Insights into egg coat assembly and egg-sperm interaction from the X-ray structure of full-length ZP3.

ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.

Effective use of legacy data in a genome-wide association studies improves the credibility of quantitative trait loci detection in rice.

Genome-wide association studies (GWASs) are used to detect quantitative trait loci (QTL) using genomic and phenotypic data as inputs. While genomic data are obtained with high throughput and low cost, obtaining phenotypic data requires a large amount of effort and time. In past breeding programs, researchers and breeders have conducted a large number of phenotypic surveys and accumulated results as legacy data. In this study, we conducted a GWAS using phenotypic data of temperate japonica rice (Oryza sativa) varieties from a public database. The GWAS using the legacy data detected several known agriculturally important genes, indicating reliability of the legacy data for GWAS. By comparing the GWAS using legacy data (L-GWAS) and a GWAS using phenotypic data that we measured (M-GWAS), we detected reliable QTL for agronomically important traits. These results suggest that an L-GWAS is a strong alternative to replicate tests to confirm the reproducibility of QTL detected by an M-GWAS. In addition, because legacy data have often been accumulated for many traits, it is possible to evaluate the pleiotropic effect of the QTL identified for the specific trait that we focused on with respect to various other traits. This study demonstrates the effectiveness of using legacy data for GWASs and proposes the use of legacy data to accelerate genomic breeding.

Extracellular vesicle-mediated MFG-E8 localization in the extracellular matrix is required for its integrin-dependent function in mouse mammary epithelial cells.

Milk fat globule-EGF factor 8 (MFG-E8) is a divalent-binding secretory protein possessing an Arg-Gly-Asp (RGD) motif and a phosphatidylserine (PS)-binding motif. This protein has been shown to be involved in mammary gland development and morphogenesis. Integrin-binding activity is necessary for these MFG-E8-dependent cell processes. Although the target cells and molecules of MFG-E8 in the cellular microenvironment are important to understand its physiological function, its localization is largely unclear. Here, we found that mouse MFG-E8 localized to the basal lamina of the mammary gland during involution. In a model system of mammary COMMA-1D cells, exogenously and endogenously expressed MFG-E8 was deposited in the extracellular matrix (ECM) with membranous particles dependently on the PS-binding motifs in the discoidin domains that were essential for association ability to extracellular vesicles (EVs). These data showed the basal MFG-E8 localization mechanism in which EVs served as a scaffold. Such an immobilized MFG-E8 associating with cell substrata but not soluble one in the culture media promoted integrin-dependent suppression of ß-casein expression. These results suggest that MFG-E8 requires EVs to transduce cellular signals from the basolateral side of the adhesion cells by accumulating in ECM.

The mRNA-binding protein Serbp1 as an auxiliary protein associated with mammalian cytoplasmic ribosomes.

While transcription plays an obviously important role in gene expression, translation has recently been emerged as a key step that defines the composition and quality of the proteome in the cell of higher eukaryotes including mammals. Selective translation is supposed to be regulated by the structural heterogeneity of cytoplasmic ribosomes including differences in protein composition and chemical modifications. However, the current knowledge on the heterogeneity of mammalian ribosomes is limited. Here, we report mammalian Serbp1 as a ribosome-associated protein. The translated products of Serbp1 gene, including the longest isoform, were found to be localized in the nucleolus as well as in the cytoplasm. Subcellular fractionation indicated that most of cytoplasmic Serbp1 molecules were precipitated by ultracentrifugation. Proteomic analysis identified Serbp1 in the cytoplasmic ribosomes of the rodent testis. Polysome profiling suggested that Serbp1, as a component of the small 40S subunit, was included in translating ribosomes (polysomes). Cosedimentation of Serbp1 with the 40S subunit was observed after dissociation of the ribosomal subunits. Serbp1 was also included in the ribosomes of human cancer cells, which may lead to a mechanistic understanding of an emerging link between Serbp1 and tumour progression. SIGNIFICANCE OF THE STUDY: In mammalian cells, the final protein output of their genetic program is determined not only by controlling transcription but also by regulating the posttranscriptional events. Although mRNA-binding proteins and the cytoplasmic ribosome have long been recognized as central players in the posttranscriptional regulation, their physical and functional interactions are still far from a complete understanding. Here, we describe the intracellular localization of Serbp1, an mRNA-binding protein, and the inclusion of this protein in actively translating ribosomes in normal and cancer cells. These findings shed a new light into molecular mechanisms underlying Serbp1 action in translational gene regulation and tumour progression.

