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.

Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4.

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1-ZP4). The functions of the three proteins present in mice (ZP1-ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Sperm-binding regions on bovine egg zona pellucida glycoprotein ZP4 studied in a solid supported form on plastic plate.

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. The bovine ZP consists of the glycoproteins ZP2, ZP3, and ZP4. Sperm-binding mechanisms of the bovine ZP are not yet fully elucidated. In a previous report, we established the expression system of bovine ZP glycoproteins using Sf9 insect cells and found that the ZP3/ZP4 heterocomplex inhibits the binding of sperm to the ZP in a competitive inhibition assay, while ZP2, ZP3, ZP4, the ZP2/ZP3 complex, and the ZP2/ZP4 complex do not exhibit this activity. Here, we show that bovine sperm binds to plastic plates coated with ZP4 in the absence of ZP3. We made a series of ZP4 deletion mutants to study the sperm-binding sites. The N-terminal region, Lys-25 to Asp-136, and the middle region, Ser-290 to Lys-340, of ZP4 exhibit sperm-binding activity. These results suggest that among the three components of bovine ZP glycoproteins, ZP4 contains the major potential sperm-binding sites, and the formation of a multivalent complex is necessary for the sperm-binding activity of ZP4.

The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex.

The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.

Posttranslational modifications of zona pellucida proteins.

The zona pellucida (ZP), which surrounds the mammalian oocyte, functions in various aspects of fertilization. The ZP consists of three or four glycoproteins, which are derived from transmembrane proteins that lack the ability to self-assemble. Following posttranslational processing at specific sites, ectodomains of ZP precursor proteins are released from the membrane and begin to form a matrix. Glycosylational modification is thought to be involved in species-selective sperm recognition by ZP proteins. However, in mice, the supramolecular structure of the zona matrix is also important in sperm recognition. One ZP protein, ZP2, is processed at a specific site upon fertilization by ovastacin, which is released from cortical granules inside the oocyte. This phenomenon is involved in the block to polyspermy. The proteolysis of ubiquitinated ZP proteins by a sperm-associated proteasome is involved in penetration of the zona matrix by sperm, at least in the pigs. Thus, the posttranslational modification of ZP proteins is closely tied to ZP formation and the regulation of sperm-oocyte interactions.

Calcium-dependent structural changes in human reticulocalbin-1.

Human reticulocalbin-1 (hRCN1) has six EF-hand motifs and binds Ca(2+). hRCN1 is a member of the CREC family localized in the secretory pathway, and its cellular function remains unclear. In this study, we established a new bacterial expression and purification procedure for hRCN1. We observed that hRCN1 binds Ca(2+) in a cooperative manner and the Ca(2+) binding caused an increase in the α-helix content of hRCN1. On the other hand, hRCN1 did not change the structure with Mg(2+) loading. hRCN1 is a monomeric protein, and its overall structure became more compact upon Ca(2+) binding, as revealed by gel-filtration column chromatography and small-angle X-ray scattering. This is the first report of conformational changes in the CREC family upon Ca(2+) binding. Our data suggest that CREC family member interactions with target proteins are regulated in the secretory pathway by conformational changes upon Ca(2+) binding.

Coordination structures of Mg2+ and Ca2+ in three types of tobacco calmodulins in solution: Fourier-transform infrared spectroscopic studies of side-chain COO- groups.

Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak ß-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+)-binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+)] increases under low [Ca(2+)] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+)]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+)-binding manner and needs higher [Ca(2+)] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+)-binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs.

Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle.

Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm-oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for ß-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from ß-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm-oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR.

Performing short tandem repeat (STR) analysis from degraded DNA is a challenge for forensic biologists. For assessing the quality and quantity of DNA, we developed quantitative PCR assays to determine the extent of DNA degradation. Quantitative PCR assays using primers that generate two sizes of amplicons from the same region of genomic DNA were used to determine the extent of DNA degradation. These quantitative PCR assays were used with artificially degraded DNA and degraded DNA extracted from aged bloodstains. Increased DNA degradation correlated with a decrease in the number of detectable loci in STR analysis. The extent of DNA degradation and the number of loci detected by STR analysis varied depending on the method of degradation. The extent of degradation of DNA extracted from aged bloodstains correlated well with that of DNA artificially degraded by DNase I in the presence of Mn(2+). Thus, determination of the extent of DNA degradation was helpful for estimating the number of detectable loci. Furthermore, this estimation method is expected to save time and labor, and is particularly suitable when only a limited amount of DNA can be extracted from casework samples.

Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex.

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.

