Sex at Atomic Resolution.

Interspecies fertilization is rare, partly due to species separation enforced at the molecular level. In this issue, Raj et al. now reveal the crystal structures of mollusk egg coat protein, VERL, complexed with cognate sperm protein lysin. Given that VERL is structurally similar to mammalian ZP2, the mechanism elucidating species-specific gamete recognition likely exists in mammals.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization.

Recognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1-3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

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.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Implications of intracrystalline OC17 on the protection of lattice incorporated proteins.

Biogenic CaCO3 formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be incorporated into the crystal lattice. As a result of incorporation, these intracrystalline proteins are protected in the lattice, an example of which is ancient eggshell proteins that have persisted in CaCO3 for thousands of years even under harsh environmental conditions. OC17 is an eggshell protein known to interact with CaCO3 during eggshell formation during which OC17 becomes incorporated into the lattice. Understanding protein incorporation into CaCO3 could offer insights into protein stability inside crystals. Here, we study the protection of OC17 in the CaCO3 lattice. Using thermogravimetric analysis we show that the effect of temperature on intracrystalline proteins of eggshells is negligible below 250 °C. Next, we show that lattice incorporation protects the OC17 structure despite a heat-treatment step that is shown to denature the protein. Because incorporated proteins need to be released from crystals, we verify metal chelation as a safe crystal dissolution method to avoid protein denaturation during reconstitution. Finally, we optimize the recombinant expression of OC17 which could allow engineering OC17 for engineered intracrystalline entrapment studies.

Integrating Computational and Experimental Methods to Identify Novel Sweet Peptides from Egg and Soy Proteins.

Sweetness in food delivers a delightful sensory experience, underscoring the crucial role of sweeteners in the food industry. However, the widespread use of sweeteners has sparked health concerns. This underscores the importance of developing and screening natural, health-conscious sweeteners. Our study represents a groundbreaking venture into the discovery of such sweeteners derived from egg and soy proteins. Employing virtual hydrolysis as a novel technique, our research entailed a comprehensive screening process that evaluated biological activity, solubility, and toxicity of the derived compounds. We harnessed cutting-edge machine learning methodologies, specifically the latest graph neural network models, for predicting the sweetness of molecules. Subsequent refinements were made through molecular docking screenings and molecular dynamics simulations. This meticulous research approach culminated in the identification of three promising sweet peptides: DCY(Asp-Cys-Tyr), GGR(Gly-Gly-Arg), and IGR(Ile-Gly-Arg). Their binding affinity with T1R2/T1R3 was lower than -15 kcal/mol. Using an electronic tongue, we verified the taste profiles of these peptides, with IGR emerging as the most favorable in terms of taste with a sweetness value of 19.29 and bitterness value of 1.71. This study not only reveals the potential of these natural peptides as healthier alternatives to traditional sweeteners in food applications but also demonstrates the successful synergy of computational predictions and experimental validations in the realm of flavor science.

Advances in Understanding the Antioxidant and Antigenic Properties of Egg-Derived Peptides.

Pepsin, trypsin and proteinase K were used in the present study to hydrolyse the proteins from whole eggs, yolks or whites, and the resulting hydrolysates were characterised in terms of antioxidant and IgE-binding properties, using a combination of in vitro and in silico methods. Based on the degree of hydrolysis (DH) results, the egg yolk proteins are better substrates for all the tested enzymes (DH of 6.2-20.1%) compared to those from egg whites (DH of 2.0-4.4%). The SDS-PAGE analysis indicated that pepsin and proteinase K were more efficient compared to trypsin in breaking the intramolecular peptide bonds of the high molecular weight egg proteins. For all the tested substrates, enzyme-assisted hydrolysis resulted in a significant increase in antioxidant activity, suggesting that many bioactive peptides are encrypted in inactive forms in the parent proteins. The hydrolysates obtained with proteinase K exhibited the highest DPPH radical scavenging activity (124-311 µM Trolox/g protein) and the lowest residual IgE-binding capacity. The bioinformatics tools revealed that proteinase K is able to break the integrity of the main linear IgE-binding epitopes from ovalbumin and ovomucoid. It can be concluded that proteinase K is a promising tool for modulating the intrinsic properties of egg proteins.

