numbered considering translational initiation at Met as 1.The schematic diagram of bZP4 (136-464) mutant proteins shows the pZP4 (333) (region 1), pZP4 (326-329) (region 2), pZP4 (321) (region 3), pZP4 (312-315) (region 4), and pZP4 (299-302) (region 5) that were replaced in the corresponding bovine sequences.(B) SDS-PAGE of bZP4 (25-464), bZP4 (136-464), pZP4 (137-462), and bZP4 (136-464) fragments with the indicated sites mutated with corresponding porcine sequences.Red arrowheads indicate protein bands of bZP4 (25-464) and bZP4 (136-464) and its mutants.Gels were silver-stained.Molecular mass standards (kDa) are indicated on the left side of each panel.Western blot original images can be found in Supplementary Materials.(C) Adsorption of each recombinant ZP4 protein to plastic wells shown in (B).The amount of each recombinant ZP4 protein added to a plastic well is indicated under each group of bars.The amounts of protein necessary for adsorption saturation were examined by detecting the adsorbed proteins with an anti-His tag antibody.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of the recombinant ZP4 proteins.Plastic wells were coated with each protein (0.8 µg) shown in (C).The number of sperm bound to the wells coated with bZP4 (25-464) varied from 29 to 58 but was designated as 100% for each experiment.Assays were repeated five times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.

Figure 2 .
Figure 2. Sperm-binding sites in the middle region of bZP4.(A) Comparison of the amino acid sequences of bZP4 (290-340) and its corresponding pZP4 (291-341) counterpart and schematic diagrams of pZP4 (137-462), bZP4 (136-464), and bZP4 (136-464) mutant proteins.The non-identical amino acid residues between bovine and porcine sequences are highlighted in yellow and purple, respectively.Amino acids are numbered considering translational initiation at Met as 1.The schematic diagram of bZP4 (136-464) mutant proteins shows the pZP4 (333) (region 1), pZP4 (326-329) (region 2), pZP4 (321) (region 3), pZP4 (312-315) (region 4), and pZP4 (299-302) (region 5) that were replaced in the corresponding bovine sequences.(B) SDS-PAGE of bZP4 (25-464), bZP4 (136-464), pZP4 (137-462), and bZP4 (136-464) fragments with the indicated sites mutated with corresponding porcine sequences.Red arrowheads indicate protein bands of bZP4 (25-464) and bZP4 (136-464) and its mutants.Gels were silver-stained.Molecular mass standards (kDa) are indicated on the left side of each panel.SDS-PAGE original images can be found in Supplementary Materials.(C) Adsorption of each recombinant ZP4 protein to plastic wells shown in (B).The amount of each recombinant ZP4 protein added to a plastic well is indicated under each group of bars.The amounts of protein necessary for adsorption saturation were examined by detecting the adsorbed proteins with an anti-His tag antibody.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of the recombinant ZP4 proteins.Plastic wells were coated with each protein (0.8 µg) shown in (C).The number of sperm bound to the wells coated with bZP4 (25-464) varied from 29 to 58 but was designated as 100% for each experiment.Assays were repeated five times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.

Figure 3 .
Figure 3.The N-glycosylation site on bZP4 (290-340) is involved in the sperm-binding activity of bZP4.(A) Schematic of bZP4 (136-464) with the mutation of Gln-311 and Asn-314 sites.(B) SDS-PAGE of bZP4 (136-464) Q311H, bZP4 (136-464) N314P, and bZP4 (136-464) N314D mutant proteins.Red arrowheads indicate protein bands of bZP4 (136-464) and its mutants.Gels were silverstained.Molecular mass standards (kDa) are indicated on the left side of each panel.Western blot original images can be found in Supplementary Materials.(C) Adsorption of each bZP4 mutant protein to plastic wells shown above.The amount of each bZP4 protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with an anti-His tag antibody.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each bZP4 protein shown above.Plastic wells were coated with each bZP4 protein (0.8 µg) indicated in the graph.The number of sperm bound to the wells coated with bZP4 (25-464) varied from 23 to 42 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.(E) Comparisons of mobilities on SDS-PAGE gels between bZP4 (136-464) and its N-glycosylation site mutants.Left panel: mobilities were compared between bZP4 (136-464), left and right lanes, and bZP4 (136-464) N314D, middle lane.Right panel: mobilities were compared between bZP4 (136-464), left and right lanes, and bZP4 (136-464) N314P, middle lane.Gels were silver-stained.Only the parts around the protein bands are shown.Western blot original images can be found in Supplementary Materials.

