Vitellogenin Ab structure of the amazonian Arapaima gigas.

The vitellogenin is composed by polypeptides that are precursors of egg yolk proteins that provides embryo and larvae nutrition. The mRNA encoding for vitellogenin Ab (Vtg-Ab; 4,536 bp long and 1,512 amino acids) were obtained by RNA-Seq library sequencing of pirarucu gonads. The Vtg-Ab sequences had high homology with Vtgs of other three teleosts species of the order Osteoglossiformes. The transcript of ovarian Vtg was identified based on structural criteria, and so we classify the Vtg of pirarucu as Vtg-Ab due to the truncated or shortened phosvitin (N-terminal end) and phosvitinless domain (C-terminal end). The Vtg-Ab of pirarucu present two major deletions with 133 amino acids in the Lipovitellin I domain and 89 amino acids in the truncated or shortened Phosvitin domain, both located in the N-terminal end region. The three-dimensional (3-D) structure Vtg-Ab protein shows the presence of a typical 4α-helices bundle protein that runs in anti-parallel. In general, the characterization of Vtg-Ab may be the useful elucidation of the hormonal regulation of vitellogenesis and improve the production of pirarucu for broodstock management in aquaculture and preparation of Vtg antibody production (species-specific) for sex identification.

Combination of High Hydrostatic Pressure and Ultrafiltration to Generate a New Emulsifying Ingredient from Egg Yolk.

Egg yolk granule phosvitin (45 kDa) is a phosphoprotein known for its emulsifying properties. Recently, high hydrostatic pressure (HHP) treatment of granule induced the transfer of phosvitin to the soluble plasma fraction. This project evaluated the performance of the ultrafiltration (UF) used to concentrate phosvitin from the plasma fraction to produce a natural emulsifier. Phosvitin was characterized in plasma from a pressure-treated granule (1.73 ± 0.07% w/w) and in its UF retentate (26.00 ± 4.12% w/w). The emulsifying properties of both retentates were evaluated. The emulsion prepared with phosvitin-enriched retentate was more resistant to flocculation and creaming. Confocal laser scanning microscopy showed a network of aggregated protein similar to a gel, which encapsulated oil droplets in emulsions made with UF-retentate of plasma from pressure-treated granule. However, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that ß-phosvitin is recovered in the cream, it is difficult to attribute the improved emulsifying properties of the UF-retentate of plasma from pressure-treated granules only to phosvitin.

Anti-elastase and anti-hyaluronidase activity of phosvitin isolated from hen egg yolk.

1. Phosvitin, a major phosphoprotein found in egg yolk, has strong antioxidant activity. Activation of elastase, collagenase, and hyaluronidase by reactive oxygen species are related to the degradation of ECM and skin aging. The objective of this study was to determine the anti-elastase and anti-hyaluronidase activity of phosvitin.2. Elastase from porcine pancreas and hyaluronidase from bovine testes were used to study the inhibitory activity of phosvitin. To elucidate the mechanism of enzyme inhibition, a Lineweaver-Burk plot was constructed.3. Phosvitin inhibited elastase and hyaluronidase activity in a dose-dependent manner. The IC50 value of phosvitin was 31.6 µg/ml and 1,270 µg/ml against elastase and hyaluronidase, respectively. The analysis of elastase and hyaluronidase kinetics indicated that the apparent Michaelis constant (appKm) was increased by phosvitin but the Vmax value was not affected.4. In conclusion, phosvitin exhibited competitive inhibitory activity against elastase and hyaluronidase. Thus, phosvitin could be used as a natural anti-aging agent in the cosmetics industry.

Type-I-hypersensitivity to 15 kDa, 28 kDa and 54 kDa proteins in vitellogenin specific to Gadus chalcogrammus roe.

BACKGROUND: Fish roe allergy is a common health problem in countries where sea food is a major part of the diet, such as Japan. ß'-component (ß'-c) in fish roe has been identified as a major antigen for patients who show hypersensitivity to various fish roes. However, little is known about causative antigens for patients reactive to fish roe of specific species. METHODS: Serum and basophils were obtained from patients who had reactivity to roes of Gadus chalcogrammus (GC) and/or other fish species. GC roe specific antigens were analyzed by immunoblotting, histamine release assay (HRA) and mass spectrometry. Recombinant-fragments of vitellogenin (Vg) were obtained by the Escherichia coli expression system. RESULTS: Serum IgE of a patient with specific reactions to GC roe bound to 15, 28, 40 and 70 kDa-proteins in GC roe extract. Mass spectrometry analysis revealed that proteins in these bands contained fragments corresponding to Vg. Immunoblotting of Vg immunoprecipitated by rabbit anti-Vg antiserum from the extract revealed 15, 28 and 54 kDa fragments bound by the patient's IgE. These bindings were inhibited by the pretreatment of recombinant phosvitin (rPv) and ß'-c (rß'-c). Fractions obtained by native gel electrophoresis containing 15, 28 and 54 kDa proteins, but not the other fractions, induced significant histamine release from the patient's basophils. Sera of the other patients with GC roe specific-IgE showed IgE binding to rPv and/or rß'-c. CONCLUSIONS: The 15, 28 and 54 kDa-fragments of Vg which include structures of Pv and ß'-c, could be antigens for GC roe specific type-I-hypersensitivity.