Effect of salting and dehydration treatments on the physicochemical and gel properties of hen and duck egg yolks, plasma and granules.

BACKGROUND: Salted hen egg yolks are less oily and less flavorful than salted duck egg yolks. However, hen eggs have a more adequate market supply and have a broader application prospect than duck eggs. In the present study, egg yolks, plasma, and granules were dehydrated by adding 1% NaCl to simulate traditional curing process of salted egg yolk. The changes in the pickling process of hen egg yolks (HEY) and duck egg yolks (DEY) plasma and granules were compared to reveal the gelation mechanism and the underlying causes of quality differences in salted HEY and DEY. Salted HEY can be compared with the changes in DEY during the pickling process to provide a theoretical basis for the quality improvement of salted HEY to salted DEY. RESULTS: The results showed that both plasma and granules were involved in gel formation, but exhibited different aggregation behaviors. Based on the intermolecular forces, the HEY proteins achieved aggregation mainly through hydrophobic interactions and DEY proteins mainly through covalent binding. According to spin-spin relaxation time, HEY gels immobilized a large amount of lipid and interacted strongly with lipids. DEY gels showed much free lipid and had weak interaction with lipid. The microstructure showed that HEY proteins were easily unfolded to form a homogeneous three-dimensional gel network structure after salting, whereas heterogeneous aggregates were formed to hinder the gel development in DEY. Changes in protein secondary structure content showed that pickling can promote the transformation of the α-helices to ß-sheets structure in HEY gels, whereas more α-helices structure was formed in DEY gels. CONCLUSION: The present study has demonstrated that different gelation behaviors of hen and duck egg yolk proteins (especially in plasma) through salting treatment led to the difference in the quality of salted HEY and DEY. © 2024 Society of Chemical Industry.

Domain Expansion and Functional Diversification in Vertebrate Reproductive Proteins.

The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1-6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C), which together form a "ZP module." All modular ZP-N domains are phylogenetically distinct from nonmodular or free ZP-N domains. Machine learning-based classification identifies eight residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection is identified in some free ZP-N domains. Our findings support that strong purifying selection has conserved an essential structural core in modular ZP-N domains, with the relaxation of this structural constraint allowing free N-terminal domains to functionally diversify.

The Impact of Processing and Extraction Methods on the Allergenicity of Targeted Protein Quantification as Well as Bioactive Peptides Derived from Egg.

This review article discusses advanced extraction methods to enhance the functionality of egg-derived peptides while reducing their allergenicity. While eggs are considered a nutrient-dense food, some proteins can cause allergic reactions in susceptible individuals. Therefore, various methods have been developed to reduce the allergenicity of egg-derived proteins, such as enzymatic hydrolysis, heat treatment, and glycosylation. In addition to reducing allergenicity, advanced extraction methods can enhance the functionality of egg-derived peptides. Techniques such as membrane separation, chromatography, and electrodialysis can isolate and purify specific egg-derived peptides with desired functional properties, improving their bioactivity. Further, enzymatic hydrolysis can also break down polypeptide sequences and produce bioactive peptides with various health benefits. While liquid chromatography is the most commonly used method to obtain individual proteins for developing novel food products, several challenges are associated with optimizing extraction conditions to maximize functionality and allergenicity reduction. The article also highlights the challenges and future perspectives, including optimizing extraction conditions to maximize functionality and allergenicity reduction. The review concludes by highlighting the potential for future research in this area to improve the safety and efficacy of egg-derived peptides more broadly.

Storage impact on egg white powder's physical and functional properties.

