Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 11 (november 2021) : 1286-1292

The Effect of Non-enzymatic Glycosylation on the Physicochemical Properties and Immunoreactivity of Chicken Egg Ovomucoid

Lyayla K. Bupebayeva2, Miciñski Bartosz3, Miciñski Jan1,*, Milewski Stanisław1
1Department of Sheep and Goat Breeding, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-719 Olsztyn, Poland.
2Department Technology of Livestock Production, Kazkh National Agrarian University, Abaja 8, Almaty, Kazakhstan.
3Department of Clinical Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-719 Olsztyn.
Cite article:- Bupebayeva K. Lyayla, Bartosz Miciñski, Jan Miciñski, Stanisław Milewski (2021). The Effect of Non-enzymatic Glycosylation on the Physicochemical Properties and Immunoreactivity of Chicken Egg Ovomucoid . Indian Journal of Animal Research. 55(11): 1286-1292. doi: 10.18805/ijar.B-1232.

ABSTRACT

Background: Research studies conducted with the involvement of various molecular techniques have demonstrated that ovomucoid, a protein found in chicken egg whites, is also a major food allergen.  Many studies have shown that about 40% of patients with allergy against egg proteins were sensitive to ovomucoid. Egg whites are popularly used in the world to enhance the nutritional value of food products (as additives in pastry, meats, sauces, salads and creams) and the production and use of eggs in the world is increasing. Their complete elimination from the diet is highly challenging. Ovomucoid comprises 186 amino acids and has antibacterial properties, and inhibits the activity of microbial enzymes. In this experiment, chicken egg ovomucoid was isolated and polyclonal antibodies were obtained by immunizing a rabbit with the ovomucoid solution to analyze the effect of non-enzymatic glycosylation on the physicochemical properties and immunoreactivity of the studied ovomucoid.

Methods: The study analyzed ovomucoid – a protein isolated from the whites of the analyzed eggs. Ovomucoid was isolated by the method proposed by Roy et al. (2003). The next procedure was rabbit immunization. Polyclonal antibodies were obtained by subcutaneously immunizing a male rabbit with a solution of chicken egg glycated or native (a 100 ìg protein dose) in 1 ml PBS with pH 7.2. All measurements were performed in triplicate. The Fisher’s test was conducted to analyze variations in immunoreactivity and changes in glycation degree.

Result: Research has shown that it is needed to research the toxicity of proteins, in this case ovomucoid. Glycation may be recommended as a way to reduce or inhibit immunoreactivity. In case of allergies, such action is necessary and purposeful and it is essential to choose such reaction conditions which do not cause products harmful to human’s health.

INTRODUCTION

Research studies conducted with the involvement of various molecular techniques have demonstrated that ovomucoid (No. P01005 in the UniProt database) (Jain et al., 2009; The UniProt 2015), a protein found in chicken egg whites (Mine and Yang, 2008), is also a major food allergen.  Experiment performed by Aabin et al., (1996) has shown that 38% of patients with allergy against egg proteins were sensitive to ovomucoid. Egg whites are popularly used to enhance the nutritional value of food products (as additives in pastry, meats, sauces, salads and creams) and their complete elimination from the diet is highly challenging (Worobiej et al., 2006). Forecasts indicate that the production and use of eggs in the world is increasing, e.g. in India it is estimated that in 2019-20 may exceed 75 billion eggs (Chaudhari and Tingree, 2015). The ovomucoid allergen is listed under the name Gal d 1 in the IUIS/WHO Allergen Nomenclature (IUIS - International Union of Immunological Societies) and with the Code 359 in the Allergome database, (Mari et al., 2009). Epitopes of chicken ovomucoid were extensively studied and are annotated in the Immune Epitope Database (IEDB) (Vita et al., 2015).
       
Ovomucoid, a serine protease inhibitor, has antibacterial properties and inhibits the activity of microbial enzymes. Ovomucoid is a major food allergen that has an estimated 11% share of all chicken egg white proteins. Its molecular weight is estimated at 28 kDa. Ovomucoid comprises 186 amino acids that form three homologous domains. The structure of the analyzed protein is stabilized by nine disulfide bridges which enable ovomucoid to retain its allergenic properties even after pepsin digestion (Kovacs-Nolan et al., 2000). Zhang and Mine (1999) have demonstrated that a long carbohydrate chain in ovomucoid’s third domain may act as an antigenic determinant aimed against IgE antibodies. According to a different study, the hydrophilic residues of the third domain show greater affinity for Ig whereas hydrophobic residues more easily bind to immunoglobulin E. Epitopes may also include sugar residues that undergo structural modification under high temperatures to alter the immunoreactivity of proteins (Zhang and Mine, 1999; Vita et al., 2010).
       
