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.

Immune-enhancing activity of phosvitin by stimulating the production of pro-inflammatory mediator.

Egg yolk phosvitin is one of the most phosphorylated proteins in nature, and the extraordinarily high concentration of phosphate groups in its structure provides a strong metal-binding ability. Phosvitin is known to possess various functional activities, including metal-chelating, antioxidant, emulsifying, antimicrobial, and cytotoxic activities. However, little is known about the immune-enhancing activity of phosvitin. The objective of this study was to evaluate the immune-enhancing activity of phosvitin in murine RAW 264.7 macrophages. Griess reagents and quantitative real-time PCR were used to determine the effect of phosvitin (at 12.5, 25, 50, and 100 µg/mL) on the levels of pro-inflammatory mediators NO and inducible nitric oxide synthase (iNOS), cytokines TNF-α and IL-1ß in RAW 264.7 macrophages. The effect of phosvitin on the phagocytic activity of RAW 264.7 macrophages was also measured using the Neutral-Red Uptake method. Lipopolysaccharides was used as a positive control. Phosvitin significantly (P < 0.05) increased the production of NO in RAW 264.7 macrophages in a dose-dependent manner, but did not show any cytotoxicity. The amounts of NO produced were 3.47, 7.12, 10.23, and 14.57 µM in 12.5 to 100 µg/mL range of phosvitin (control: 0.46 µM). Compared with the untreated group, phosvitin treatment at a 100 µg/mL level increased the production of NO by 31.67 times. Phosvitin also significantly increased the mRNA expression of the RAW 264.7 macrophages: 100 µg/mL of phosvitin treatment increased the expression of mRNA for iNOS, TNF-α, and IL-1ß by 46.25, 9.09, and 85.18 times of the control, respectively. The phagocytic activity of RAW 264.7 macrophages was also increased significantly by phosvitin treatment. These results demonstrated that phosvitin dramatically improved the immune functions RAW 264.7 macrophages by enhancing the production of immune mediators and increasing phagocytic activity. Therefore, phosvitin has a potential to be used as an immune-enhancing agent by food or nutraceutical industries.

Antiviral activity of phosvitin from zebrafish Danio rerio.

Lymphocystis disease virus (LCDV), a virus of Iridoviridae, can infect numerous teleost species, causing serious losses of aquaculture industry, and thus effective ways of prophylaxis and treatment are demanded. Previous studies have shown that phosvitin (Pv) is an antimicrobial agent in zebrafish, and vitellogenin, the precursor of yolk proteins including Pv, is able to neutralize virus, we thus hypothesize that Pv may have an antiviral activity. Here we clearly demonstrated that recombinant Pv (rPv) purified was capable of inhibiting the cytopathic effect in LCDV-infected cells and reducing the virus quantities in the infected cells as well as in the infected zebrafish. These data indicate that Pv possesses an antiviral activity and participates in immune defense of host against the infection by viruses like LCDV.

Maternal immunity in fish.

Both innate and adaptive immune-relevant factors are transferred from mother to offspring in fishes. These maternally-transferred factors include IgM, lysozymes, lectin, cathelicidin and complement components. Recently, yolk proteins, phosvitin and lipovitellin, have been shown to be maternally-transferred factors, functioning in the defense of teleost larvae against pathogens. Among these factors, the mode of action of complement components and yolk proteins has been explored, whereas that of all the other factors remains elusive. At present, the transfer mechanisms of maternally-derived immune factors are largely unknown although those of IgM and yolk protein transmission from mother to offspring have been reported in some fishes. Maternal transfer of immunity is affected by many elements, including biological factors, such as age and maturation, and environmental conditions experienced by brood fish, such as pathogens and nutritional supply. Practically, the manipulation of maternal immunity transfer can be used to enhance the survival rate of fish larvae.

Antimicrobial-immunomodulatory activities of zebrafish phosvitin-derived peptide Pt5.

A phosvitin (Pv)-derived peptide, Pt5, which consists of the C-terminal 55 residues of Pv in zebrafish, has been shown to function as an antimicrobial agent capable of killing microbes in vitro. However, its in vivo role in zebrafish remains unknown. In this study, we clearly demonstrated that Pt5 protected adult zebrafish from pathogenic Aeromonas hydrophila attack, capable of significantly enhancing the survival rate of zebrafish after the pathogenic challenge. Pt5 also caused a marked decrease in the numbers of A. hydrophila in the blood, spleen, kidney, liver and muscle, suggesting that Pt5 was able to block multiplication/dissemination of A. hydrophila in zebrafish. Additionally, Pt5 markedly suppressed the expression of the proinflammatory cytokine genes IL-1ß, IL-6, TNF-α and IFN-γ in the spleen and head kidney of A. hydrophila-infected zebrafish, but it considerably enhanced the expressions of the antiinflammatory cytokine genes IL-10 and IL-4 in the same tissues. Taken together, these data indicate that Pt5 plays a dual role in zebrafish as an antimicrobial and immunomodulatory agent, capable of protecting zebrafish against pathogenic A. hydrophila through its antimicrobial activity as well as preventing zebrafish from the detrimental effects of an excessive inflammatory response via modulating immune functions.

