Ovalbumin Structural Changes by Phenolic Compounds Interactions

Ovalbumin (OVA) is the most prevalent protein in egg white and, represents the major allergen from avian egg white that causes IgE-mediated food allergic reactions particularly in children. It has been shown that phenolic compounds interact with proteins by a single or multipoint mechanism, promoting structural and functional changes. Moreover, the interaction of some allergens with polyphenols, led to permanent modification of the tertiary structure of the allergen, which can result in a reduction in its IgE-binding capacity. This work aimed to analyse the effect of phenolic compounds (Gallic, Caffeic, Ferulic, Chlorogenic and Tannic Acids, Resveratrol and Quercetin) on the native structure of OVA, using Circular Dichroism (CD), fluorescence and Fourier transform infrared spectroscopy (FTIR). The phenolic compounds were incubated with OVA at different times, concentrations and temperatures. Changes in OVA secondary and tertiary structures were increasingly induced with increasing temperatures by the phenolic compound. Also, for a constant temperature, the changes found in OVA structure increased with the phenolic compounds concentration. The results show that the interactions between phenolic compounds and OVA result in complexes (phenolics – OVA) where OVA native structure is changed. This is likely to affect epitopes and hence OVA allergenicity.N/

Interactions of Phenolic Compounds with Ovalbumin

Ovalbumin (OVA) is the major protein in egg white and can cause allergy mainly in infants and young children [1]. Egg allergy is an IgE mediated reaction and is one the most common food allergies. So far, the avoidance of the egg has been the unique way to prevent this allergy [2]. It is well known that phenolic compounds can bind to proteins promoting structural and functional changes. In this work, the interactions between OVA and the phenolic compounds were studied through spectroscopic techniques (fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR)) and docking. OVA solutions were incubated at different temperatures and times, with different phenolic compounds prepared with the same buffer solution (Gallic, Caffeic, Ferulic, Chlorogenic and Tannic Acids, Resveratrol and Quercetin). Results indicate that OVA's structure was affected by the binding of phenolic compounds. CD and FTIR experiments showed changes in the secondary structure of OVA, originated by the conversion of α-helix into β-sheets [1]. Fluorescence spectra demonstrated that phenolics are quenchers of fluorescent aminoacids (Tyrosine, Phenylalanine and Tryptophan) meaning that interactions occur directly or near these aminoacids. Fluorescence results also suggest that these interactions are electrostatic and thermodynamically favorable (∆G<0). Docking studies showed that the tested phenolic compounds can interact directly with OVA epitopes, or with its neighbors, thus avoiding the IgE binding. Therefore, the phenolic compounds can be used as a strategy for reducing egg allergy in foods.info:eu-repo/semantics/publishedVersio

Phenolic compounds: a novel approach to reduce egg allergenicity?

Hen's egg allergy has been climbing in all countries due to eggs ubiquity in foodstuffs. This allergy is an IgE mediated reaction that affects mainly infants and young children. So far, the processes used to decrease the allergenicity of egg proteins, such as cooking, thermal processing, storage and enzymatic digestion have not been totally effective. Previous studies demonstrated that proteins form complexes with phenolic compounds, so the aim of this work was to analyze the effect of these compounds in ovalbumin (OVA) conformation and its possible application to reduce eggs allergenicity. OVA was incubated at different temperatures with phenolic compounds (caffeic, chlorogenic, ferulic, gallic and tannic acids; resveratrol and quercetin) and was analyzed by circular dichroism (CD), Attenuated Total Reflection–Fourier Transform Infra-Red (ATRFTIR) spectroscopy and fluorescence. Changes in the secondary structure of OVA were evidenced by CD and ATR-FTIR. Also, protein fluorescence decreased with increasing concentrations of phenolic compounds. The thermodynamic analysis suggested that electrostatic interactions are important in the binding process, and the quenching mechanism occur by contact. This was confirmed by docking where the phenolic compounds bind specifically to some regions of the protein, including those with the allergenic epitopes.info:eu-repo/semantics/publishedVersio

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases

Funding Information: This project was supported by the University of Beira Interior— Health Sciences Research Centre (CICS-UBI) supported by FEDER funds through the POCI—COMPETE 2020—Operational Programme Competitiveness and Internationalisation in Axis I—Strengthening research, technological development, and innovation Project (POCI-01- 0145-FEDER-007491). CNB-CSIC proteomics lab is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I +D+i 2013–2016, funded by ISCIII and FEDER. Publisher Copyright: Copyright © 2023 Santos, Ciordia, Mesquita, Cruz, Sousa, Passarinha, Tomaz and Paradela.Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD.  Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4).  Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).publishersversionpublishe

Interactions of phenolic compounds with ovalbumin: a spectroscopic approach

Ovalbumin (OVA) is the major protein in egg white and can cause allergy mainly in infants and young children. Egg allergy is an IgE mediated reaction and is one the most common food allergies. So far, the avoidance of the egg has been the unique way to prevent this allergy. It is well known that phenolic compounds can bind to proteins promoting structural and functional changes and, phenolic compounds are been recognized as having a potent antioxidant, anti-inflammatory and antitumoral activity. In this work, the interactions between OVA and the phenolic compounds were studied through spectroscopic techniques (fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR)) and docking. spectroscopy (FTIR)) and docking.MESCTI and INAGBE-Angolainfo:eu-repo/semantics/publishedVersio

Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation

The deeper understanding of retinal detachment (RD) pathogenesis may improve the visual outcome after surgery. Given the main role of the vitreous in retinal eye diseases, two strategies were explored to identify its proteome in RD. Fractionation techniques such as anion exchange chromatography (IEX) and SDS-PAGE combined with MALDI-TOF/TOF analysis allowed to identify 127 proteins in vitreous of RD patients. From these proteins, 19 were identified using only the IEX fractionation strategy, and 117 using a bidimensional (IEX and SDS-PAGE) fractionation. Of these proteins, 68 had not yet been found in other vitreous proteomic studies. The fractionation with IEX and SDS-PAGE largely improved the number of identified proteins proving that it is crucial to combine several methodologies to cover vitreous proteome.CENTRO-07-ST24-FEDER-002014;Novartis Farma-Produtos Farmacêuticos, SA; Project POCI-01-0145-FEDER-007491info:eu-repo/semantics/publishedVersio

VEGF-B Levels in the Vitreous of Diabetic and Non-Diabetic Patients with Ocular Diseases and Its Correlation with Structural Parameters

Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples (n = 33) from diabetic (n = 25) and non-diabetic (n = 8) patients. Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL ) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (μm) was positive and moderate: rs = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm3) was positive and robust: rs = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications

Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices.This project was supported by the University of Beira Interior—Health Sciences Research Centre (CICS). Santos FM received a fellowship (CENTRO-07-ST24-FEDER-002014) and a doctoral fellowship (SFRH/BD/112526/2015) from FCT. Gaspar LM received a fellowship from Novartis Farma-Produtos Farmacêuticos, SA. This work is supported by FEDER funds through the POCI—COMPETE 2020—Operational Programme Competitiveness and Internationalisation in Axis I—Strengthening research, technological development and innovation Project (POCI-01-0145-FEDER-007491) and National Funds by FCT—Foundation for Science and Technology Project (UID/Multi/ 00709/2013). This work was also supported by the Applied Molecular Biosciences Unit- UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). CNB-CSIC proteomics lab is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I +D+i 2013–2016, funded by ISCIII and FEDER