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.     

Influence of processing factors on the stability of model mayonnaise with whole egg during long-term storage

Mayonnaise-like oil-in-water emulsions with different stabilities—evaluated from the degree of macroscopic defects, e.g., syneresis—were prepared by different formulations and processing conditions (egg yolk weight, homogenizer speed, and vegetable oil temperature). Emulsions prepared with lower egg yolk content were destabilized for shorter periods. The long-term stability of emulsions was weakly related to initial properties, e.g., oil droplet distribution and protein coverage at the interface. Protein aggregation between oil droplets was observed and would be responsible for the instability of emulsions exhibited by the appearance defects. SDS-PAGE results for adsorbed and unadsorbed proteins at the O/W interface suggested that predominant constituents adsorbed onto the interface were egg white proteins as compared with egg yolk components when the amount of added egg yolk was low. In present condition, egg white proteins adsorbed at the O/W interface could be a bridge of neighboring oil droplets thereby causing flocculation in emulsions.     

Absorption, transportation and digestion of egg white in quail embryos

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor-mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid-phase endocytosis took place in the cells generally referred to collectively as the 'albumen sac'. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross-reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.     

The chicken egg yolk plasma and granule proteomes

Using 1-D SDS-PAGE, LC-MS/MS, and MS(3), we identified 119 proteins from chicken egg yolk, 86 of which were not identified in yolk previously. Proteins were roughly quantitated by calculating their exponentially modified protein abundance index (emPAI) to classify them as major or minor yolk components, and to estimate their distribution between yolk plasma and yolk granular fraction. The proteins with highest abundance were serum albumin, the vitellogenin cleavage products, apovitellenins, IgY, ovalbumin, and 12 kDa serum protein with cross-reactivity to beta2-microglobulin. In addition yolk contained many other serum and egg white proteins, the proteases nothepsin and thrombin, numerous protease inhibitors, and antioxidative enzymes, such as superoxide dismutase and glutathione peroxidase. Among the moderately abundant proteins were two alpha2-macroglobulin-like proteins different from egg white alpha2-macroglobulin, and the major biotin-binding protein of yolk. An unexpected identification was that of the eggshell matrix protein ovocleidin-116, which was previously thought to be eggshell-specific. The list of chicken egg yolk proteins provided in this report is by far the most comprehensive at present and may serve as a starting point for the characterization of less well-known yolk proteins.     

Heat Resistance of Salmonella in Various Egg Products

The heat-resistance characteristics of Salmonella typhimurium Tm-1, a reference strain in the stationary phase of growth, were determined at several temperatures in the major types of products produced by the egg industry. The time required to kill 90% of the population (D value) at a given temperature in specific egg products was as follows: at 60 C (140 F), D = 0.27 min for whole egg; D = 0.60 min for whole egg plus 10% sucrose; D = 1.0 min for fortified whole egg; D = 0.20 min for egg white (pH 7.3), stabilized with aluminum; D = 0.40 min for egg yolk; D = 4.0 min for egg yolk plus 10% sucrose; D = 5.1 min for egg yolk plus 10% NaCl; D = 1.0 min for scrambled egg mix; at 55 C (131 F), D = 0.55 min for egg white (pH 9.2); D = 1.2 min for egg white (pH 9.2) plus 10% sucrose. The average Z value (number of degrees, either centigrade or fahrenheit, for a thermal destruction time curve to traverse one logarithmic cycle) was 4.6 C (8.3 F) with a range from 4.2 to 5.3 C. Supplementation with 10% sucrose appeared to have a severalfold greater effect on the heat stabilization of egg white proteins than on S. typhimurium Tm-1. This information should be of value in the formulation of heat treatments to insure that all egg products be free of viable salmonellae.     

Avian SERPINB11 gene: characteristics, tissue-specific expression, and regulation of expression by estrogen

