Chicken Ovotransferrin Research Articles

Sequence comparison of the lactoferrin of various animal species and its prospects as an animal feed additive

Lactoferrin as a feed additive is worth to be explored. However, reports on its application are contradictory, which might be associated with the genetic relatedness. Here we compare the lactoferrin nucleotide and its deduced amino acid sequence. Sequence data for lactoferrin of various animal and human were download from GenBank. The phylogenetic relatedness was inferred and the amino acid sequences were aligned to identify conserved and polymorphic sites. The protein three-dimension structures were estimated using online software. The result showed the lengths of lactoferrin, lactotransferrin or ovotransferrin are 703–711 residues. The phylogeny showed that the lactoferrins of buffalo, cow, goat, pig, camel, and horse formed one group; monkey, human, and gorilla formed a second group; and dog and cat formed a third group. Chicken ovotransferrin was an outgroup. Genetic distances between groups were 0.242–0.061, while smallest span between taxa was 0.016 (human to gorilla) and the highest was 0.612 (chicken to goat). The conserved residues spanned from the amino terminus to the carboxy terminus. There are 27 conserved cysteine residues. N-link glycosylation of the “NXS” and “NXT” motives of lactoferrin diverge between species. A species specific or group specific lactoferrin supplement should be beneficial to animal production.

Functional properties of ovotransferrin from chicken egg white and its derived peptides: a review

With emerging trends in the food and pharmaceutical industries, potential applications of egg-derived bioactive compounds were recognized. Ovotransferrin is a major egg white functional protein responsible for multiple bioactivities. The objectives of this review are to provide scientific evidence of the functional properties of chicken ovotransferrin and its derived peptides and to identify future research approaches and applications. Various easy, economical, and non-toxic methods have been reported to produce ovotransferrin with high yield and purity, and chemical and enzymatic approaches have been employed to release bioactive peptides. The native ovotransferrin is known to have antimicrobial, antioxidant, anticancer, and immunomodulatory activities. The peptides produced from ovotransferrin also are reported to have antioxidant, antimicrobial, antihypertensive, and anticancer properties. However, little or no application of these compounds in the food and pharmaceutical areas is available yet. Therefore, the practical application of OTF in nutraceutical and pharmaceutical areas are among the emerging areas of research.

Chicken Ovotransferrin Variants OTFB and OTFC Harboring Substitution of GAT (Asp) to AAT (Asn) in the Codon 500 and their Antimicrobial Activity.

Chicken egg white ovotransferrin (OTF) has been reported to exist in three electrophoretic variants (OTFA, OTFB and OTFC). In this report, we identified a causal polymorphism between the OTFB and OTFC variants in Japanese and Taiwanese native chickens and compared the antibacterial activity between these two variants. The cDNA sequence analyses from Satsumadori oviducts revealed that three non-synonymous SNPs T1809G (Ser52Ala), A2258G (Ile96Val) and G7823A (Asp500Asn) corresponded to the OTF electrophoretic phenotypes. Of the three SNPs, the G7823A mutation perfectly corresponded to the electrophoretic phenotypes OTFB (G/G, Asp500Asp), OTFB/C (G/A, Asp500Asn) and OTFC (A/A, Asn500Asn) in three chicken populations. The variants OTFB and OTFC exhibited similar antibacterial potency against Gram-positive and Gram-negative bacteria. This study provides, for the first time, molecular information on polymorphism of OTFB and OTFC variants of chicken ovotransferrin and its effect on the antimicrobial activity of the respective variants.

Purification and characterization of tinamou egg white ovotransferrin as an antimicrobial agent against foodborne pathogenic bacteria