Inhibitory effects of dietary soyasaponin on 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Soyasaponins (SSs) abundant in soybean have anti-inflammatory activities; however, their therapeutic effects on allergic contact dermatitis (ACD) remain unknown. To assess the effects of SS-enriched diets on ACD, we used a mouse model of contact hypersensitivity (CHS). Mice were fed low-dose or high-dose SS-containing diets for 3 weeks prior to CHS induction with 2,4-dinitrofluorobenzene (DNFB). The low-dose SS diet attenuated DNFB-induced ear swelling and tissue oedema, and reduced the number of infiltrating Gr-1-positive myeloid cells. Low-dose, but not high-dose, SSs decreased chemokine (C-X-C motif) ligand 2 (CXCL2) and triggering receptor expressed on myeloid cells (TREM)-1 production in ear tissues, compared to a control. Taxonomic 16S rRNA analysis revealed significant alterations in faecal microbiota caused by CHS, which were reversed by low-dose SSs. The low-dose SS and non-CHS groups clustered together, while the high-dose SS group split between CHS and non-CHS clusters. Our results demonstrated that low-dose SSs alleviated CHS symptoms by attenuating inflammation and improving the intestinal microbiota composition, suggesting that dietary SSs may have beneficial effects on ACD.

Fertilization 1: Sperm-Egg Interaction.

In birds in the reproductive season, an egg is ovulated without cumulus cells from the largest follicle with the highest hierarchy in the ovary. The outermost part of the ovulated eggs is the perivitelline layer, a glycoprotein matrix consisting of a few ZP-glycoproteins. The fertilization starts from sperm penetration of the perivitelline layer predominantly in the germinal disc region, followed by uptake of the sperm into the egg, and goes through by the fusion of sperm male pronucleus with the female pronucleus in the egg. A series of these fertilization steps occurs in the infundibulum of the oviduct within a short period after ovulation. Some pioneering microstructural studies using electron microscopy and supporting biochemical data from later studies indicate that, in avian fertilization, sperm interacts with the perivitelline layer covering the germinal disc, locally degrade and dissolve the matrix of the perivitelline layer, and penetrate it through the hole made proteolytically at the sperm-binding site on the perivitelline layer. Several molecules and structures presumably involved in the sperm-perivitelline interaction have been characterized, especially sperm proteases and their targets in the egg perivitelline layer. On the other hand, no molecules involved in the sperm-egg membrane fusion for the male pronucleus uptake into the egg have yet been identified or characterized and, moreover, no orthologue but one have been annotated so far in the chicken genome for the mouse genes involved in the sperm-egg membrane fusion.

Impaired O-linked N-acetylglucosaminylation in the endoplasmic reticulum by mutated epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine transferase found in Adams-Oliver syndrome.

Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 µm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT(R377Q) were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT(R377Q) was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins.

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28-58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

Acid resistance contributes to the high-pressure carbon dioxide resistance of Escherichia coli K-12.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Multispecificity of immunoglobulin M antibodies raised against advanced glycation end products: involvement of electronegative potential of antigens.

