Reduction of antigenicity and allergenicity of genetically modified egg white allergen,ovomucoid third domain

Ovomucoid (Gal d1) is a major allergen in hen egg white, consisting of three tandem domains. In this study, five genetically modified third domain (DIII) mutants, which were substituted single or double amino acids within its IgE and IgG epitopes were compared with those prepared and their antigenicity and allergenicity with native analogue using Western immunoblot and enzyme-linked immunosorbent assay. The replacement of phenylalanine at 37 (F37) position with methionine caused drastical loss of IgG and IgE binding activities of human sera derived from egg allergic patients as well as disruption of the alpha-helix structure which comprises a part of the IgG and IgE epitopes. Substituting glycine at 32 position in conjunction with F37 showed a synergistic effect of decreasing antigenicity. The present study indicated that glycine 32 and phenylalanine 37 have an important role on its antigenicity and allergenicity as well as structural integrity of ovomucoid DIII.     

Interaction of ovomucoid from duck egg white with serine proteinases

The interaction of highly purified duck egg white ovomucoid with trypsin and chymotrypsin was studied. It was found that the ovomucoid molecule contains two equally effective trypsin-binding sites which, in their turn, comprise lysine residues and one independent chymotrypsin-binding site. The values of inhibition constants were determined and the changes in free energy, enthalpy and entropy during the ovomucoid interaction with trypsin and chymotrypsin were established. It was shown that the inhibitor affinity for the enzymes does not change at low degrees of free amino groups modification.     

Identification and isolation of a bactericidal domain in chicken egg white lysozyme

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fractionation of clostripain-digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I-V-S-D-G-N-G-M-N-A-W-V-A-W-R (amino acids 98-112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. The peptide I-V-S-D-G-N-G-M consisting of the eight amino acids of the N-terminal side had no bactericidal properties, whereas the peptide N-A-W-V-A-W-R of the C-terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R-A-W-V-A-W-R) increased the bactericidal activity considerably. The D enantiomer of R-A-W-V-A-W-R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.     

Isolation and characterization of thiamin-binding protein from chicken egg white.

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.     

Producing a low ovomucoid egg white preparation by precipitation with aqueous ethanol

A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20% (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20% ethanol (6.25 ml each), with about 65% of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70% of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.     

Human IgE antibody to the carbohydrate-containing third domain of chicken ovomucoid

Two kinds of the third domain, either with or without a carbohydrate chain, were prepared from chicken ovomucoid. The immunoreactivity of the domain preparations to human IgE antibody directed against ovomucoid was examined by using the sera from patients of egg allergy. About 30% of the serum antibody to ovomucoid reacted with the carbohydrate-containing domain, whereas little or no antibody with reactivity to the carbohydrate-free domain was detected, suggesting that the carbohydrate chain attached to the third domain played an important role in antigenic determinants of the carbohydrate-containing third domain against the human IgE antibody.     

The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases.

The crystal structure of chicken egg white cystatin has been solved by X-ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X-ray data between 6 and 2.0A. The molecule consists mainly of a straight five-turn alpha-helix, a five-stranded antiparallel beta-pleated sheet which is twisted and wrapped around the alpha-helix and an appending segment of partially alpha-helical geometry. The 'highly conserved' region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta-hairpin loop which on opposite sides is flanked by the amino-terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I - Trp104I. These loops and the amino-terminal Gly9I - Ala10I form a wedge-shaped 'edge' which is quite complementary to the 'active site cleft' of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino-terminal segment Gly9I - Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin-like inhibitors seems to be fundamentally different from the 'standard mechanism' defined for serine proteinases and most of their protein inhibitors.     

Antimicrobial activity of chicken egg white cystatin

The cysteine protease inhibitor cystatin was purified from chicken egg white and its antimicrobial activity determined for a series of pathogenic bacteria. The results indicate that Acinetobacter lwoffii, Escherichia coli, Oligella sp. and Pseudomonas aeruginosa are highly sensitive to low concentrations of cystatin, which possesses bactericidal activity. No inhibition was observed with a Citrobacter freundii strain. Fifty percent growth inhibition (IC₅₀) was observed at cystatin concentrations in the range of 80 and 100g/ml, and the growth was completely inhibited at concentrations in the range of 100 and 200g/ml. Fifty percent growth inhibition (IC₅₀) for Staphylococcus aureus, Staphylococcus gallinarum, and Staphylococcus xylosus strains was observed at 150 and 200 g of cystatin/ml respectively, and growth was completely inhibited at cystatin concentrations in the range of 300 and 1000 g/ml. The activity of cysteine proteases was significantly decreased in the culture supernatant of microorganisms when chicken cystatin was added. In this study, we observed that chicken cystatin may be a candidate for antibacterial drug development aiming at controlling bacterial pathogens including Escherichia coli, Pseudomonas aeruginosa, and another possible application might be as a therapeutic agent for health improvement.     

Preventive effect of egg yolk phosvitin on heat-insolubilization of egg white protein and its application to heat-induced egg white gel

The effects of phosvitin (PV) on insolubilization of egg white protein (EWP) and ovotransferrin (OT) were examined by measuring turbidity after heating at 80 degrees C in a pH range of 5 to 8. PV showed preventive ability against heat-insolubilization of EWP, especially heat-labile OT. The preventive ability of PV was reduced by adding NaCl to a PV-OT mixture on heating. Native PAGE and gel filtration analyses showed that PV prevented an insolubilization of heat-denatured OT through ionic interactions. The preventive effects of PV on insolubilization of EWP and OT resulted in the formation of a firm, transparent gel from EWP in coexistence with PV on heating. The addition of PV might make possible the preparation of liquid egg white without insoluble products even on heat-treatment at high temperatures.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Exploring the chicken egg white proteome with combinatorial peptide ligand libraries

The use of two types of peptide ligand libraries (PLL), containing hexapeptides terminating either with a primary amine or modified with a terminal carboxyl group, allowed the discovery and identification of a large number of previously unreported egg white proteins. Whereas the most comprehensive list up to date ( Mann, K. , Proteomics 2007, 7, 3558- 3568 ) tabulated 78 unique gene products, our findings have almost doubled that value to 148 unique protein species. From the initial nontreated egg, it was possible to find 41 protein species; the difference (107 proteins) was generated as a result of the use of PLLs from which a similar number of species (112 and 109, respectively) was evidenced. Of those, 35 proteins were the specific catch of the amino-terminus PLL, while 33 were uniquely captured by the carboxy-terminus PLL. While a number of these low-abundance proteins might have a biological role in maintaining the integrity of the egg white and protecting the yolk, others might be derived from decaying epithelial cells lining the oviduct and/or represent remnants of products from the magnum and eggshell membrane components secreted by the isthmus, which might ultimately be incorporated, even if in trace amounts, into the egg white. The list of egg white components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.     

One-electron reduction of flavoproteins: pulse radiolysis of chicken egg white riboflavin binding protein

Reduction of the chicken egg white riboflavin binding protein by the hydrated electron results in competitive formation of both a disulfide-electron adduct and an anionic flavin semiquinone bound to the protein. The former decays to products that cannot be observed under the conditions of our experiments. The latter is rapidly protonated to the stable neutral semiquinone. The pH dependence of the rate constant associated with this protonation suggests that an acid/base group on the protein donates a proton to the anionic semiquinone.     

Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin.     

Structural studies on periodate-oxidized chicken ovomucoid. Spectral behaviour of the tyrosine residues.

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2-5 glucosamines and 1-2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodic oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as 'dissolved' in the 'carbohydrate solvent'. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the 'sugar solvent' and disturbs the conformation of the sugar chain.