Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Isotope-coded protein label based quantitative proteomic analysis reveals significant up-regulation of apolipoprotein A1 and ovotransferrin in the myopic chick vitreous.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching -10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing -10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

Purification, characterization, cloning and structural analysis of Crocodylus siamensis ovotransferrin for insight into functions of iron binding and autocleavage.

Ovotransferrin (OTf), the major protein constituent of egg white, is of great interest due to its pivotal role in biological iron transport and storage processes and its spontaneous autocleavage into peptidic fragments with alternative biological properties, such as antibacterial and antioxidant activities. However, despite being well-investigated in avian, a detailed elucidation of the structure-function relationship of ovotransferrins in the closely related order of Crocodilia has not been reported to date. In this study, electron paramagnetic resonance (EPR) confirmed the presence of two spectroscopically distinct ferric iron binding sites in Crocodylus siamensis OTf (cOTf), but implied a five-fold lower quantity of bound iron than in hen OTf (hOTf). In addition, quantitative estimation of free sulfhydryl groups revealed slight differences to hOTf. To gain a better structural understanding of cOTf, we found a cOTf gene consisting of an open reading frame of 2040bp and encoding a protein of 679 amino acids. In silico prediction of the three-dimensional structure of cOTf and comparison with hOTf revealed four evolutionarily conserved iron-binding sites in both N- and C-lobes, as well as the presence of only 13 of the 15 disulfide bonds in hOTf. This evolutionary loss of disulfide linkages in conjunction with the lack of hydrogen bonding from a dilysine trigger in the C-lobe are presumed to affect the iron binding and autocleavage character of cOTf. As a result, cOTf may be capable of exerting a more diverse array of functions compared to its avian counterparts; for instance, ion buffering, antioxidant and antimicrobial activities.

Absolute quantification of a very virulent Marek's disease virus dynamic quantity and distributions in different tissues.

Chickens infected with Marek's disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors and increases its potential for horizontal transmission. There is a positive correlation between very virulent (vv) MDV quantity and the pathology. The purpose of this study was to determine the vvMDV loads dynamics in different phases, and the correlation between the viral quantity and tumor development. We used a SYBR Green duplex real-time quantitative PCR (q-PCR) assay to detect and quantify MDV loads and distributions in different tissues, targeting the Eco-Q protein gene (meq) of the virus and the house-keeping ovotransferrin (ovo) gene of chickens. The q-PCR was performed using different tissue DNA preparations derived from chickens which were infected with 1,000 pfu of the SDWJ1302 strain and tissue samples were collected from control and MDV-infected birds on 7, 10, 15, 21, 28, 40, 60, and 90 d post-infection (DPI). The data indicated that the MDV genome was almost quantifiable in immune organs of infected chickens as early as 7 DPI, and the number of MDV genome copies in the blood and different organs peaked by 28 DPI, but then gradually decreased by 40 DPI. The levels of viral quantity in the lymphocytes, liver, and spleen were all higher than those in other organs, and that in the feather follicles was the highest among different phases of MDV infection. The vvMDV could still be detected in peripheral blood and tissues by 90 DPI, and the vast existence of virus will stimulate tissue destruction. The data provided further evidence of viral infection involving multi-organ distribution and mainly involving immune organ proliferation, resulting in immunosuppression.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli.

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients' sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Expression of ovotransferrin enhances tolerance of yeast cells toward oxidative stress.

Recently, we found that ovotransferrin (OTf) undergoes distinct self-cleavage in a redox-dependent process and exhibited in vitro superoxide dismutase (SOD)-like activity. In this study, we explore that the expression of OTf confers high tolerance to oxidative stress in yeast cells. The OTf gene was cloned into the vector pPICZB and was successfully expressed in methylotrophic yeast, Pichia pastoris KM71H. There was no growth difference between the non-transformed strain and recombinant strains harboring a mock vector (pPICZB) or the OTf gene carrying a vector (OTf-pPICZB). Intracellularly expressed OTf was found to undergo self-cleavage, producing a major fragment of 15 kDa, which corresponded to the disulfide kringle domain of the N-terminal lobe. The yeast OTf transformants exhibited strong tolerance to oxidative stress induced by either hydrogen peroxide (H2O2) or diethyl maleate (DEM). Further, OTf transformants showed higher intracellular reducing capacity and enhanced cytosolic reductase activity. This study is the first to describe the ability of OTf to confer in vivo antioxidative stress function within a complicated milieu of eukaryotic cells and provide novel insights for the potential of the OTf gene for molecular breeding of industrial yeast strains with high tolerance to oxidative stress.

Molecular evolution of the transferrin family and associated receptors.

