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Ovotransferrin (OVT) is a multifunctional protein related to lactoferrin (LF), sharing similar characteristics and considered a cost-effective alternative. Researchers were intrigued by the differences in iron saturation between native-LF and native-OVT, but whether iron saturation affected the cost-effectiveness of the ligand of OVT compared to LF was still uncertain. This study investigated the structure, physicochemical properties, and potential functions of an OVT with varying iron saturation levels, aiming to clarify the impact of iron saturation on an OVT. The findings showed that increased iron saturation altered the structure of an OVT, leading to changes in its physicochemical properties, such as larger particle size and better thermal stability. The findings from peptidomics indicated that iron saturation affects the resistance of OVT to digestion, resulting in the generation of peptides with distinct molecular weights and diverse potential functionalities. Overall, this study provided evidence to support that iron saturation was a limiting factor for the functional performance of the OVT.

As the demand for lactoferrin increases, the search for cost-effective alternative proteins becomes increasingly important. Attention naturally turns to other members of the transferrin family such as ovotransferrin. The iron-binding abilities of these proteins influence their characteristics, although the underlying mechanisms remain unclear. This overview systematically summarizes the effects of the iron-binding ability on the fate of food-derived transferrins (lactoferrin and ovotransferrin) and their potential applications. The findings indicate that iron-binding ability significantly influences the structure of food-derived transferrins, particularly their tertiary structure. Changes in structure influence their physicochemical properties, which, in turn, lead to different behaviors in response to environmental variations. Thus, these proteins exhibit distinct digestive characteristics by the time they reach the small intestine, ultimately performing varied physiological functions in vivo . Consequently, food-derived transferrins with different iron-binding states may find diverse applications. Understanding this capability is essential for developing food-derived transferrins and driving innovation in lactoferrin-related industries.

The impact of covalent or non-covalent bound gallic acid (GA) on the formation, physicochemical properties, and digestion of ovotransferrin (OTF) nanofibrils was comprehensively studied. Thioflavin T fluorescence results revealed that bound GA could inhibit OTF nanofibrillation and that the fibril-inhibitory activity of bound GA was dose dependent. Covalent bound GA exerted stronger inhibition on OTF nanofibrillation than an equal amount of non-covalent bound GA. Atomic force microscopy revealed that covalent bound GA shortened OTF nanofibrils significantly, while non-covalent bound GA did not change the contour length of OTF fibrils remarkably. Bound GA altered diameter of OTF nanofibrils. Both covalent and non-covalent bound GA could alter the zeta potential, surface hydrophobicity, and rheological properties of OTF nanofibrils. Bound GA endowed OTF nanofibrils with a strong antioxidant activity. In vitro gastrointestinal digestion results showed that covalent bound GA elevated the fibril digestion rate better than non-covalent bound GA. Polyphenol binding provided a new approach to modulating the physicochemical properties of protein nanofibrils.

The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal proliferation, oxidative stress, and inflammation in vascular smooth muscle cells (VSMCs) that contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on VSMCs are still unclear. In the present study, we evaluated the antiproliferative, antioxidant, and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production, and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-κB pathway. These findings could further our understanding on the antihypertensive mechanism of IRW beyond vascular endothelium.

Oxygen radical absorbance capacity (ORAC) is a widely used method of measuring antioxidant capacities of various antioxidant components. Surprisingly, 16 antioxidant peptides previously identified from egg protein ovotransferrin using the ORAC method did not show any anti-inflammatory and antioxidant activities in cells. After simulated gastro-intestinal digestion (GID), several peptide digests significantly reduced the expression of tumor necrosis factor-α (TNF-α)-induced pro-inflammatory intercellular cell adhesion molecule-1 (ICAM-1) by 65.7 ± 10.4% and vascular cell adhesion molecule-1 (VCAM-1) by 53.5 ± 9.6% to 61.0 ± 14.5%, but only GWNI reduced TNF-α-activated superoxide generation by 71.0 ± 12.9% when tested with dihydroethidium (DHE) assay. Mass spectrometer analysis identified two new peptides, GWN and GW, in the GWNI digest; however, only GW reduced TNF-α-induced VCAM-1 expression (64.3 ± 20.6%) significantly compared to the TNF-α treated cells. Our study suggested that ORAC lacked biological relevance in assessing bioactive peptides.