Serpins, a group of proteins with similar structural and functional properties, were first identified based on their unique mechanism of action: their inhibition of proteases. While most serpins have inhibitory roles, certain serpins are not involved in canonical proteolytic cascades but perform diverse functions including storage of ovalbumin in egg white, transport of hormones (thyroxine- and cortisol-binding globulin), and suppression of tumors. Of these, serpin peptidase inhibitor, clade B, member 11 (SERPINB11) is not an inhibitor of known proteases in humans and mice, and its function is unknown. In the present study, the SERPINB11 gene was cloned, and its expression profile was analyzed in various tissues from chickens. The chicken SERPINB11 gene has an open reading frame of 1346 nucleotides that encode a protein of 388 amino acids that has moderate homology (38.8%-42.3%) to mammalian SERPINB11 proteins. Importantly, SERPINB11 mRNA is most abundant in the chicken oviduct, specifically luminal and glandular epithelia, but it was not detected in any other chicken tissues of either sex. We then determined effects of diethylstilbestrol (DES; a synthetic nonsteroidal estrogen) on SERPINB11 expression in the chicken oviduct. Treatment of young chicks with DES induced SERPINB11 mRNA and protein only in luminal and glandular epithelial cells of the oviduct. Collectively, these results indicate that the novel estrogen-induced SERPINB11 gene is expressed only in epithelial cells of the chicken oviduct and implicate SERPINB11 in regulation of oviduct development and differentiated functions.     

Carbohydrate compositional effects on tissue distribution of chicken riboflavin-binding protein

Riboflavin-binding proteins (RBP) purified from chicken egg white, yolk and the serum of laying hens differ in their carbohydrate compositions reflecting tissue-specific modifications of a single gene product. All three are complex glycoproteins having more than twice as many N-acetylglucosamine residues (>12) as mannose residues (approx. 6). Egg white RBP is distinctive in having only one sialic acid and two galactose residues. Serum RBP contains approx. five sialic acid and seven galactose residues. In addition there is one residue of fucose. The carbohydrate composition of yolk RBP indicates the hydrolysis, respectively, of one, one, two and 3 residues of sialic acid, fucose, galactose, and N-acetylglucosamine from its precursor, serum RBP. The effect of these differing levels of glycosylation on plasma clearance, ovarian uptake and tissue distribution of ¹²⁵I-labeled riboflavin-binding proteins in laying hens were compared. 2 h after intravenous injection, 19% of the egg white RBP, 29% of the yolk RBP, and 37% of the serum RBP remained in circulation. The kinetics of plasma clearance was distinctly biphasic for each of the radioiodinated proteins. The initial rapid-turnover component ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=13}\text{min}$) ranged from 27% of the serum RBP sample to 48% of the egg white RBP sample. The remaining slow-turnover components were cleared with half-lives fo 81 min (egg white RBP), 101 min (yokl RBP), and 121 min (serum RBP). 16 h after injection, only 4% of the egg white RBP was deposited in the yolk of developing oocytes while about 12% of the serum RBP and yolk RBP was deposited. This higly significant difference is apparently due to preferential, carbohydrate-dependent clearance of egg white RBP by the liver rather than preferential uptake of serum and yolk RBP by the ovarian follicle. We find no evidence for carbohydrate-directed uptake of riboflavin-binding protein by the ovarian follicle.     

Flow of egg white ovalbumin into the yolk sac during embryogenesis

Western blot analyses of yolk proteins of the White Leghorn hens showed that an ovalbumin-like molecule was included in day 16 eggs but not in the ovarian follicles, and very little in newly deposited eggs. Northern hybridization, as well as in vitro translation, of poly(A)+ RNAs prepared from the yolk sac membranes of developing embryos gave no signal for ovalbumin messages. These results imply that the present ovalbumin-like protein of yolk has its origin in egg white, not being a de novo synthesis product in the yolk sac membranes.     

Separation and Formation of Egg Yolk Oil by Solubilizing the Lipoproteins of Spray-dried Egg Yolk into Polypeptides

Egg yolk oil was formed from a hen’s egg without using the traditional charring method and organic solvents. By treating a spray-dried egg yolk suspension with a commercial crude enzyme preparation having protease and lipase activities, the lipids (egg yolk oil) could be easily separated, and a transparent solution of soluble polypeptides was obtained. When the enzyme preparation (4 mg) was added to a 5% spray-dried egg yolk (100 mg) suspension, the reaction mixture became transparent, and the egg yolk oil floated to the surface of the reaction mixture within 3 h. In the case of a 10% spray-dried egg yolk suspension, a transparent solution and egg yolk oil could be successfully obtained within 4 to 5 h by using a larger reaction vessel and a 0.2M lactate buffer. About 87% of the total polypeptides in the initial reaction mixture was recovered from the transparent solution, while about 83% of the total phospholipids was recovered from the floating egg yolk oil.     