31 P NMR assessment of the phosvitin-iron complex in mayonnaise.

Lipid oxidation is the main reason for the limited shelf life of mayonnaise. One of the main catalysts of this process is iron, which is introduced in its ferric (Fe(III)) form via phosvitin, an egg yolk phosphoprotein rich in phosphoserines. The binding of Fe(III) to phosvitin and its ability to establish a redox couple with Fe(II) is believed to determine the oxidation rate of unsaturated lipids. In this work, a 31 P NMR based method was developed to quantify loading of phosvitin with Fe(III) and its reductive release. Both features could be quantified in model phosvitin solutions by exploiting the paramagnetic broadening of 31 P NMR signal of phosphoserine residues by Fe(III). This method was then successfully applied to quantify the phosvitin-Fe(III) loading in mayonnaise water phase by liquid NMR, whereas 31 P NMR MAS could only provide a qualitative measure. The 31 P NMR method showed a direct relation between loading of the Fe(III)-phosvitin complex and lipid oxidation.

Augmentation of the antibacterial activities of Pt5-derived antimicrobial peptides (AMPs) by amino acid substitutions: Design of novel AMPs against MDR bacteria.

The ever-growing concerns on multi-drug resistant (MDR) bacteria lead to urgent demands for novel antibiotics including antimicrobial peptides (AMPs). Pt5, a peptide consisting of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. Here we used Pt5 as a template to design new AMPs by shortening the sequence and substituting with tryptophan (W) and lysine (K) at selected positions. Among the resultant Pt5-derived peptides, Pt5-1c showed the strongest antimicrobial activity against both Gram-negative and Gram-positive bacteria, including MDR bacteia, with the minimum inhibitory concentrations (MICs) ranging from 1.2 µM to 4.8 µM. Electron microscopic examination showed that Pt5-1c was able to kill the bacteria directly. ELISA revealed that Pt5-1c possessed high affinity to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Importantly, Pt5-1c was able to disrupt the bacterial membrane by a combined action of membrane depolarization and permeabilization, with little cytotoxicity to mammalian cells. Taken together, these findings suggest that Pt5-1c has considerable potential for future development as novel peptide antibiotics against MDR bacteria.

Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes.

Egg yolk proteins are mainly derived from vitellogenin (Vtg), and serve as essential nutrients during early development in oviparous organisms. Vertebrate Vtgs are predominantly synthesized in the liver of maturing females, and are internalized by the oocyte after binding to specific surface receptors (VtgR). Here, we clarify the evolutionary history of vertebrate Vtgs, including the teleost VtgC, which lacks phosvitin, and investigate the repertoire of Vtgs and VtgRs in the tetraploid Atlantic salmon (Salmo salar). Conserved synteny of the vtg genes in elephant fish (Callorhinchus milii) strongly indicates that the vtg gene cluster was present in the ancestor of tetrapods and ray-finned fish. The shortened phosvitin in the VtgC ortholog of this chondrichthyean fish may have resulted from early truncation events that eventually allowed the total disappearance of phosvitin in teleost VtgC. In contrast, the tandem-duplicated VtgCs identified in the spotted gar (Lepisosteus oculatus) both contain the phosvitin domain. The Atlantic salmon genome harbors four vtg genes encoding the complete VtgAsa1, phosvitin-less VtgC, and truncated VtgAsb proteins; vtgAsa2 is a pseudogene. The three vtg genes were mainly expressed in the liver of maturing females, and the vtgAsa1 transcript predominated prior to spawning. The splice variant lacking the O-linked sugar domain dominated ovarian expression of vtgr1 and vtgr2. Strongly increased vtgAsa1 expression during vitellogenesis contrasted with the peaks of vtgr1 and vtgr2 in the previtellogenic oocytes, which gradually decreased over the same period. Recycling of the oocyte VtgRs is probably not sufficient to maintain receptor number during vitellogenesis.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid.