Species-specific ovotransferrin features a highly conservative protein sequence, but it varies in the structure of the attached oligosaccharides, which may contribute to the differences observed in its bioactivity and nutritional value. Herein, chicken ovotransferrin (COT) and pheasant ovotransferrin (POT) isolated by repeated ethanol precipitation of egg white were digested with peptide N-glycosidase F to release N-glycans. The obtained N-glyans were isotopically labeled with aniline and analyzed via electrospray ionization mass spectrometry and online hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS). Relative quantitation based on isotopic aniline labeling and HILIC-MS/MS analysis revealed in detail the conspicuous difference between COT and POT in the abundance of their N-glycan compositions and isomers. In total, 16 COT N-glycans were observed, including 1 core structure (3.18%), 3 hybrid type (5.42%), and 12 complex type (91.40%), whereas 21 POT N-glycans were found, including 1 truncated structure (1.88%), 1 core structure (6.26%), 3 high mannose type (5.20%), 6 hybrid type (19.14%), and 10 complex type (67.52%). To our knowledge, this study is the first qualitative and quantitative comparison of COT and POT N-glycosylation patterns. These results suggest that POT has a different glycosylation pattern compared to that of COT and thus the effect of its glycosylation pattern on its bioactivity is worthy of further exploration.

Ovotransferrin (OTF), representing 12-13% of the total egg white, is a member of transferrin family with antimicrobial and antioxidant activities. Catechin is a polyphenolic antioxidant found in green tea. The objective of the study was to conjugate ovotransferrin with catechin to improve the antioxidant activity of OTF. Conjugates were prepared either by the free radical method using hydrogen peroxide-ascorbic acid as the initiator or by the alkaline method at pH of 9.0. The oxygen-radical-scavenging effect was increased from 3.95 mol trolox equivalent (TE)/mol of ovotransferrin to 22.80 and 17.14 mol TE/mol sample, respectively, in radical and alkaline prepared conjugates, which indicated that conjugation with catechin is an effective way to improve antioxidant activity of the protein. Conjugation between ovotransferrin and catechin was analyzed by fluorescence analyses, ultra performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography coupled online to a tandem mass spectrometer. Catechin was covalently bound to lysine (residues 327) and glutamic acid (residues 186) in ovotransferrin. The ovotransferrin-catechin conjugate may have a potential application as a functional food and nutraceutical ingredient.

Egg protein ovotransferrin derived peptides (IRW and IQW) can attenuate tumor necrosis factor (TNF) induced inflammatory responses and oxidative stress in endothelial cells. The present study investigates the structural requirements and molecular mechanisms underlying these events. Whereas IRW significantly inhibited TNF-induced up-regulation of intercellular cell adhesion molecule-I (ICAM-1) and vascular cell adhesion molecule-I (VCAM-1), IQW could inhibit only the up-regulation of ICAM-1. The anti-inflammatory effects of these peptides appeared to be mediated by the nuclear factor-κB (NF-κB) pathway, which was differentially regulated by IRW and IQW. Both IRW and IQW exhibited antioxidant effects as shown by reduction of TNF-induced superoxide generation. The structural integrity of these peptides was essential for their activities, because dipeptides or the combination of constituent amino acids did not exhibit the same effect. This study demonstrated the significance of the structural integrity of these two tripeptides in attenuating endothelial inflammation and oxidative stress, indicating their potential as nutraceuticals.

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.

Ovotransferrin is a glycoprotein well-known for its iron-binding property. Ovotransferrin was reported to have antioxidative properties, but the presence of antioxidant peptides within the protein has not been reported. The purpose of the study was to characterize the antioxidant peptides within ovotransferrin. Ovotransferrin was sonicated and hydrolyzed by thermolysin, and peptides from the hydrolysate were fractionated by ion-exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Fourteen peptides derived from ovotransferrin were characterized using LC-MS/MS, and their oxygen radical absorbance capacity (ORAC) values were determined using synthetic peptides. Two tetrapeptides (Trp-Asn-Ile-Pro and Gly-Trp-Asn-Ile) showed the highest antioxidant activity. Interestingly, the addition of amino acid residues to either the N or C terminus of the two peptides decreased the antioxidant activity, suggesting that the motif of Trp-Asn-Ile is responsible for the high antioxidant activity.