Detection of four distinct groups of hen egg allergens binding IgE in the sera of children with egg allergy.

BACKGROUND: There appears to be a lack of agreement in the literature on the allergenicity of hen egg proteins. This may be partly due to the use of impure proteins in some cases. Egg yolk proteins have also been largely ignored in such studies. We therefore set out to determine, using especially purified proteins, their relative allergenicity, and to observe whether there were any relationships between their potency and the sensitivity of patients to them. METHODS AND RESULTS: The sera of 40 patients with clinically observed hen egg hypersensitivity were tested for specific IgE binding to purified egg white and egg yolk proteins using the radioallergosorbent test (RAST). Statistical treatment by correspondence analysis of the percent radioactive uptakes in the RAST to the 8 proteins demonstrated that there were four distinct groups of patients reacting in a similar way to four discrete sets of proteins. CONCLUSIONS: The first three sets of allergens consisted of egg white proteins as follows: firstly, lysozyme and ovalbumin; secondly, ovomucoid; and thirdly, ovomucin. The fourth set contained the egg white protein ovotransferrin and the egg yolk proteins apovitellenins I and VI and phosvitin. The existence of patient groups may explain why various workers have reported different allergens to be important in egg hypersensitivity. A sufficiently large number of patients must be examined so as to give a representative distribution across each group, otherwise the results may be biased towards one allergen.

Relationship between vitellogenin and its related egg yolk proteins in Sakhalin taimen (Hucho perryi).

Vitellogenin (Vg) and its three egg yolk protein products, lipovitellin (Lv), phosvitin (Pv) and beta'-component, were isolated from mature female Sakhalin taimen (Hucho perryi). Vg had an apparent molecular weight of 540 kDa and appeared as a major 240 kDa band in SDS-PAGE, which resolved into two major bands (165 and 125 kDa) after reduction. The estimated molecular weights of purified Lv, Pv, and beta'-component were 330, 23, and 30 kDa, respectively. Lv appeared as a main band of 150 kDa in SDS-PAGE which resolved into two smaller bands (92 and 29 kDa) after reduction. beta'-component appeared as a 34 kDa band before and as a 17kDa band after reduction. Except for Pv, the purified proteins all reacted with an antiserum to Vg. In SDS-PAGE, Pv appeared as a 23 kDa band and a second < 6.5 kDa diffuse band. An antiserum to Pv dephosphorylated by alkaline phosphatase (Ap) was prepared. In Western blots, the antiserum reacted with dephosphorylated Pv and Vg, but not with Lv and beta'-component. This is the first immunological proof that three egg yolk proteins (Lv, Pv, and beta'-component) are derived from Vg in fish.

New allergens from hen's egg white and egg yolk. In vitro study of ovomucin, apovitellenin I and VI, and phosvitin.

Three hen egg yolk proteins, apovitellenins I and VI and phosvitin, and one egg white protein, ovomucin, were purified and tested for their ability to bind IgE in the sera of patients hypersensitive to egg. All of the proteins bound IgE from the sera of egg-allergic individuals in the radioallergosorbent test, and they also inhibited binding of IgE to the parent fractions-either egg yolk (apovitellenins I and VI and phosvitin) or egg white (ovomucin). It appears that apovitellenins I and VI are major allergens for some of the individuals tested. This is the first report of the in vitro allergenicity of these proteins.

An anti-acetylcholine receptor monoclonal antibody cross-reacts with phosvitin.

Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.

Estrogen induction of plasma vitellogenin in the cockerel: studies with a phosvitin antibody.

The effects of estrogen on plasma vitellogenin have been studied in the cockerel by immunoprecipitation techniques using an antiserum prepared against the egg yolk phosphoprotein, phosvitin. The antiserum gave precipitin lines of complete identity to phosvitin and to vitellogenin which was isolated from hen plasma by DEAE-cellulose chromatography and by affinity chromatography using anti-phosvitin coupled to Sepharose 4B. The cross-reactivity of vitellogenin and phosvitin adds support to the concept that plasma vitellogenin is the precursor phosphoprotein of egg yolk phosvitin. In the three-week old cockerel, anti-phosvitin produced no detectable immunoprecipitate in the plasma. However, after a single sc injection of diethylstilbestrol (2.5 mg), plasma vitellogenin levels began to increase at 4 h and reached a maximum 20-30 h after hormone administration. The increase in plasma levels of triglyceride paralleled those of vitellogenin. These studies suggest that there is no significant time lag in the estrogenic induction of plasma vitellogenesis in the cockerel, the longer lag periods observed by other investigators may be a function of the sensitivity of the assays used for detecting vitellogenin.