Biochemical and immunological aspects of riboflavin carrier protein

Riboflavin carrier protein which is obligatorily involved in yolk deposition of the vitamin in the chicken egg, is a unique glycophosphoprotein present in both the yolk and white compartments. The yolk and egg white proteins are products of a single estrogen-inducible gene expressed in the liver and the oviduct respectively of egg laying birds. Despite the fact that the carbohydrate composition of the yolk and white riboflavin carrier proteins differ presumably due to differential post-translational modification, the proteins are immunologically similar and have identical amino acid sequence (including a cluster of 8 phosphoser residues towards the C-terminus) except at the carboxy terminus where the yolk riboflavin carrier protein lacks 13 amino acids as a consequence of proteolytic cleavage during uptake by oocytes. The protein is highly conserved throughout evolution all the way to humans in terms of gross molecular characteristics such as molecular weight and isoelectric point, and in immunological properties, preferential affinity for free riboflavin and estrogen inducibility at the biosynthetic locusviz., liver. Obligatory involvement of the mammalian riboflavin carrier protein in transplacental flavin transport to subserve fetal vitamin nutrition during gestation is revealed by experiments using pregnant rodent or subhuman primate models wherein immunoneutralisation of endogenous maternal riboflavin carrier protein results in fetal wastage followed by pregnancy termination due to selective yet drastic curtailment of vitamin efflux into the fetoplacental unit. Using monoclonal antibodies to chicken riboflavin carrier protein, it could be shown that all the major epitopes of the avian riboflavin carrier protein are highly conserved throughout evolution although the relative affinities of some of the epitopes for different monoclonal antibodies have undergone progressive changes during evolution. Using these monoclonal antibodies, an attempt is being made to map the different epitopes on the riboflavin carrier protein molecule with a view to delineate the immunodominant regions of the vitamin carrier to understand its structure-immunogenicity relationship. Presented at the 3rd National Symphosium on Bioorganic Chemistry, 1987, Hyderabad.     

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.     

Low environmental selenium availability as an additional determinant for goiter in East Java, Indonesia?

Iodine deficiency, which is most visibly indicated by goiter, is highly prevalent in Indonesia. Since 1994, Indonesia has a decree that all salt used for human, livestock, and industry must be iodized. However, despite the increased distribution of iodized salt, pockets with significantly higher prevalence of goiter still remain. This situation may be consequence of selenium (Se) deficiency. This study aimed to assess the Se level in the environment of goiter prevalent areas. Five hundred eleven school children participated in this study. Goiter was measured using both ultrasound and palpation. Ninety-nine eggs were collected from free-living chicken in 11 villages, and the Se contents of egg yolk and egg white were determined by neutron activation analysis. In the villages studied, Se concentration in egg yolk ranged from 0.15 to 1.52 Μg/g and in egg white from 0.18 to 2.97 Μg/g. The prevalence of goiter measured by palpation ranged from 18.4% to 70% and by ultrasound from 0% to 100%. Because of the inconsistency of goiter rate measured by palpation and ultrasonography, the question remains whether low availability of Se in the environment might be an additional contributing factor for goiter.     

Avidin traps biotin diffusing out of chicken egg yolk

1. The unequal distribution of biotin and biotin-binding proteins between the yolk and albumen of freshly laid chicken eggs provides the potential for time-dependent redistribution of biotin that could affect egg quality, biotin availability, and hatchability. 2. Avidin-bound biotin was measured in albumen next to the shell and next to the yolk in eggs stored up to 23 days. 3. Biotin bound to biotin-binding proteins (BBP-I and BBP-II) was measured at the center and periphery of yolk from the same eggs. 4. After 11 days of storage, significant amounts of biotin from the yolk began to accumulate in the albumen adjacent to the yolk. 5. This transfer is attributed to a change in the vitelline membrane that permits diffusion of biotin, not BBP-I or BBP-II, out of the yolk. 6. The dynamics of this phenomenon suggest that in addition to its antimicrobial role, avidin may be involved in the utilization of biotin by the chick embryo.     