BACKGROUND: Advanced glycation end products (AGEs) can act as neoantigens to trigger immune responses. RESULTS: Natural IgM antibodies against AGEs recognize multiple molecules, including DNA and chemically modified proteins. CONCLUSION: There is a close relationship between the formation of AGEs and innate immune responses. SIGNIFICANCE: Our findings highlight AGEs and related modified proteins as a source of multispecific natural antibodies Advanced glycation end products (AGEs) are a heterogeneous and complex group of compounds that are formed when reducing sugars, such as dehydroascorbic acid, react in a nonenzymatic way with amino acids in proteins and other macromolecules. AGEs are prevalent in the diabetic vasculature and contribute to the development of atherosclerosis. The presence and accumulation of AGEs in many different cell types affect the extracellular and intracellular structure and function. In the present study, we studied the immune response to the dehydroascorbic acid-derived AGEs and provide multiple lines of evidence suggesting that the AGEs could be an endogenous source of innate epitopes recognized by natural IgM antibodies. Prominent IgM titers to the AGEs were detected in the sera of normal mice and were significantly accelerated by the immunization with the AGEs. Patients with systemic lupus erythematosus (SLE), a potentially fatal systemic autoimmune disease characterized by the increased production of autoantibodies, showed significantly higher serum levels of the IgM titer against the AGEs than healthy individuals. A progressive increase in the IgM response against the AGEs was also observed in the SLE-prone mice. Strikingly, a subset of monoclonal antibodies, showing a specificity toward the AGEs, prepared from normal mice immunized with the AGEs and from the SLE mice cross-reacted with the double-stranded DNA. Moreover, they also cross-reacted with several other modified proteins, including the acetylated proteins, suggesting that the multiple specificity of the antibodies might be ascribed, at least in part, to the increased electronegative potential of the proteins. These findings suggest that the protein modification by the endogenous carbonyl compounds, generating electronegative proteins, could be a source of multispecific natural antibodies.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs.

Lyar, a cell growth-regulating zinc finger protein, was identified to be associated with cytoplasmic ribosomes in male germ and cancer cells.

Translational control is a basic mechanism for gene regulation in cells and important for tissue growth and development in mammals. Deregulation of the mechanism thus causes diseases such as cancer. Considering the importance of the ribosome as a factory of polypeptide synthesis, some new factors have been expected to be associated with the ribosome and involved in translational control. Our proteomic survey for these factors identified a zinc finger protein, Lyar, in cytoplasmic ribosomes of the rodent testis. Subcellular fractionation of the testis provided data supporting association of Lyar with ribosomes. Lyar was then suggested to be included in the 60S large subunit, but not in polysomes, by ultracentrifugation of testicular ribosomes. While analysis of tissue distribution of Lyar has indicated its testis-predominant expression, Lyar mRNA was expressed in the cancer cells originated from tissues other than testis, and Lyar promoted proliferation of NIH-3T3 cells. Furthermore, translation was increased by Lyar in vitro, pointing out the first experimental link between this protein and translation. Taken together, Lyar seems to be a new player in translational control and a potential target for cancer therapy.

Vitamin C deficiency alters the transcriptome of the rat brain in a glucocorticoid-dependent manner, leading to microglial activation and reduced neurogenesis.

Vitamin C (VitC) is maintained at high concentrations in the brain and is an essential micronutrient for brain function. VitC deficiency leads to neuropsychiatric scurvy, which is characterized by depression and cognitive impairment. However, the molecular mechanism by which mild VitC deficiency impairs brain function is currently unknown. In the present study, we conducted RNA sequencing analysis and found that a short-term VitC deficiency altered the brain transcriptome in ODS rats, which cannot synthesize VitC. Bioinformatic analysis indicated that VitC deficiency affected the expression of genes controlled by the glucocorticoid receptor in the brain. We confirmed an increased secretion of glucocorticoids from the adrenal gland during VitC deficiency. We found that non-neuronal cells, including microglia, which are resident immune cells in the brain, changed their transcriptional patterns in response to VitC deficiency. Immunohistochemical analysis revealed that the quiescent ramified microglia transform into the activated amoeboid microglia during three weeks of VitC deficiency. The morphological activation of microglia was accompanied by increased expression of proinflammatory cytokines such as interleukin-6 in the hippocampus. Furthermore, VitC deficiency decreased the number of newly born neurons in the dentate gyrus of the hippocampus, suggesting that VitC was required for adult neurogenesis that plays a crucial role in learning and memory. Our findings may provide insights into the molecular mechanisms underlying the maintenance of normal brain function by adequate levels of VitC.

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons.