BACKGROUND: Changes in storage temperature and time alter the functional properties of egg white powder (EWP) and determine its quality and shelf-life, finally affecting the consumer acceptance of the products made from EWP. In the present study, the EWP samples were stored at four different temperatures (-20, 4, 25 and 37 °C) for 60 days, and then the protein structural, physical and functional properties of EWP were measured and assessed further for correlation with storage conditions using heatmap. RESULTS: The viscosity of the EWP solution increased after 30 days. Foaming ability and rheological properties increased first and then decreased compared to untreated samples with the prolonged storage time. Correlation analysis results indicated that the gel hardness, water holding capacity, foaming ability, emulsifying ability, particle size, dispersibility and viscosity of EWP were significantly related to storage time (P < 0.05). Only the gelation properties of EWP stored at 37 °C for 60 days changed significantly and were negatively related to its moisture content (P < 0.05). Additionally, the random coil content of EWP was positively correlated with particle size, moisture content, solubility and gel properties, whereas ß-sheet was negatively correlated with them. CONCLUSION: Compared to other temperatures, the functional properties of EWP were relatively stable under 4 °C. Therefore, the low temperature (4 °C) was selected as the most suitable storage temperature for EWP. The results of the present study could provide a theoretical basis for the shelf-life extension of EWP. © 2022 Society of Chemical Industry.

Changes in partial properties of glycosylated egg white powder during storage.

BACKGROUND: Glycosylation is an effective method to modify protein. However, there is a lack of research on the property changes of glycosylated protein during storage. In the present study, the changes in the physicochemical, functional, and structural properties of xylo-oligosaccharide (XOS) glycosylated egg white powder (EWP) (XOS-EWP conjugates) prepared with different glycosylation conditions (XOS/EWP ratio and reaction time) were investigated when stored at 25 °C and 60% relative humidity. RESULTS: In the 12 weeks of storage, the degree of grafting, browning, and the formation of Maillard reaction products of XOS-EWP conjugates increased. The increase in XOS/EWP ratio and reaction time led to an increase in protein aggregation, though a decrease in solubility, due to increased degree of glycosylation and structural changes. Furthermore, improved gel hardness of XOS-EWP conjugates deteriorated, while improved emulsification ability was kept stable during storage. For the sample with a lower XOS/EWP ratio and reaction time, the gel hardness and emulsifying properties underwent little or no deterioration even improving during storage. The results could be attributed to the limited degree of glycosylation, further unfolding of the protein structure, increased surface hydrophobicity of protein, and improved thermal characteristics. CONCLUSION: During storage, the Maillard reaction would continue to occur in the glycosylated EWP, further affecting the performance of modified EWP. Modified EWP prepared under different glycosylation conditions performed differently during storage. Modified EWP with a larger XOS/EWP ratio and reaction time meant it was harder to maintain good performance. Modified EWP with a smaller XOS/EWP ratio and reaction time changed significantly to better performances. © 2022 Society of Chemical Industry.

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.

Fabrication and Characterization of the Egg-White Protein Chitosan Double-Layer Emulsion.

Egg-white protein has an abundance of hydrophobic amino acids and could be a potential emulsifier after modification. Here, egg-white protein was modified via ultrasonic and transglutaminase treatments to destroy the globular structure. The egg-white protein gel particles (EWP-GPs) were prepared and then a novel highly stable EWP-chitosan double-layer emulsion was constructed. When ultrasonic treatment was applied at 240 W and TGase (20 U/g EWP) treatment, the EWP-GPs had a low particle size and good emulsification performance. The particle size of EWP-GPs was a minimum of 287 nm, and the polymer dispersity index (PDI) was 0.41. The three-phase contact angle (θo/w) of EWP-GPs was 79.6° (lower than 90°), performing with good wettability. Based on these results, the EWP-chitosan double-layer emulsion was prepared through the EWP-GPs being treated with 240 W ultrasound, TGase, and chitosan in this study. When the double-layer emulsion had 0.6% (v/v) chitosan, the zeta potential of the double-layer emulsion was -1.1 mV and the double-layer emulsion had a small particle size (56.87 µm). The creaming index of double-layer emulsion at 0.6% (v/v) chitosan was 16.3% and the droplets were dispersed uniformly. According to the rheological results, the storage modulus (G') was larger than the loss modulus (G″) in the whole frequency, indicating the formation of an elastic gel network structure in the emulsion. It is hoped to develop a novel food-grade stabilizer and a stable double-layer emulsion, providing new environment-friendly processing in hen egg products and delivery systems.