As a glycoprotein with an acidic pH, ovomucoid contains up to 25% carbohydrates. Sugar chains comprise 14% glucosamine, 7% mannose and around 1% sialic acid (Kovacs-Nolan et al., 2000; Whitton, 2000; Gołąb and Warwas, 2005). In its native form, ovomucoid is highly resistant to the denaturing effects of temperature, more so in an acidic than an alkaline environment. It has also been found to demonstrate pH-dependent antitrypsin activity (Frederico and Deutsch, 1949; Konisi et al., 1985).
       
Glycation is a protein modification process (Cazacu-Davidescu, 2005; Ashraf et al., 2015; Gupta et al., 2016). During the reaction, sugars bind to other biologically active compounds, such as proteins and lipids.
 
In this experiment, chicken egg ovomucoid was isolated and polyclonal antibodies were obtained by immunizing a rabbit with the ovomucoid solution to analyze the effect of non-enzymatic glycosylation on the physicochemical properties and immunoreactivity of the studied ovomucoid.

MATERIALS AND METHODS

The experimental material comprised seven fresh chicken eggs (Gallus domesticus) intended for human consumption purchased in supermarket. The study analyzed ovomucoid- a protein isolated from the whites of the analyzed eggs. Ovomucoid was isolated by the method proposed by Roy et al., (2003). Briefly: The whites of raw eggs were separated from the yolks, homogenized and lyophilized. The resulting 16 g of the material was dissolved in 25 mM citrate starter buffer with pH 5.5. The sample was subjected to ion exchange chromatography in a column filled with SP Sephadex C-25 gel (GE Healthcare). The column was left in a horizontal shaker working at 80 rpm for 1 hour. The top fraction was removed and the column was rinsed with the elution buffer (5% ammonium carbonate with 1 M NaCL, pH 7.4) to remove adsorbed fractions. The column was regenerated by rinsing with the starter buffer three times. The pH of collected fraction was adjusted to 3.5 using 5M sulfuric (VI) acid. One volume of 10% TCA, pH 3.0, was then added and the resulting solution was centrifuged for 15 minutes at 15000 × g at a temperature of 4°C (Eppendorf Centrifuge 5804 R). The ovomucoid was precipitated from the supernatant by addition of ethanol followed by centrifugation as described above. The precipitate was dissolved in a minimal quantity of water and freeze dried. Amounts of chemicals and volumes of solutions were used in proportions recommended by Roy et al., (2003), taking into account larger amount of egg white lyophilizate. Protein purity was verified by SDS-PAGE electrophoresis.
 
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify protein purity at each purification stage and to evaluate changes in ovomucoid’s physical and chemical properties after glycation (Laemmli, 1970). The separation process was carried out at two acrylamide gel concentrations: 4% stacking gel and 12% running gel. Staining was performed with colloidal Coomassie Brilliant Blue R-250. The molecular weight of separated proteins was determined using a Sigma-Aldrich low-range marker (catalogue No. M3913).
 
Non-enzymatic glycation (Cazacu-Davidescu et al., 2005; Bielikowicz et al., 2012) was performed using solutions containing 1 mg/ml of ovomucoid and 1 mg/ml of glucose in 0.2 M PBS (phosphate buffered saline; pH 7.4). The samples were freeze dried and subjected to non-enzymatic glycosylation at 37°C and 60°C for 3, 7 and 14 days. Temperature 37°C corresponds to physiological conditions whereas 60°C to drying process (Falade et al., 2003). Every tested variant comprised an experimental sample and a control sample. A native protein sample was additionally prepared and it was not subjected to any modifications.
 
The content of free amino groups was determined by the method proposed by Nielsen et al., (2001), based on the reaction between o-phthaldieldehyde (OPA) and primary amine groups. 30 µl of the sample and 200 µl of the reagent were transferred to a polystyrene assay plate and left at room temperature for 20 minutes. Absorbance was identified in the ASYS UVM 340 microplate reader at 340 nm wave length.
 
