Naoto Yonezawa

Characterization of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs

Bovine zona pellucida (ZP) glycoproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-beta-galactosidase (E beta G) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (E beta G-76), 68 kDa (E beta G-68), 63 kDa (E beta G-63), 47 kDa (E beta G-47) and 21 kDa (E beta G-21) under the same conditions. The N-terminal amino acid sequences of E beta G-76 and E beta G-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that E beta G-21 (N-terminal region) is linked to E beta G-63 (C-terminal region) through disulfide bond to form E beta G-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine E beta G-76, E beta G-68 and E beta G-47 correspond to pig PZP2, PZP3 alpha and PZP3 beta glycoproteins, respectively. The E beta G-76 and E beta G-68 components were shown to be specifically cleaved during fertilization.

Localization of Sperm Ligand Carbohydrate Chains in Pig Zona pellucida Glycoproteins

Neutral N-linked carbohydrate chains from pig ZPB/ZPC mixture are shown to possess sperm ligand activity. Of these complex-type chains, triantennary/tetraantennary chains exhibit the activity stronger than that of diantennary chains. Intact ZPB and ZPC cannot be separated from each other unless acidic N-acetyllactosamine regions of their carbohydrate chains are removed by endo-β-galactosidase digestion. The endo-β-galactosidase-digested ZPB and its N-terminal fragment of 111 residues retain the sperm ligand activity. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, are obtained by lysylendopeptidase digestion of the heat-solubilized zonae containing intact ZPB, and lysylendopeptidase and chymotryptic digestions of endo-β-galactosidase-digested ZPB. On the basis of sugar mapping analysis of the N-linked chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N-linked chains of ZPB/ZPC, the triantennary and tetraantennary chains are shown to be localized mainly at Asn220 of ZPB, whereas diantennary chains are present on all the three N-glycosylation sites (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm-egg binding. ZPC has been shown to support the expression of sperm ligand activity of ZPB. Three glycopeptides, each having one of the N-glycosylation sites, are obtained by tryptic digestion of endo-β-galactosidase-digested ZPC. Triantennary and tetraantennary chains are found mainly at Asn271 of ZPC, whereas diantennary chains are present at all three N-glycosylation sites (Asn124, Asn146 and Asn271). Thus, the localization of triantennary and tetraantennary chains in ZPC is different from that in ZPB.

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.

A D19S433 Primer Binding Site Mutation and the Frequency in Japanese of the Silent Allele It Causes

Abstract:  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.

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 prelmplantatlon. 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 β-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. α-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 β-structure as the main constituent. Attenuated total reflectlon-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the β-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.

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.

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.

The presence of a glycosyl phosphatidylinositol-anchored α-mannosidase in boar sperm

alpha-Mannosidase and beta-galactosidase were released from boar sperm into the medium by treatment with calcium ionophore A23187 or by 0.2% Brij-35/2% acetic acid. About half as much alpha-mannosidase activity as that in the acid extract was recovered by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the liberation rate of beta-galactosidase treated with PI-PLC was low. These results suggest that some alpha-mannosidase is anchored in the plasma membrane of the acrosomal region by attachment to the lipid phosphatidylinositol and that beta-galactosidase is localized mainly in the acrosome or integrated in the plasma membrane by a spanning stretch of hydrophobic peptides. beta-Galactosidase, which is present as an oligomers in the acid extract of sperm, dissociated into monomers under weakly alkaline conditions; under acidic conditions, the monomers associated again. No pH-sensitive association-dissociation of alpha-mannosidase was observed.

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.