Characterisation of Flavour Attributes in Egg White Protein Using HS-GC-IMS Combined with E-Nose and E-Tongue: Effect of High-Voltage Cold Plasma Treatment Time.

Egg white protein (EWP) is susceptible to denaturation and coagulation when exposed to high temperatures, adversely affecting its flavour, thereby influencing consumers' decisions. Here, we employ high-voltage cold plasma (HVCP) as a novel nonthermal technique to investigate its influence on the EWP's flavour attributes using E-nose, E-tongue, and headspace gas-chromatography-ion-mobilisation spectrometry (HS-GC-IMS) due to their rapidness and high sensitivity in identifying flavour fingerprints in foods. The EWP was investigated at 0, 60, 120, 180, 240, and 300 s of HVCP treatment time. The results revealed that HVCP significantly influences the odour and taste attributes of the EWP across all treatments, with a more significant influence at 60 and 120 s of HVCP treatment. Principal component analyses of the E-nose and E-tongue clearly distinguish the odour and taste sensors' responses. The HS-GC-IMS analysis identified 65 volatile compounds across the treatments. The volatile compounds' concentrations increased as the HVCP treatment time was increased from 0 to 300 s. The significant compounds contributing to EWP characterisation include heptanal, ethylbenzene, ethanol, acetic acid, nonanal, heptacosane, 5-octadecanal, decanal, p-xylene, and octanal. Thus, this study shows that HVCP could be utilised to modify and improve the EWP flavour attributes.

Fabrication and characterization of Pickering emulsions stabilized by desalted duck egg white nanogels and sodium alginate.

BACKGROUND: The waste of salted egg white resources has always been a serious problem in the food industry. In this current study, we report on a kind of Pickering emulsion system, which was stabilized by duck egg white nanogels (DEWNs) and sodium alginate (SA), followed by which this system was crosslinked by calcium carbonate (CaCO3 ) via controlling the gluconolactone (GDL) concentrations, aiming to open up a promising route for making full use of these protein resources. RESULTS: The droplet size of the emulsion exhibited a reduction with an increase in SA concentrations, indicating that higher negative charges and steric hindrance was useful for a stable emulsion system. Meanwhile, the result of rheology measurement showed that storage modulus (G') values were higher than loss modulus (G″) values of the samples at higher GDL concentration, revealing the formation of elastic gel-like networks in the system, which was fabricated by SA and Ca2+ released by the CaCO3 particles. The gel-like network structure in the continuous phase improved both the freeze-thaw and thermal stability of the obtained Pickering emulsion system. Encouragingly, the Pickering high internal phase emulsions (HIPEs, φ = 0.75) stabilized by DEWN/SA3 -GDL3 were prepared, which could be stored at 4 °C for at least 30 days without oiling-off and creaming. CONCLUSION: These findings not only develop a green ultra-stable Pickering emulsion system but also extend the potential commercial applications of duck egg white proteins in the food, cosmetics, and pharmaceutical industries. © 2021 Society of Chemical Industry.

Phosphoproteomic analysis of duck egg yolk provides novel insights into its characteristics and biofunctions.