The next procedure was rabbit immunization. Polyclonal antibodies were obtained by subcutaneously immunizing a male rabbit with a solution of chicken egg glycated or native (a 100 μg protein dose) in 1 ml PBS with pH 7.2. Four vaccination doses were administered at three week intervals. The first vaccination involved Freund’s complete adjuvant and incomplete adjuvants were used in the following immunizations.
 
Immunization was directly controlled by the modified competitive ELISA assay. Variations in the immunogenicity of glycated protein were evaluated using the ELISA immunoenzymatic test. The optimal testing conditions were determined. A 96-well polystyrene assay plate (Costar 3591 Medium Binding, Corning Incorporated) was coated with 10 μg/ml native protein solution in a carbonate buffer with pH 9.6, 100 μl per well. The plate was placed in the SKY-LINE incubator (SHAKER DTS-4, ELMI) for 60 minutes at 37°C and stirred at 250 rpm. The plate was once rinsed with 200 μl of 0.5% Tween-PBS buffer. Unbound sites on the plate were blocked with 200 μl of 1% gelatin solution for 60 minutes in the above conditions and the plate was rinsed three times with Tween-PBS. 50 μl of primary antibodies and 50 μl of glycated protein in serial dilutions (0.01 μl/ml to 200 μl/ml) were added. The plate was incubated in identical conditions for 60 minutes and it was rinsed three times with 0.5% Tween-PBS. 100 μl of anti-rabbit secondary antibodies marked with 1:10000 horseradish peroxidase was placed in each well and the plate was incubated for 1 hour. The plate was rinsed four times with Tween-PBS and each well was filled with 100 μl of 0.5% o-phenylenediamine substrate (Sigma-Aldrich) in citrate buffer with 0.06% addition of hydrogen peroxide. The plate was incubated for 30 minutes and the reaction was blocked with 50 μl of 5 M HCl. Absorbance was measured at a wavelength of 492 nm in a microplate reader (ASYS UVM 340) and the results were analyzed in the GraphPad Prism 4 program. The competitive ELISA assay, modified for the needs of this study, was used.
 
All measurements were performed in triplicate. The Fisher’s test was conducted to analyze variations in immunoreactivity and changes in glycation degree.

RESULTS AND DISCUSSION

Each stage of the protein purification process is compared against the molecular weight marker (Sigma-Aldrich, catalogue No. M3913) in Fig 1.
 

Fig 1: SDS-PAGE - protein purification stages (WM - molecular weight marker, WZ - trypsin inhibitor - ovomucoid, 1 - whole chicken egg, 2 - protein isolated by ion exchange chromatography, 3 - 10% TCA - precipitated fraction, 4 - ethanol-precipitated fraction).


 
The high-molecular-weight-fraction (in excess of 66 kDa) in lanes 1, 2 and 3 was identified as ovotransferin. The presence of fractions with molecular weight in the range of 45 to 30 kDa (fraction C) was also determined in lanes 1, 2 and 3. The absence of lysozyme in the analyzed fraction (extract from the entire chicken egg white) and the resulting differences in electrophorogram and chromatogram results could be attributed to the clogging of gel pores and excessive ovalbumin content in the sample (ovalbumin accounts for 50% of total proteins in chicken egg white). A 28 kDa fraction (electrophoretic mobility the same as mobility of 28 kDa standard) which corresponds to the molecular weight of ovomucoid was reported in lane 4.
 
The trypsin inhibitor (Fig 2) and the ethanol-precipitated fraction (Fig 3) were separated by HPLC. Two peaks were reported for the trypsin inhibitor. Peak 1 with retention time of approximately 24 minutes was associated with ovomucoid and peak 2 with estimated retention time of 32 minutes was identified as lysozyme. The purified ovomucoid solution in the ion exchange column revealed one peak. Based on the obtained chromatograms, electrophoresis results and glycoprotein staining, it was identified as pure ovomucoid (peak 1 in Fig 3).
 

Fig 2: Chromatogram of isolated trypsin inhibitor.


 

Fig 3: Chromatogram of purified ovomucoid.


       
The electrophoretic separation of glycated protein vs. native protein is presented in Fig 4. As regards samples incubated at 37°C for 3 and 7 days, no significant differences were determined between the apparent molecular weight of the studied proteins (lanes 1 and 2) and native protein (lane N). An estimated c.a. 2 kDa increase in molecular weight was reported for proteins glycated over 14 days (lane 3).
 