BACKGROUND: Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. RESULTS: Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. CONCLUSION: Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation. © 2017 Society of Chemical Industry.

Zebrafish phosvitin-derived peptide Pt5 inhibits melanogenesis via cAMP pathway.

Zebrafish phosvitin-derived peptide Pt5, consisting of the C-terminal 55 residues of phosvitin, has been shown to have an antimicrobial-immunomodulatory activity comparable to phosvitin. Here, we showed clearly that Pt5 had the capacity to inhibit tyrosinase (TYR) activity and melanin biosynthesis, and this inhibition was independent of cell proliferation and cytotoxic effects. Incubation of fluorescein isothiocyanate (FITC)-labeled Pt5 with B16F10 melanoma cells revealed that Pt5 was localized in the cytoplasm of the cells. In addition, Pt5 inhibited the expression of TYR, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells and reduced the intracellular cyclic adenosine monophosphate (cAMP) concentration in the cells, but it did not affect the cellular contents of pERK1/2 and ß-catenin, suggesting that Pt5 regulates melanin biosynthesis via cAMP signaling pathway rather than Wnt and MAPK pathways. Collectively, these data indicate that Pt5 has the potential to be used as a melanogenesis inhibitor in medical and cosmetic industry, a novel role ever reported.

Antibacterial activity and modes of action of phosvitin-derived peptide Pt5e against clinical multi-drug resistance bacteria.

Pt5e, a mutant peptide derived from the C-terminal 55 residues of zebrafish phosvitin, has been suggested to be a novel antibacterial peptide. However, if it is applicable to clinical MDR bacteria remains to be tested. In this study, high-purity Pt5e was first expressed and purified by fusion with cationic elastin-like polypeptide. Pt5e was then shown to be capable of effectively killing all the five clinical MDR bacteria tested. Pt5e kill the MDR bacteria at several levels, including inserting into the bacterial membranes, causing the membrane depolarization and permeabilization, and inducing the intracellular apoptosis/necrosis. All these data suggest that Pt5e is a promising therapeutic potential as an antibiotics against clinical MDR bacteria.

Vitellogenin is an immunocompetent molecule for mother and offspring in fish.

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. Initially, Vg was regarded as a female-specific reproductive protein, which is cleaved into yolk proteins such as phosvitin (Pv) and lipovitellin (Lv), stored in egg, providing the nutrients for developing embryos. Recently, Vg is shown to be an immune-relevant molecule involved in the defense of the host against the microbes including bacterium and virus. Furthermore, Pv and Lv, that both are proteolytically cleaved products of Vg, play a defense role in developing embryos. Importantly, yolk protein-derived small peptides also display antimicrobial activity. These data together indicate that Vg, in addition to being involved in yolk protein formation, plays a non-reproductive role via functioning as an immune-relevant molecule in both parent fishes and their offspring. It also shows that yolk proteins and their degraded peptides are novel players in maternal immunity, opening a new avenue to study the functions of reproductive proteins.

Zebrafish phosvitin is an antioxidant with non-cytotoxic activity.

Antioxidants, or anti-oxidant agents, have attracted a great deal of attention in recent years because of their roles in prevention of chronic diseases and utilization as preservatives in food and cosmetics. In this study, we clearly demonstrated that zebrafish recombinant phosvitin (rPv) is an antioxidant agent capable of inhibiting the oxidation of the linoleic acid, and scavenging the 2,2-diphenyl-1-picrylhydrazyl radical. We also showed that zebrafish rPv is a cellular antioxidant capable of protecting radical-mediated oxidation of cellular biomolecules. Importantly, zebrafish rPv is non-cytotoxic to murine macrophage RAW264.7 cells. It is the first report that showed the antioxidant activities of Pv in fishes, suggesting that zebrafish Pv can be an important antioxidant, which can be used as preservatives in food and cosmetics and even as supplementary mediator in different diseased states.

Thermal-aided phosvitin extraction from egg yolk.

BACKGROUND: Phosvitin is the principal phosphoprotein in egg yolk and has great potential for use as a functional food ingredient in improving bone health. This study reports a thermal-aided extraction method without using organic solvents or non-food-compatible chemicals. RESULTS: Egg yolk was two times diluted with water and then extracted by 100 g L(-1) NaCl. Effects of pH and heating temperature on the extract were examined. The phosvitin purity increased from 75.7% at pH 8.0 to 80.1% at pH 5.0 and then started to decrease, but the yield decreased at decreasing pHs. The phosvitin purity increased at increasing temperature up to 90 °C and then started to decrease at 95 °C, while the yield increased from 70 to 80 °C and then started to decline at 85 °C. CONCLUSION: A purity of 88.0% and a yield of 23.5 g kg(-1) yolk dry matter were obtained at 90 °C. The purity and yield were comparable to or higher than those of previously methods. The method developed in this study is simple, including mainly two steps, i.e. water dilution of egg yolk and NaCl extraction with heating, and can be scaled up for industrial production.

Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin. In our resorption model live bone cultures were grown in the absence of ascorbate and were stimulated by parathyroid hormone (PTH) to undergo rapid osteoclast formation/differentiation with bone resorption. In this resorption model native phosvitin potently inhibited PTH-induced osteoclastic bone resorption with simultaneous new osteoid/bone formation in the absence of ascorbate (vitamin C). These surprising and critical observations were extended using the bone formation model in the absence of ascorbate and in the presence of phosvitin which supported the above results. The results were corroborated by analyses for calcium release or uptake, tartrate-resistant acid phosphatase activity (marker for osteoclasts), alkaline phosphatase activity (marker for osteoblasts), collagen and hydroxyproline composition, and histological and quantitative histomorphometric evaluations. The data revealed that the discovered bioactivity of phosvitin mirrors that of ascorbate during collagen synthesis and the formation of new osteoid/bone. Complementing those studies use of the synthetic collagen peptide analog and cultured calvarial osteoblasts in conjunction with mass spectrometric analysis provided results that augmented the bone organ culture work and confirmed the capacity of phosvitin to stimulate differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization. There are striking implications and interrelationships of this affect that relates to the evolutionary inactivation of the gene of an enzyme L-gulono-γ-lactone oxidase, which is involved in the final step of ascorbate biosynthesis, in many vertebrate species including passeriform birds, reptiles and teleost fish whose egg yolk contain phosvitin. These represent examples of how developing ex-vivo embryos of such species can achieve connective tissue and skeletal system formation in the absence of ascorbate.

Phosvitin phosphorus is involved in chicken embryo bone formation through dephosphorylation.

The aim of this study was to investigate the role of phosvitin in bone formation in chicken embryos. The yolk P content, P/N ratio and secondary structure of phosvitin, alkaline phosphatase (ALP) activity of the tibia, and body length were determined during incubation. A high correlation was found between the phosphate group content of phosvitin and both secondary structure and bone metabolism (ALP activity in the tibia, body length). The ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. The phosphate content of phosvitin decreased, the γ-random coil and ß-turn gradually transformed into α-helixes, and the secondary structure of protein tended to become more orderly; these changes mainly occurred on d 13 to 16. Bone formation of the chicken embryos occurred primarily on d 14 to 18, whereas ALP activity and body length growth increased substantially. The results indicate that phosvitin P is involved in chicken embryo bone formation through dephosphorylation.

Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents.

A study was conducted to develop a simple sequential separation protocol to separate phosvitin and IgY from egg yolk without using organic solvents. Egg yolk was diluted with 2 volumes of distilled water (DW), homogenized, and centrifuged. The precipitant was collected and homogenized with 4 volumes of 10% NaCl (wt/vol) in 0.05 N NaOH solution to extract phosvitin. The pH of the homogenate was adjusted to 4.0 and the precipitate was removed by centrifugation. The supernatant was collected and then heat-treated at 70°C for 30 min and centrifuged to remove impurities. The supernatant containing phosvitin was collected, had salts removed, and was concentrated and then freeze-dried. The supernatant from the centrifugation of diluted egg yolk was diluted again with 3 volumes of DW, and the precipitate was removed by centrifugation. The resulting supernatant was concentrated using ultrafiltration and then IgY was precipitated using 20% saturated (NH4)2SO4+ 15% NaCl (wt/vol). The precipitant was collected after centrifugation at 3,400 × g for 30 min at 4°C and dissolved with DW, had salts removed, and then was freeze-dried. The purity of separated phosvitin and IgY was checked using SDS-PAGE and the proteins were verified using Western blotting. The purity of phosvitin and IgY was 97.2 and 98.7%, and the yield was 98.7 and 80.9%, respectively. The ELISA results indicated that the activities of separated IgY and phosvitin were 96.3 and 98.3%, respectively. This study proved that both phosvitin and IgY can be separated in sequence from egg yolk without using an organic solvent. Also, the method is very simple and has a high potential for scale-up processing.

Cytotoxic and antigenotoxic activities of phosvitin from egg yolk.