BACKGROUND: Although the importance of phosphorylation in the function of proteins is known, investigation of the protein phosphorylation of duck egg yolk (DEY) is still very limited. This study aimed to conduct a detailed phosphoproteomic study of DEY using immobilized metal affinity chromatography and ultra-high liquid chromatography tandem mass spectrometry. RESULTS: A total of 253 phosphorylation sites assigned to 66 phosphoproteins were identified in DEY, of which VTG-1, VTG-2, and fibrinogen alpha chain were found to be the highly phosphorylated proteins in DEY. The biological functions of the identified phosphoproteins were illuminated through gene ontology analysis, which showed that they were mainly involved in binding, catalytic, immune response, and metabolic activity. S-X-E and S-X-S were found to be the most conserved serine motifs of phosphorylation in DEY. The comparison of DEY phosphoproteins with those of chicken egg yolk (CEY) revealed that differences mostly involved molecular functions and biological processes. The comparison also revealed a higher phosphorylation level in DEY proteins. CONCLUSION: The higher phosphorylation level in DEY proteins than that in CEY proteins are supposed to help enhance duck growth performance and biological activities (e.g. antibacterial and antioxidant ability) for better adapting the humid environment the duck lived. © 2021 Society of Chemical Industry.

Modification methods and applications of egg protein gel properties: A review.

Egg protein (EP) has a variety of functional properties, such as gelling, foaming, and emulsifying. The gel characteristics provide a foundation for applications in the food industry and research on EP. The proteins denature and aggregate to form a dense three-dimensional gel network structure, with a process influenced by protein concentration, pH, ion type, and strength. In addition, the gelation properties of EP can be altered to varying degrees by applying different treatment conditions to EP. Currently, modification methods for proteins include physical modification (heat-induced denaturation, freeze-thaw modification, high-pressure modification, and ultrasonic modification), chemical modification (glycosylation modification, phosphorylation modification, acylation modification, ethanol modification, polyphenol modification), and biological modification (enzyme modification). Pidan, salted eggs, egg tofu, and other egg products have unique sensory properties, due to the gel properties of EP. In accessions, EP has also been used as a new ingredient in food packaging and biopharmaceuticals due to its gel properties. This review will further promote EP gel research and provide guidance for its full application in many fields.

Facile modification of polycaprolactone nanofibers with egg white protein.

Synthetic polymers remain to be a major choice for scaffold fabrication due to their structural stability and mechanical strength. However, the lack of functional moieties limits their application for cell-based therapies which necessitate modification and functionalization. Blending synthetic polymers with natural components is a simple and effective way to achieve the desired biological properties for a scaffold. Herein, nanofibrous mats made of polycaprolactone (PCL) and egg white protein (EWP) blend were developed and further evaluated for use as a scaffold for tissue engineering applications. Homogeneous distribution of EWP was achieved throughout the nanofibrous mats, as shown by immunohistochemistry. ATR-FTIR analysis and contact angle measurements have further confirmed the presence of EWP on the surface of the samples. The swelling test showed that PCL/EWP nanofibers have higher water uptake than PCL nanofibrous mats. Also, EWP addition on the nanofibrous mats resulted in an increase in the tensile strength and Young's modulus of the mats, indicating that the presence of protein can greatly enhance the mechanical properties of the mats. A significantly higher, more uniform, and dispersed cell spreading was observed on days 7 and 14 than that on neat PCL mats, demonstrating the importance of providing the required cues for cell homing by the availability of EWP. Hence, EWP is shown to be a simple and low-cost source for the functionalization of PCL nanofibrous mats. EWP is, therefore, a facile candidate to enhance cellular interactions of synthetic polymers for a wide range of tissue engineering applications.

Comparative Studies of Yolkin Preparations Isolated from Egg Yolks of Selected Bird Species.

The results of our research have proven that yolkin preparations isolated from eggs of different bird species show a high similarity in polypeptide composition. Despite the small differences in protein patterns, all of yolkin preparations showed also strong immunomodulatory activity, comparable with yolkin obtained previously from hen egg yolk. It can therefore be deducted that the presence of this polypeptide complex in the egg is not accidental and performs an important biological function for developing embryo.