Chilean tinamou (Nothoprocta perdicaria) egg white ovotransferrin was purified with anion-exchange Fast Protein Liquid Chromatography and identified by peptide mass fingerprinting. Tinamou egg white ovotransferrin was compared to chicken (Gallus gallus) and emu (Dromaius novaehollandiae) ovotransferrin by SDS-PAGE analysis. Though all three species contained similar molecular weight ovotransferrins, tinamou egg white contained 20% more ovotransferrin than chicken, but 30% less than emu.The effectiveness of tinamou ovotransferrin as a natural antimicrobial agent was compared to chicken ovotransferrin against two food related pathogens, Escherichia coli O157:H7 and Staphylococcus aureus. The bacteriostatic and bactericidal activities of the native, apo and holo forms of the proteins were dependent on the presence of 50mM bicarbonate and protein iron saturation. Native ovotransferrins were the most effective at a concentration of 10mg/ml with 50mM bicarbonate, exhibiting bacteriostatic and bactericidal activities against both pathogens. Holo ovotransferrins exhibited moderate antimicrobial activity in the presence of bicarbonate. Different antimicrobial activities were observed between chicken and tinamou ovotransferrins, suggesting tinamou ovotransferrin may possess unique antimicrobial motifs that warrant further investigation of its amino acids composition. Further, our results suggest that tinamou ovotransferrin may represent a natural antimicrobial agent suitable for use in food matrices or on food preparation surfaces.

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 2409 nt (ORF: 2106 nt encoding a 701-amino-acid polypeptide; AJ786651), 2418 nt (ORF 2118 nt encoding a 705-amino-acid polypeptide; AJ786650), and 2397 nt (ORF: 2118 nt encoding a 705-amino-acid polypeptide; AJ966469) were determined for ostrich (OtrF), red-eared turtle (TtrF), and turkey (MtrF) ovotransferrin, respectively.

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T- ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.

Identification of Ovotransferrin as an Acute Phase Protein in Chickens

Inflammation is homeostatic process associated with a variety of cellular injuries resulting from infections, toxicosis, and physical trauma. The studies on inflammation in avian species are limited. To understand the inflammation-induced changes, 4-wk-old male broiler chickens were subjected to experimental inflammation by a subcutaneous injection of croton oil (inflammatory) with changes in serum measured over time and were compared with birds treated similarly with olive oil (injected control). Croton oil treatment significantly elevated serum interleukin (IL)-6 concentrations and heterophil counts by 6 and 16 h postinjection, respectively, which returned to the basal levels of controls at 16 and 24 h, respectively. Croton oil treatment affected the serum protein profiles of chickens as assessed by SDS-PAGE and densitometric analyses. Compared with olive oil-injected or noninjected chicken sera, there were increases in the density of protein bands corresponding to molecular weights (MW) of 42, 65, 200, and 219 kDa and decreases in bands corresponding to 49 kDa (serum albumin) and a 56-kDa protein in chickens treated with croton oil. Most of these changes were evident at 24 h and lasted through 48 h. The protein band corresponding to 65 kDa was further characterized using two-dimensional gel electrophoresis and N-terminal sequence analyses. A sequence similarity search in the Genbank database using the first 22 amino acids yielded a complete homology with chicken ovotransferrin. Western blot analysis using antichicken serum transferrin or antichicken ovotransferrin antibodies also confirmed the 65-kDa protein band to be ovotransferrin. Under nonreducing conditions, the ovotransferrin standard also showed an apparent MW corresponding to 65 kDa, like the serum transferrin. The serum ovotransferrin was found to be glycosylated using a glycoprotein stain. Although the significance of ovotransferrin in avian inflammation is not clear, these results show that it is a major acute phase protein (APP) in chickens.

Inhibition of proliferative responses of mouse spleen lymphocytes by lacto‐ and ovotransferrins

This study evaluated the influence of three transferrin family proteins (bovine lactotransfer‐rin, bLf; chicken ovotransferrin, cOf; quail ovotransferrin, qOf) at concentrations ranging from 10 to 50 fig ml‐1 on proliferative responses of mouse spleen lymphocytes stimulated by lipopolysaccharide (LPS) and phytohemagglutinin (PHA). The transferrins significantly inhibited both the mitogen‐induced proliferation in a dose‐dependent manner. Ovotransferrins, especially qOf, had cytotoxicity for the lymphocytes. There is no relationship between iron saturation and inhibitory effect of each transferrin or cytotoxicity of ovotransferrins. All these transferrins bound to CD4+ T‐lymphocytes, CD8+ T‐lymphocytes and B‐lymphocytes, but no transferrin conjugated with PHA. PHA bound to lymphocytes previously incubated with each transferrin. These results indicate that the inhibition of proliferation by the transferrins and the cytoxicity of ovotransferrins are not attributable to a function of iron transport and to inactivation of mitogen, suggesting that the hyporesponsiveness is due to interaction of lymphocytes and/or accessory cells with each transferrin.