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. The multipolar-to-bipolar transition, a critical step in establishing neuronal polarity during radial migration, occurs in the subplate/intermediate zone (SP/IZ), a distinct region of the embryonic cerebral cortex. It has been known that the extracellular matrix (ECM) molecules are enriched in the SP/IZ. However, the molecular constitution and functions of the ECM formed in this region remain poorly understood. Here, we identified neurocan (NCAN) as a major chondroitin sulfate proteoglycan in the mouse SP/IZ. NCAN binds to both radial glial-cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA in the SP/IZ. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, both TNC and NCAN promoted the morphological maturation of cortical neurons in vitro. The present results provide evidence for the cooperative role of neuron- and radial glial-cell-derived ECM molecules in cortical development.

Measurement of barley ß-glucan concentration in the plasma by sandwich ELISA using rat dectin-1.

We used recombinant rat dectin-1 proteins to newly establish sandwich ELISA for determining barley ß-glucan (BßG). The ELISA method had a working range of 15-4,000 µg/L. Plasma BßG was detectable up to 24 h after an intravenous administration of BßG (1 mg/kg of body weight) to rats. This method may be an effective tool for investigating the immune modulatory effects of BßG.

Intracellular retention and subsequent release of bovine milk lactoferrin taken up by human enterocyte-like cell lines, Caco-2, C2BBe1 and HT-29.

Lactoferrin (LF) is an iron-binding glycoprotein contained in milk and other exocrine fluids, and is believed to have multiple biological functions. We investigated the intracellular dynamics of LF taken up by three lines of human enterocytes and the subsequent release of internalized LF by using two-site ELISA and confocal microscopy. LF taken up by Caco-2 cells was kept partially intact within the cells and subsequently released to the medium as degraded fragments of 30-50 kDa. The retention and subsequent release of LF by Caco-2 cells were much more abundant than those of ovalbumin, ovomucoid and lysozyme. Such results characteristic of LF were also similarly observed in C2BBe1 and HT29 cells more markedly. LF was detected as punctate signals and partially colocalized with the lactoferrin receptor, intelectin-1, in the respective cytoplasm and nuclei of Caco-2 and C2BBe1 cells. In contrast, LF within the HT-29 cells was detected as much smaller punctate signals scattered in the cytoplasm.

New insights into the role of microheterogeneity of ZP3 during structural maturation of the avian equivalent of mammalian zona pellucida.

The egg coat including mammalian zona pellucida (ZP) and the avian equivalent, i.e., inner-perivitelline layer (IPVL), is a specialized extracellular matrix being composed of the ZP glycoproteins and surrounds both pre-ovulatory oocytes and ovulated egg cells in vertebrates. The egg coat is well known for its potential importance in both the reproduction and early development, although the underlying molecular mechanisms remain to be fully elucidated. Interestingly, ZP3, one of the ZP-glycoprotein family members forming scaffolds of the egg-coat matrices with other ZP glycoproteins, exhibits extreme but distinctive microheterogeneity to form a large number of isoelectric-point isoforms at least in the chicken IPVL. In the present study, we performed three-dimensional confocal imaging and two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE) of chicken IPVLs that were isolated from the ovarian follicles at different growth stages before ovulation. The results suggest that the relative proportions of the ZP3 isoforms are differentially altered during the structural maturation of the egg-coat matrices. Furthermore, tandem mass spectrometry (MS/MS) analyses and ZP1 binding assays against separated ZP3 isoforms demonstrated that each ZP3 isoform contains characteristic modifications, and there are large differences among ZP3 isoforms in the ZP1 binding affinities. These results suggest that the microheterogeneity of chicken ZP3 might be regulated to be associated with the formation of egg-coat matrices during the structural maturation of chicken IPVL. Our findings may provide new insights into molecular mechanisms of egg-coat assembly processes.

Diverse lectin-binding specificity of four ZP3 glycoprotein isoforms with a discrete isoelectric point in chicken egg coat.

The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galß1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((ß-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galß1-4GlcNAcß1-2Manα1-6) and RCA I (terminal Galß1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1.

O-linked-ß-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.