Egg yolk phosvitin is one of the most phosphorylated proteins in nature, and thus has a strong metal-binding ability. The objective of this study was to evaluate the cytotoxic and antigenotoxic activities of phosvitin in vitro. Using the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of phosvitin was evaluated in human cancer cell lines of various tissue origins, including the cervix (HeLa), breast (MCF-7), stomach (AGS), lung (A549 and SK-MES-1), liver (HepG2), and larynx (Hep-2). The growth of all cancer cell lines was inhibited in a dose-dependent manner by phosvitin. Among the cancer cell lines tested, MCF-7 and SK-MES-1 were the least sensitive and HeLa, AGS, and HepG2 were the most sensitive to phosvitin. The 50% inhibition of cell viability values of phosvitin were 5.38, 11.57, 4.78, 6.98, 11.82, 3.93, and 9.97 mg/mL for HeLa, MCF-7, AGS, A549, SK-MES-1, HepG2, and Hep-2, respectively. The protective effects of phosvitin against DNA damage in human leukocytes indicated that phosvitin showed protective effects against the oxidative stress-induced DNA damages in human leukocytes. These results suggested that phosvitin has a high potential to be used as an anticancer agent for humans.

Phosvitin-based hydrogels prepared in AmimCl under magnetic field treatment: Structural characteristics, biological functions, and application in skin wound healing.

Due to the serious bacterial infection of skin and the waste of petroleum-based materials, there is an urgent need to develop natural biodegradable wound dressings with high antibacterial activity. Phosvitin (PSV) has shown its natural antioxidant and antibacterial properties, making it an excellent material for preparing wound healing dressings. In this study, we investigated the effect of magnetic field on the preparation of PSV-Microcrystalline Cellulose (MCC) composite hydrogels in 1-Allyl-3-methylimidazolium chloride (AmimCl) system. The results showed that the prepared hydrogels exhibited homogeneous surface structure, suitable swelling capacity and elasticity modulus, and sufficient thermal stability. The excellent antibacterial and antioxidant activities of hydrogels were mainly resulting from AmimCl and PSV, respectively, and the properties were enhanced after magnetic field treatment. The proteomics analysis indicated that AmimCl can readily penetrate the biological membranes of Staphylococcus aureus (S. aureus), upsetting the metabolism and reducing the virulence. The hydrogels showed great blood compatibility. Compared with the commercial materials, the 5 mT-treated hydrogels presented a comparable wound healing rate in the full-thickness skin injury model. On day 7, the wound healing rate of the 5 mT group reached approximately 84.40 %, which was significantly higher than that of the control group, 72.88 % (P < 0.05). In conclusion, our work provides experience for the development of biodegradable materials combined in ionic liquids and magnetic field, and explores their applications in wound healing dressings.

Promoting effect of phosvitin in the mineralization of eggshell inner membrane with the application in osteogenic induction scaffold.

Exploring affordable and easily prepared inorganic-organic hybrid membrane materials has attracted a great interest in the bone repair field. This study is based on biomimetic mineralization technique to study the role of phosvitin (PV) in the mineralized process of eggshell inner membrane. Results showed that PV promoted the formation of hydroxyapatite on the eggshell inner membrane surface, and the phosvitin content in the simulated body fluid was decreased during the mineralization process. Besides, in vitro preosteoblast experiments indicated that mineralized membrane with PV exhibited more conducive to cell proliferation and differentiation than that mineralized membrane without PV. Interestingly, with the increase of mineralization time, the stimulating ability of mineralized membranes with PV on adhesion, proliferation, alkaline phosphatase activity and collagen type I content gradually improved. In summary, the eggshell inner membrane composites mineralized with PV obtained by biomimetic mineralization might be potential scaffold materials for bone repair.

Separation of Phosvitin from Egg Yolk without Using Organic Solvents.

The objective of this study was to develop a new method to separate phosvitin from egg yolk without using organic solvents. Phosvitin was extracted from yolk granules using 10% NaCl or 10% (NH4)2SO4 (final concentration) and then treated with heat to precipitate the lipoproteins from the extracted solution. The optimal pH for the phosvitin extraction from yolk granules was determined, and the iron-binding ability of the extracted phosvitin (final product) was tested. Adding 10% (NH4)2SO4 disrupted the granules, and the subsequent thermal treatment at 90°C for 1 h precipitated low density and high density lipoproteins, which enabled separation of phosvitin by centrifugation. The phosvitin concentration in the extract was significantly higher when the pH of the solution was adjusted to pH ≥9. The purity and recovery rate of phosvitin at the end of the separation process were approximately 78% and 56%, respectively. The separated phosvitin was confirmed to have ferrous and ferric iron binding ability. The advantages of this new method compared with the traditional methods include no organic solvents and high-priced equipment are needed for the separation. Also, this method is more environment and consumer friendly than that of the traditional methods.