BACKGROUND: In vertebrates, serum transferrins are essential iron transporters that have bind and release Fe(III) in response to receptor binding and changes in pH. Some family members such as lactoferrin and melanotransferrin can also bind iron while others have lost this ability and have gained other functions, e.g., inhibitor of carbonic anhydrase (mammals), saxiphilin (frogs) and otolith matrix protein 1 (fish). SCOPE OF REVIEW: This article provides an overview of the known transferrin family members and their associated receptors and interacting partners. MAJOR CONCLUSIONS: The number of transferrin genes has proliferated as a result of multiple duplication events, and the resulting paralogs have developed a wide array of new functions. Some homologs in the most primitive metazoan groups resemble both serum and melanotransferrins, but the major yolk proteins show considerable divergence from the rest of the family. Among the transferrin receptors, the lack of TFR2 in birds and reptiles, and the lack of any TFR homologs among the insects draw attention to the differences in iron transport and regulation in those groups. GENERAL SIGNIFICANCE: The transferrin family members are important because of their clinical significance, interesting biochemical properties, and evolutionary history. More work is needed to better understand the functions and evolution of the non-vertebrate family members. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.

Primary structure of potential allergenic proteins in emu (Dromaius novaehollandiae) egg white.

The emu (Dromaius novaehollandiae) egg is considered promising as an alternative egg product. To obtain basic biochemical information on emu egg white, the major protein compositions in emu and chicken egg whites and the primary structures of potential allergenic proteins were compared. The dominant protein in emu egg white was ovotransferrin (OVT), followed by ovalbumin (OVA) and TENP protein. The OVA and ovomucoid (OVM) levels in emu egg white were estimated as significantly lower than those in chicken egg white by Western blotting and enzyme-linked immunosorbent assays using anti-chicken OVA or OVM antibodies. Lysozyme and its enzymatic activity were not detected in emu egg white. OVT, OVA, and OVM genes were also cloned, and their nucleotide and amino acid sequences were determined. The protein sequences of OVT, OVA, and OVM from emu showed lower similarities to those of chicken than other avian species, such as quail and turkey. These results emphasize the low allergenicity of emu egg white and its potential as an alternative to chicken egg white.

Estimations of repeatability and heritability of egg albumen antimicrobial activity and of lysozyme and ovotransferrin concentrations.

1. The repeatability and heritability of growth inhibition by egg albumen of two major pathogenic bacteria, a Gram-negative (Salmonella Enteritidis) and a Gram-positive (Staphyloccocus aureus) and of two antimicrobial albumen proteins, lysozyme and ovotransferrin, were estimated in commercial pedigree hens. 2. Repeatability was evaluated in 100 egg-type hens at the beginning, middle and end of the laying cycle on eggs collected for 3 weeks. Heritabilities were estimated at 36 to 40 weeks of age on 400 pedigree hens (2 eggs/hen), which were the offspring of 25 sires each mated with 4 dams. Ovotransferrin and lysozyme were quantified by ELISA. Salmonella Enteritidis (S.E.) and Staphyloccocus aureus (S.A.) were inoculated into a sample of sterilised albumen and enumerated after incubation. 3. Total protein content in albumen decreased with age of laying hens, whereas there were increases in lysozyme or ovotransferrin concentrations and in the bacteriostatic effect of albumen. 4. Repeatability for bacterial growth in albumen ranged from 0.29 to 0.39 for the number of S.E. (log cfu/ml) one day post inoculation (p.i.) but was lower and more variable at 5 d p.i. or for S.A. number. It ranged from 0.27 to 0.38 for S.E. and S.A. number at the mid period of the laying cycle. Repeatabilities were low and variable for total egg albumen protein or lysozyme and ovotranferrin concentrations (0 to 0.22). 5. Negative phenotypic correlations were observed between lysozyme concentrations and S.E. number but that between lysozyme and S.A. number was not significant. 6. Heritabilities were low (0.01 to 0.09) for protein traits. They were 0.11 for S.A. number and 0.16 for S.E. number one day p.i. 7. It appears to be more efficient to select on global bacterial growth than on specific antimicrobial proteins. The most promising trait is the number of S.E. one day p.i.

Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich, and turkey.

Transferrins form an important class of iron-binding proteins widely distributed in the physiological fluids of vertebrates and invertebrates. In vertebrates they are present mostly in serum as serotransferrins. In birds and reptiles transferrins are also found in eggs as ovotransferrins. However, until now only chicken and duck ovotransferrin sequences have been published. This paper presents data on the purification, biochemical analysis, cloning and sequencing of ovotransferrins from red-eared turtle, African ostrich and turkey, revealing their significant homology with other known ovotransferrin sequences. The proteins were purified by size-exclusion and anion-exchange chromatography. Isoelectric points, iron-saturated and iron-free spectra, as well as the mRNA nucleotide sequences of 2,409 nt (ORF: 2,106 nt encoding a 701-amino-acid polypeptide; ), 2,418 nt (ORF 2,118 nt encoding a 705-amino-acid polypeptide; ), and 2,397 nt (ORF: 2,118 nt encoding a 705-amino-acid polypeptide; ) were determined for ostrich (OtrF), red-eared turtle (TtrF), and turkey (MtrF) ovotransferrin, respectively.

Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants.

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).

Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer.

The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80 kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

Differential sensitivity to mutations in a single peptide by two TCRs having identical beta-chains and closely related alpha-chains.

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.