Proteins of avian egg albumin have been suggested to play various biological roles during the development of chick embryo to confer protection. Recently, we have shown that ovotransferrin (OTf), the second major protein in egg albumin, undergoes thiol-linked autocleavage at distinct sites upon reduction. This study explores the physiological significance of OTf autocleavage by examining the effect of the reduced autocleaved OTf (termed rac-OTf) on modulation of cell proliferation, lethality, and apoptosis in two human cancer cell lines, colon cancer (HCT-116) and breast cancer (MCF-7). The rac-OTf was prepared by reduction of OTf with a non-thiol reductant (TCEP), to avoid reductive alkylation and produce highly soluble fragments. Unlike OTf, rac-OTf remarkably inhibited the proliferation of cancerous MCF-7 and HCT-116 cells in a dose-dependent manner, with the greatest effect on HCT-116, but had no effect on normal human mammary epithelial cells (HMEC). Cytofluorometric and trypan blue exclusion analyses indicated that rac-OTf exhibits cytotoxicity to HCT-116 in a dose-dependent fashion. The cytotoxic mechanism of rac-OTf against cancer cells was found to be induction of apoptosis as judged by changes in cell morphology, annexin-V binding, collapse of mitochondrial membrane potential, and caspase-9 and -6 activation, indicating the involvement of the mitochondrial pathway. This finding is the first to describe the reduction-dependent autocleaved OTf as an anticancer molecule, providing insights into a novel physiological function of OTf, suggesting its therapeutic potential in the treatment of human cancers and health benefit in nutraceuticals.

The effects of alphas-casein on heat aggregation of ovotransferrin (OT) were studied by heating at 80 degrees C for 20 min in 10 mM phosphate buffer, pH 7.0. The heat interactions between alphas-casein and OT were followed by turbidity development and polyacrylamide gel electrophoresis. We found that alphas-casein can effectively suppress the heat-induced aggregation of heat-labile OT. The suppressive ability of alphas-casein was reduced by the presence of NaCl on heating. Dephosphorylated alphas-casein had less ability to suppress the aggregation of OT than native alphas-casein. Our results indicate that alphas-casein interacts with the heat-denatured OT through its exposed hydrophobic surface and phosphoserine residue. Such interactions seem to be important in helping to suppress the aggregation of heated OT. The suppressive effects of alphas-casein on heat aggregation of OT would be partially ascribed to the formation of transparent gel from egg white by the addition of alphas-casein., (Copyright 2004 American Chemical Society)

Structural modifications of ovalbumin, ovotransferrin, and lysozyme at the air-water interface have been investigated using SDS-PAGE, both intrinsic and ANS fluorometry, and circular dichroism experiments. Ovalbumin contact with an interface induced an exposure of aromatic residues, a slight decrease in alpha-helix structures (-1.7%), and an increase in both beta-sheet (+3.4%) and beta-turn (+7.9%) structures. Moreover, these conformational changes led to the formation of insoluble polymers of ovalbumin through intermolecular disulfide bonds. Ovotransferrin contact with an interface led to an increase in its surface hydrophobicity (+30%) and modifications of its secondary structure (-33% of alpha-helices, +96.4% of beta-sheets, +13.2% of beta-turns, and +21.2% of random coils), characteristic of major conformational changes. On the other hand, lysozyme did not undergo any structural modification. These results clearly underscore that at the air-water interface proteins are susceptible to denaturation.

Ovalbumin (OVA) was aggregated stepwise by dry heating at 120 degrees C with a gradual increase in its heating times (10 min-6 h). The inhibiting effects of DHOVAs (OVAs dry-heated for various times up to 6 h) on the heat coagulation of ovotransferrin (OT) were studied. DHOVAs and OT were solubilized at 5% (w/w) concentration with 10 mM sodium phosphate buffer, pH 7.4. Their solutions were mixed at the volume ratio of 1:1 and reheated at 60 degrees C for 3. 5 min. Some remarkable differences according to dry-heating time were observed: coagulum formations were greatly inhibited in the solutions mixed with DHOVAs treated for more than around 2.0 h, with decreasing turbidity as dry-heating time increased. In addition, the effects of reheating time and temperature, as well as those of pH and ionic strength, were also examined on coagulum formation and turbidity development in connection with dry-heating time. Thus, the inhibiting effects of dry-heated egg white on the heat coagulation of fresh egg white previously described were confirmed on the molecular level of OVA and OT.