Figure 3 .
Figure 3.The N-glycosylation site on bZP4 (290-340) is involved in the sperm-binding activity of bZP4.(A) Schematic of bZP4 (136-464) with the mutation of Gln-311 and Asn-314 sites.(B) SDS-PAGE of bZP4 (136-464) Q311H, bZP4 (136-464) N314P, and bZP4 (136-464) N314D mutant proteins.Red arrowheads indicate protein bands of bZP4 (136-464) and its mutants.Gels were silver-stained.Molecular mass standards (kDa) are indicated on the left side of each panel.SDS-PAGE original images can be found in Supplementary Materials.(C) Adsorption of each bZP4 mutant protein to plastic wells shown above.The amount of each bZP4 protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with an anti-His tag antibody.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each bZP4 protein shown above.Plastic wells were coated with each bZP4 protein (0.8 µg) indicated in the graph.The number of sperm bound to the wells coated with bZP4 (25-464) varied from 23 to 42 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.(E) Comparisons of mobilities on SDS-PAGE gels between bZP4 (136-464) and its N-glycosylation site mutants.Left panel: mobilities were compared between bZP4 (136-464), left and right lanes, and bZP4 (136-464) N314D, middle lane.Right panel: mobilities were compared between bZP4 (136-464), left and right lanes, and bZP4 (136-464) N314P, middle lane.Gels were silver-stained.Only the parts around the protein bands are shown.SDS-PAGE original images can be found in Supplementary Materials.

Figure 4 .
Figure 4. N-glycosylation sites in the middle region of both bZP3 and bZP4 are involved in spermbinding activity.(A) Schematic diagrams of bZP4 (136-464) with mutation of Asn-314 site and bZP3 (32-178) with mutation of Asn-146 site.The Asn-314 and Asn-146 were mutated to Asp (N314D and N146D, respectively).(B) SDS-PAGE images of bZP3 (32-178)/bZP4 (136-464), bZP3 (32-178) N146D/bZP4 (136-464), bZP3 (32-178)/bZP4 (136-464) N314D, and bZP3 (32-178) N146D/bZP4 (136-464) N314D.Blue arrows indicate protein bands of bZP4 (136-464) and bZP4 (136-464) N314D.Red arrows indicate protein bands of bZP3 (32-178) and bZP3 (32-178) N146D.Gels were silverstained.Molecular mass standards (kDa) are indicated on the left side of each panel.Western blot original images can be found in Supplementary Materials.(C)Adsorption of each complex protein to plastic wells shown above.The amount of each complex protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with a mixture of anti-His tag and anti-FLAG tag antibodies.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each protein complex shown above.Plastic wells were coated with each protein complex (0.8 µg for each protein complex).The number of sperm bound to wells coated with bZP4 (25-464) varied from 21 to 39 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.

Figure 4 .
Figure 4. N-glycosylation sites in the middle region of both bZP3 and bZP4 are involved in spermbinding activity.(A) Schematic diagrams of bZP4 (136-464) with mutation of Asn-314 site and bZP3 (32-178) with mutation of Asn-146 site.The Asn-314 and Asn-146 were mutated to Asp (N314D and N146D, respectively).(B) SDS-PAGE images of bZP3 (32-178)/bZP4 (136-464), bZP3 (32-178) N146D/bZP4 (136-464), bZP3 (32-178)/bZP4 (136-464) N314D, and bZP3 (32-178) N146D/bZP4 (136-464) N314D.Blue arrows indicate protein bands of bZP4 (136-464) and bZP4 (136-464) N314D.Red arrows indicate protein bands of bZP3 (32-178) and bZP3 (32-178) N146D.Gels were silverstained.Molecular mass standards (kDa) are indicated on the left side of each panel.SDS-PAGE original images can be found in Supplementary Materials.(C)Adsorption of each complex protein to plastic wells shown above.The amount of each complex protein added to a plastic well is indicated under each group of bars.The amounts of proteins necessary for adsorption saturation were examined by detecting the adsorbed proteins with a mixture of anti-His tag and anti-FLAG tag antibodies.The experiment was performed thrice, and the average ± standard deviation (SD) of absorbance at 405 nm is shown.(D) Sperm-binding activity of each protein complex shown above.Plastic wells were coated with each protein complex (0.8 µg for each protein complex).The number of sperm bound to wells coated with bZP4 (25-464) varied from 21 to 39 but was designated as 100% for each experiment.Assays were repeated at least four times.Data are presented as the mean ± SD, with statistical significance between two bars indicated as p < 0.01 (**) on each line connecting the two bars.