A forensic method for the simultaneous analysis of biallelic markers identifying Y chromosome haplogroups inferred as having originated in Asia and the Japanese archipelago.

Information regarding the ancestral and geographical origins of biological evidence samples may be useful for crime investigators as they narrow down the possible donors of the sample. A method for simultaneous analysis of seven biallelic markers (M130, M131, M57, M125, M175, M122 and M134) was developed for forensic application. M57, M125 and M131 are included to identify haplogroups inferred as having originated in the Japanese archipelago. Our method employs allele-specific PCR and fragment analysis using fluorescently labeled primers and capillary electrophoresis. This method can be used to assign a haplogroup from both of degraded male DNA samples and DNA samples containing a mixture of female and male DNA by designing PCR primers that generate small amplicons and are highly specific for targets on the Y chromosome. A total of 1346 samples from Japanese males collected from the four major islands and Okinawa island were classified into seven Y binary haplogroups i.e., C-M130, C-M131, D-M57, D-M125, O-M175, O-M122 and O-M134, and a "no-mutation detected" group and their frequencies were 0.0617, 0.0565, 0.1441, 0.182, 0.3418, 0.11, 0.0847 and 0.0193, respectively. Samples of "no-mutation detected" were further analyzed by direct sequencing for identification of the major haplogroup to which they belong. Along with the haplogroup data, we report haplotype data for the 16 Y-STR markers included in the AmpFlSTR Yfiler PCR amplification kit (Applied Biosystems). These data will be useful in the prediction of haplogroups based on Y-STR haplotypes.

A D19S433 primer binding site mutation and the frequency in Japanese of the silent allele it causes.

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler kit failed to amplify an allele at the D19S433 locus, producing a silent ("null") allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3' end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent-child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an "apparent opposite homozygosity" and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.

Structure of calcium-bound human S100A13 at pH 7.5 at 1.8 A resolution.

S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins. S100A13 plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1 alpha, two pro-angiogenic factors released by the nonclassical endoplasmic reticulum/Golgi-independent secretory pathway. The X-ray crystal structure of human S100A13 at pH 7.5 was determined at 1.8 A resolution. The structure was solved by molecular replacement and was refined to a final R factor of 19.0%. The structure revealed that human S100A13 exists as a homodimer with two calcium ions bound to each protomer. The protomer is composed of four alpha-helices (alpha(1)-alpha(4)), which form a pair of EF-hand motifs. Dimerization occurs by hydrophobic interactions between helices alpha(1) and alpha(4) and by intermolecular hydrogen bonds between residues from helix alpha(1) and the residues between alpha(2) and alpha(3) of both chains. Despite the high similarity of the backbone conformation in each protomer, the crystal structures of human S100A13 at pH 7.5 (this study) and at pH 6.0 [Li et al. (2007), Biochem. Biophys. Res. Commun. 356, 616-621] exhibit recognizable differences in the relative orientation ( approximately 2.5 degrees) of the protomers within the dimer and also remarkable differences in the side-chain conformations of several amino-acid residues.

Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex.

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glycoproteins (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-terminal 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-complexes 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.

Crystallization and preliminary X-ray analysis of human S100A13.

S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1alpha, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 A resolution and the space group was assigned as primitive orthorhombic P2(1)2(1)2(1).

Fourier transform infrared spectroscopic analysis of the intact zona pellucida of the mammalian egg: changes in the secondary structure of bovine zona pellucida proteins during fertilization.

The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of beta-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. Alpha-helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have beta-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the beta-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.

Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm.

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull.

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.

Identification of the carboxyl termini of porcine zona pellucida glycoproteins ZPB and ZPC.

The extracellular matrix surrounding mammalian oocytes plays important roles in fertilization and is known as the zona pellucida (ZP). The ZP consists of three glycoproteins, ZPA, ZPB, and ZPC, which contain homologous regions known as ZP domains. The ZP domain is also found in many other secretory glycoproteins. Putative transmembrane domains present at the C-termini of ZP glycoprotein precursors are removed as the proteins proceed through the secretory pathway. However, the details of this processing have been unclear. In particular, the precise locations of the C-termini of mammalian zona proteins have not yet been determined. In this study, the C-terminal residues of porcine ZPB and ZPC were identified as Ala-462 and Ser-332, respectively, by mass spectrometry of C-terminal polypeptide fragments of these proteins. These results suggest that ZPB is processed at its furin consensus site, whereas ZPC is processed N-terminal to the furin consensus site. In addition, the analyses of porcine ZPB and ZPC fragments revealed that disulfide bonds within the ZP domains are divided into two groups, suggesting that the ZP domain consists of two subdomains.

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.