Proteomic analysis of the chicken egg vitelline membrane

The avian vitelline membrane (VM) is a multilayered proteinaceous structure separating egg white from yolk. The innermost layer of the VM, deposited onto the oocyte plasma membrane in the ovary, corresponds to the mammalian zona pellucida (ZP). The outer layer is produced in the infundibulum, the first section of the oviduct. Using high-throughput, high-end LC-MS(n) 137 proteins were identified, only 13 of which were known previously to be components of the VM. Depending on the washing protocol, two largely overlapping, but not identical, sets of identified proteins were produced from water-washed and salt-washed VMs. Most of the components of the VM were known previously from other egg compartments, such as, for instance, the egg white proteins lysozyme C, ovalbumin, ovotransferrin, and ovomucin. Specific components of the VM not identified previously in other egg compartments included eight ZP proteins, oviductin protease, and two ATPases. The vitelline outer membrane protein (VMO) VMO II was identified as beta-defensin-11. The list of VM proteins presented in this report is by far the most comprehensive dataset available at present and complements proteomic analyses of chicken egg compartments published previously.     

Elevated plasma corticosterone decreases yolk testosterone and progesterone in chickens: linking maternal stress and hormone-mediated maternal effects

Despite considerable research on hormone-mediated maternal effects in birds, the underlying physiology remains poorly understood. This study investigated a potential regulation mechanism for differential accumulation of gonadal hormones in bird eggs. Across vertebrates, glucocorticoids can suppress reproduction by downregulating gonadal hormones. Using the chicken as a model species, we therefore tested whether elevated levels of plasma corticosterone in female birds influence the production of gonadal steroids by the ovarian follicles and thus the amount of reproductive hormones in the egg yolk. Adult laying hens of two different strains (ISA brown and white Leghorn) were implanted subcutaneously with corticosterone pellets that elevated plasma corticosterone concentrations over a period of nine days. Steroid hormones were subsequently quantified in plasma and yolk. Corticosterone-implanted hens of both strains had lower plasma progesterone and testosterone levels and their yolks contained less progesterone and testosterone. The treatment also reduced egg and yolk mass. Plasma estrogen concentrations decreased in white Leghorns only whereas in both strains yolk estrogens were unaffected. Our results demonstrate for the first time that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk. Maternal corticosterone could therefore mediate environmentally induced changes in yolk gonadal hormone concentrations. In addition, stressful situations experienced by the bird mother might affect the offspring via reduced amounts of reproductive hormones present in the egg as well as available nutrients for the embryo.     

Calorimetric studies of flavin-binding proteins: FMN and FAD binding to hen egg riboflavin-binding proteins

Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 10⁵ and 10⁴ were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form.     

Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma

SERPINB1 (serine protease inhibitor, clade B, member1) is a member of the SERPINB family. Recent studies suggested that SERPINB1 may suppress the migration and invasion of lung and breast cancers. In this study, we investigated a possible involvement of SERPINB1 in the regulation of hepatocellular carcinoma metastasis (HCC). The expression of SERPINB1 was evaluated using western blot analysis in 8 paired fresh HCC specimens and immunohistochemistrical assay on 67 paraffin-embedded HCC slices. SERPINB1 was downregulated in HCC specimens and correlatively related with two clinicopathologic features of HCC, metastasis (P = 0.000) and vein invasion (P = 0.006). Univariate and multivariate survival analyses showed a lower level of SERPINB1 expression is associated with poor prognosis and clinical outcome (P = 0.001). In addition, small interfering RNA targeting SERPINB1 was used to knock down the expression of SERPINB1 in Huh7 and BEL-7404 cells. We showed that interference of SERPINB1 promoted migration and invasion of HCC cells, while cell proliferation was not affected. Finally, we observed an apparent increase in the level of active matrix metalloproteinase-2 (MMP2) after SERPINB1 knockdown, implying that SERPINB1 might participate in the regulation of HCC metastasis through modulating the activation of matrix metalloproteinases. Overall, our results suggested an inhibitory role of SERPINB1 in the migration and invasion of HCC, implying that SERPINB1 might be a potential prognostic indicator of HCC metastasis.     

Characterization of four substrates emphasizes kinetic similarity between insect and human C-domain angiotensin-converting enzyme.

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb-ODAIF-1(1-13)), NKLKPSQWI (Neb-ODAIF-1(1-9)), SLKPSNWLTPSE (Neb-ODAIF-2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins. An antiserum raised against Neb-ODAIF-1(1-9) immunostained one out of three yolk protein bands of SDS/PAGE-separated fly haemolymph and egg homogenate, thus confirming that these peptides originate from a yolk protein gene product. Kinetic analysis of these peptides and of the peptides Neb-ODAIF and Neb-ODAIF-1(1-7) with insect ACE and human ACE show both similar and unique properties for insect ACE as compared with human C-domain ACE.