Induction of antigen-presenting capacity in tumor cells upon infection with non-replicating recombinant vaccinia virus encoding murine MHC class II and costimulatory molecules

The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate ‘artificial’ APC able to stimulate CD4 + T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-A k and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-A k alone, pulsed with hen egg white lysozyme peptide (HEL 46–61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4 + T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-A k and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin 133–145), induced a significantly higher response in a specific Th 2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-A k molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.

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.

Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins.

A rapid and sensitive technique, involving difference spectral titration with cobalt(III), to measure the epsilon values of chicken ovotransferrin, human serum transferrin, the N-lobe of human transferrin and several single point mutants is reported. The resulting epsilon values were compared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411-2423]. The titrations with cobalt feature sharp break-points and do not destroy the protein samples. The choice of buffer was found to be important, depending on the metal-binding avidity of the proteins. Cobalt titration should prove useful for studying the comparative metal-binding properties of transferrin and mutants of transferrin being generated by recombinant technology.

Monoclonal antibodies to chicken ovotransferrin: epitopic and phylogenetic analysis

1. The ability of four domain-specific anti-chicken ovotransferrin antibodies to bind to turkey, quail, duck, pheasant and goose ovotransferrins was examined directly by an enzyme-linked immunoassay and indirectly in a competitive radioimmunoassay. 2. The ability of these same ovotransferrins to compete with radioiodinated chicken ovotransferrin for binding to transferrin receptors on chick embryo red blood cells was also tested. 3. The hypothesis that evolutionarily conserved determinants involved in receptor recognition can be predicted by monoclonal antibodies that block binding of ovotransferrin to receptor was found to be incorrect.

Thallium-205 and carbon-13 NMR studies of human sero- and chicken ovotransferrin.

We have examined the binding of Tl3+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by 205Tl and 13C NMR spectroscopy. With carbonate as the synergistic anion, one observes two 205Tl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of each protein. When the same adducts are prepared with 13C-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the 13C NMR spectrum of serotransferrin; these correspond to the labeled anion directly bound to the metal ion in both sites of the protein. The analogous resonances in ovotransferrin are completely degenerate, and only one doublet can be detected. The magnitudes of the spin-spin coupling between the bound metal ion and carbonate range from 2J(205Tl-13C) approximately 270 to 290 Hz. We have used the proteolytic half-molecules of ovotransferrin and the recombinant N-terminal half-molecule of serotransferrin to assign the 205Tl and 13C NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that Tl3+ is bound with a greater affinity at the C-terminal site of serotransferrin, whereas no site preference can be noted for ovotransferrin. When oxalate is used as the anion instead of carbonate, the 205Tl NMR signals arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex pattern of resonances is observed for bound 13C2O4(2-) in the 13C NMR spectra of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

A Scandium-45 NMR Study of Ovotransferrin and Its Half-Molecules

The binding of Sc 3+ to chicken ovotransferrin has been investigated by 45 Sc and 13 C NMR spectroscopy. In the presence of carbonate, one observes two 45 Sc and 13 C signals which can be assigned using the proteolytic half-molecules of ovotransferrin to bound Sc 3+ and 13 CO 3 2- in both metal ion binding sites of the protein. When the synergistic anion is changed to oxalate, two overlapping 45 Sc resonances are again detected

Change in glycosylation of chicken transferrin glycans biosynthesized during embryogenesis and primary culture of embryo hepatocytes.

Transferrins were isolated by immunoaffinity chromatography from chicken serum, chicken embryo serum and from the culture medium of chicken embryo hepatocytes in primary culture. The glycovariants of these three transferrins were separated by ion-exchange chromatography using a fast protein liquid chromatography (FPLC) system. The structures of the oligosaccharide-alditols released by hydrazinolysis from the glycovariants were compared after analysis by a combination of methanolysis, methylation analysis and 1H-NMR spectroscopy. In the three transferrins analysed, the oligosaccharides were of the biantennary N-acetyllactosaminic type, having several prominent features. In particular, the embryo serum transferrin glycan differed from that of chicken serum transferrin by the presence of a bisecting N-acetylglucosamine, suggesting a developmental change in glycosylation. The glycan structure of the transferrin secreted by the embryo hepatocytes in primary culture was marked by the presence of fucose (alpha 1-6) linked to the core N-acetylglucosamine, suggesting that expression of the fucosyltransferase activity is dependent on cell culture conditions. Moreover, comparative analysis of chicken serum transferrin and ovotransferrin glycans reinforces the idea that the glycosylation of two identical polypeptide chains is organ specific.

