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

2022 ◽  
Vol 23 (2) ◽  
pp. 762
Author(s):  
Kamila Dilimulati ◽  
Misaki Orita ◽  
Yoshiki Yonahara ◽  
Fabiana Lica Imai ◽  
Naoto Yonezawa
Keyword(s):  
Binding Sites ◽  
Binding Activity ◽  
Binding Region ◽  
Terminal Domain ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Selective Interaction ◽  
Egg Coat

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.

scholarly journals Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
César L. Pastrana ◽  
Ngat T. Tran ◽  
Clare. E. Stevenson ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

ABSTRACTThe tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254234
Author(s):  
Kamila Dilimulati ◽  
Misaki Orita ◽  
Ganbat Undram ◽  
Naoto Yonezawa
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Insect Cells ◽  
Competitive Inhibition ◽  
Expression System ◽  
Binding Activity ◽  
Mammalian Oocyte ◽  
Sperm Binding ◽  
Bovine Sperm ◽  
Binding Mechanisms

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.

scholarly journals Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

1993 ◽  
Vol 123 (6) ◽  
pp. 1431-1440 ◽  
Author(s):  
D J Miller ◽  
X Gong ◽  
G Decker ◽  
B D Shur
Keyword(s):  
Zona Pellucida ◽  
Binding Sites ◽  
Binding Activity ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Sperm Binding ◽  
Competitive Inhibitors ◽  
Glycosidase Inhibitor ◽  
Egg Coat

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

scholarly journals Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

2020 ◽  
Author(s):  
Alena Stsiapanava ◽  
Chenrui Xu ◽  
Martina Brunati ◽  
Sara Zamora-Caballero ◽  
Céline Schaeffer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Zona Pellucida ◽  
Binding Sites ◽  
Lateral Interaction ◽  
Biological Processes ◽  
Binding Region ◽  
Sperm Binding ◽  
Cryo Electron Microscopy ◽  
Uropathogenic Bacteria ◽  
Egg Coat

SUMMARYAssembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

scholarly journals First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

2004 ◽  
Vol 186 (14) ◽  
pp. 4620-4627 ◽  
Author(s):  
Wakao Fukuda ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Binding Sites ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cation Binding ◽  
Activity Levels ◽  
Binding Region ◽  
Hyperthermophilic Archaeon ◽  
Dependent Activity ◽  
Additional Role

ABSTRACT Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (Pck Tk ). Pck Tk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in Pck Tk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant Pck Tk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of Pck Tk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of Pck Tk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

scholarly journals Colchicine-binding protein of the liver. Its characterization and relation to microtubules.

1975 ◽  
Vol 66 (3) ◽  
pp. 609-620 ◽  
Author(s):  
C Patzelt ◽  
A Singh ◽  
Y L Marchand ◽  
L Orci ◽  
B Jeanrenaud
Keyword(s):  
High Speed ◽  
Specific Binding ◽  
Electrophoretic Analysis ◽  
Binding Activity ◽  
Low Concentrations ◽  
Electrophoretic Behavior ◽  
High Affinity Binding Site ◽  
High Concentrations

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

2012 ◽  
Vol 449 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chiara Saggioro ◽  
Anne Olliver ◽  
Bianca Sclavi
Keyword(s):  
Temperature Dependence ◽  
Binding Sites ◽  
Dna Interaction ◽  
Bacterial Dna ◽  
Dnaa Protein ◽  
High Affinity ◽  
Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

scholarly journals Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 725-735
Author(s):  
Julieta Gabriela Hamze ◽  
María Jiménez-Movilla ◽  
Raquel Romar
Keyword(s):  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
In Vitro Fertilisation ◽  
Sperm Binding ◽  
Fertilisation Rate ◽  
Gamete Recognition ◽  
Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

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