Molecular modeling of human serum transferrin for rationalizing the changes in its physicochemical properties induced by iron binding. Implication of the mechanism of binding to its receptor.

In order to rationalize the physicochemical properties of human serum-transferrin (STf) and the STf-receptor (TfR) recognition process, we have tried to predict the 3D structures of apo- and iron-loaded STf using a homology modeling technique to study the changes in the structural characteristics that take place upon the uptake of iron by STf in solution. The crystal structures of both forms for ovotransferrin were used as templates for the STf modeling. The modeled structure of STf gave a satisfactory interpretation for the typical physicochemical properties such that (1) STf has a negative electrophoretic mobility and its value increases with iron uptake, and (2) the radius of gyration Rg of Tf decreases with iron uptake. It was found that upon iron binding, interdomain closures take place with large movements of the NII and CII subdomains comprising the N- and C-lobes in STf through a hinge-bending motion, accompanied by the opening of the bridge region with a displacement of more than 15 A. Moreover, in view of the findings from our capillary electrophoresis experiments that the electrostatic interactions significantly contribute to a specific binding of Fe2-STf with TfR, it is inferred that the connecting (bridge) and its neighboring region associated with a surface exposure of negative charge play an important role in the STf-receptor recognition process.

Periplasmic secretion of functional ovotransferrin N-lobe in Escherichia coli.

The cytoplasmic and periplasmic production systems of ovotransferrin N-lobe in Escherichia coli were constructed. The periplasmic, but not cytoplasmic product, was found to have the iron-binding function.

Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins.

Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/1 oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/1 and a foreshortened oTF consisting of residues 1-682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291-295]. Electrospray mass spectrometry gives results consistent with these six changes. Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al.

Effects of insulin-like growth factor-I, estrogen, glucocorticoid, and transferrin on the mRNA contents of ovalbumin and conalbumin in primary cultures of quail (Coturnix coturnix japonica) oviduct cells.

The effects of estrogen, dexamethasone, insulin-like growth factor-I (IGF-I), and transferrin on the messenger RNA (mRNA) contents of ovalbumin and conalbumin in primary cultures of quail oviduct cells were investigated. In the absence of one of the above hormones or factors, a decrease in ovalbumin mRNA was prominent. In particular, removal of IGF-I and transferrin caused a significant effect. Studies using a combination of estrogen, dexamethasone, IGF-I and transferrin indicated that IGF-I cooperates with estrogen or dexamethasone and transferrin works with dexamethasone. Specifically, IGF-I enhanced ovalbumin synthesis or increased cellular ovalbumin mRNA content depending on its concentration in the medium in the presence of estrogen. However, the effects of estrogen, dexamethasone, IGF-I, and transferrin were not similarly observed with conalbumin mRNA. These results show that ovalbumin synthesis is controlled by estrogen or glucocorticoid with IGF-I or transferrin and that cellular ovalbumin mRNA content is also regulated by these hormones or transferrin. In contrast, conalbumin synthesis and cellular content of conalbumin mRNA are not affected by these hormones under the conditions of the present study.

Correlation between in vitro and in vivo behaviour of liposomal antigens.

Using conalbumin as a model antigen, we demonstrate in this paper that liposomal antigen differently influences the activation of the immune system depending on the mode of association of the antigen with the liposomal vehicle whether it is by encapsulation or surface linkage. This conclusion is based on in vivo data showing that encapsulated antigen induces a short-lasting response dominated by IgG1 production while surface-linked antigen has a longer-lasting effect characterized by increased production of IgM, IgG2a, IgG3 as well as of IgG1. The in vivo data were complemented by in vitro proliferation studies carried out on spleen cells or macrophage-depleted spleen cells obtained from mice sensitized in vivo and rechallenged in vitro on day 4 following sensitization. Rechallenge was carried out in the absence or presence of anti-IL1. The data indicate that, in contrast to what is generally observed in vivo, liposomes alone potentiate spleen cell proliferative response in a dose-dependent manner. This liposomal effect totally obscures the antigen-specific proliferation that was expected with encapsulated antigen without masking that induced by surface-linked antigen. The mode of antigen association also influences anti-cytokine responsiveness as demonstrated by the insensitivity of the surface-linked antigen response to the presence of anti-IL1 and the significantly decreased response observed with encapsulated antigen under identical conditions. The response to both liposomal antigenic formulations was almost totally abolished in adherent cell-depleted cultures. The overall results therefore suggest that encapsulated and surface-linked antigens activated different immune pathways.

Human HE2 (microB) and microA motifs show the same function as whole IgH intronic enhancer in transgenic mice.

In the murine IgH gene intronic enhancer (ENHiH), two major functional domains were reported. One is the E4/octomer region and another includes the microA and microB motifs. In the human ENHiH, it was reported that the HE2, which corresponds to the murine microB, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined the in vivo function of the E6, microA and HE2 motifs within the human ENHiH by using the transgenic mice technique. The microA and HE2 motifs together revealed almost the same enhancer function as the whole human ENHiH, but the E6 motif had lesser enhancer activity and tissue-specificity.