Electron spin resonance study of Gd(III) ions bound to iron binding sites in chicken ovotransferrin

Electron spin resonance (ESR) of Gd(III) ions bound to the two iron binding sites (amino and carboxy sites) in chicken ovotransferrin was studied to investigate the differences between them. From the analysis of the ESR spectrum and its concentration dependence on Gd(III), it is concluded that the symmetry of the ligand field is almost the same for both binding sites and that the magnitude of the carboxy site is larger than that of the amino site.

ニワトリ・オボトランスフェリンに結合したＧｄ（ＩＩＩ）の電子スピン共鳴

Electron spin resonance of Gd(I11) bound to the two iron specific binding sites (N- and Csites)of chicken ovotransferrin was studied to investigate the difference between these binding sites. From the analysis of the electron spin resonance data and its Gd(III) concent ration dependence, we concluded that the symmetry of the ligand field (E and D) is almost the same for two sites and that the ligand fields (E and D) of the C-site are larger than those of the N-site.

Structural organization of the mouse lactoferrin gene

The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encoded by a single copy gene of approximately 30 kilobases (kb) in size. The gene is organized into 17 exons separated by 16 introns. The exons range in size from 48 base pairs (bp) to 190 bp whereas the introns range from 0.2 kb to 4.3 kb. Structural analysis of the mouse lactoferrin gene reveals that this gene shares a similar intron-exon distribution pattern with both human transferrin and chicken ovotransferrin.

X-ray solution scattering reveals conformational changes upon iron uptake in lactoferrin, serum and ovo-transferrins

X-ray solution scattering has been used for studying the structural changes that take place upon uptake and release of iron from serum and chicken ovo-transferrin and human lactoferrin. In the case of chicken ovo-transferrin, data have been obtained for both the intact protein and the isolated N and C-lobes with and without iron. These studies reveal that both lobes undergo a change that is consistent with an opening of the inter-domain cleft when iron is removed from the protein. We suggest that the conformational change of the protein increases the specificity of receptor binding and that the closed configuration of the iron-loaded protein is one, or perhaps the, decisive step in the mechanism for receptor-mediated endocytosis.

X.a.f.s. studies of chicken dicupric ovotransferrin.

A comparison of Cu K-edge x.a.f.s. spectra with that of the equivalent Fe K-edge for chicken ovotransferrin (COT) indicates that the metal ions occupy essentially the same binding sites in the protein. However, in the case of the Cu2+ complex the metal appears to have reduced co-ordination. Changes are observed in the x.a.f.s. of 90%-saturated COT (Cu1.8COT) on freeze-drying. The three-dimensional X-ray structures of rabbit serum transferrin and human lactoferrin have shown that the ferric cations are co-ordinated by four protein ligands and a bidentate carbonate anion in a distorted octahedral arrangement [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1768-1774; Anderson, Baker, Norris, Rice and Baker (1989) J. Mol. Biol. 209, 711-734; Bailey, Evans, Garratt, Gorinsky, Hasnain, Horsburgh, Jhoti, Lindley, Mydin, Sarra & Watson (1988) Biochemistry 27, 5804-5812]. This structural information, together with the differences in e.x.a.f.s. spectra for solution and freeze-dried samples of diferric COT [Hasnain, Evans, Garratt & Lindley (1987) Biochem. J. 247, 369-375] suggests that the synergistic carbonate anion may be capable of behaving as a unidentate linkage to the Cu2+ in the dicupric complex. Data for Cu1.8COT are consistent with only three protein ligands bound to Cu2+, monodentate binding of the synergistic anion in one lobe and its bidentate binding in the other lobe. Such flexibility in the anion co-ordination may be a requirement for the uptake and release of metals by the transferrins.