Fig 4: Electrophorogram of glycated proteins (WM - molecular weight marker, WZ - trypsin inhibitor, N - unmodified protein, A-ovomucoid, B - lysozyme).


 
In samples incubated at 60°C, an apparent molecular weight increase of 5-6 kDa was observed for all time variants (lanes 4, 5 and 5). As demonstrated in the electrophorogram, the most significant variations in molecular weight were induced by a temperature of 60°C in all glycation variants. The degree of glycation (Fig 5) was identified by determining the content of free amino groups. Statistical analyses revealed significant variations in the content of free amino groups in comparison to native protein.
 

Fig 5: The content of free amino groups in glycated samples.


 
The content of free amino groups in unmodified native protein (control) was used as the 100% reference. In samples glycated at 37°C, the content of free amino groups decreased by 43% in proteins incubated for 3 days, 31% in proteins incubated for 7 days and 29% in proteins incubated for 14 days in comparison with control. Protein modification at 60°C lowered the share of free amino groups by 78% in samples glycated for 3 days, 79% in samples glycated for 7 days and 76% in samples glycated for 14 days in comparison with native protein (control). The differences in the content of free amino groups were statistically significant (p≤0.01) in every analyzed variant.
       
Quantitative variations in ovomucoid immunoreactivity were evaluated by the competitive ELISA test. Changes in immunoreactivity were determined as percentage variations in antibody-antigen binding interactions relative to unmodified protein.
       
The effect of temperature on changes in ovomucoid immunoreactivity is presented in Fig 6.
 

Fig 6: The effect of glycation on ovomucoid immunoreactivity.


  
At a temperature of 37°C, significant variations (p≤0.05) were observed between ovomucoid samples incubated for 7 and 14 days. As regards samples incubated at 60°C, significant variations were reported between the control and all time variants at p≤0.01 for samples incubated over 3 and 7 days and p≤0.05 for samples incubated over 14 days.
       
The effect of glycation on changes in ovomucoid immunoreactivity is illustrated in Fig 6. In reference to samples glycated at a temperature of 37°C, significant variations were noted between unmodified protein (control) and proteins modified for 3 days (p≤0.01). Significant differences between glycation variants lasting 3 and 7 days and 7 and 14 days were determined at p≤0.05. Statistically significant variations (p≤0.01) were also reported by comparing glycation time of 3 days and 14 days.
       
Statistically significant differences (p≤0.01) were observed between unmodified protein (control) and all variants modified at a temperature of 60°C. A comparison of glycation times of 3 and 14 days also revealed significant differences (p≤0.05).
       
A noticeable drop in ovomucoid’s electrophoretic mobility was found. Extensive glycation leads to increase of molecular weight, but observed changes of electrophoretic mobility may be enlarged by another phenomenon. Acidic character of ovomucoid could pose a problem (a higher number of acidic amino acid residues than alkaline residues and the presence of sialic acid residues). The observed molecular weight of acidic proteins may be higher than their actual weight (Chiou and Wu, 1999). There is no published explanation of molecular mechanism of this phenomenon until now. Glycation causes modification of basic residues and may affect acid-base balance in protein molecules.
       
Ovomucoid possess many epitopes indicated in the Immune Epitope Database (Vita et al., 2015). Increase of immunogenicity due to moderate glycation may be explained by the exposition of additional epitopes due to protein structure changes, induced by glycation. Some products of reaction between sugars protein form rigid structures consisting of multiple, condensed rings, connecting two lysine or arginine residues (Ashraf et al., 2015). Additional steric repulsion, caused by formation of such moieties may lead to partial disruption of protein secondary and tertiary structure and hence to exposition of epitopes, which are normally inaccessible for antibodies. This explanation may be supported by the results of experiment described by Ma et al., (2013). In the experiment of these authors moderate glycation led to increase of immunoreactivity of ovalbumin, associated with decrease of the b-sheet structure content and increase of surface hydrophobicity. These effects mean, that structural changes of ovalbumin provided exposition of additional epitopes, containing hydrophobic amino acid residues and buried within native protein structure. Similar phenomenon could occur due to moderate glycation of ovomucoid during the experiment, described here.
       
Alternative explanation of ovomucoid immunogenicity increase due to glycation at 37°C for 3 and 7 days, taking into account possibility of formation of new epitopes by modified amino acid residues itself (Gupta et al., 2016), is less likely. There was no simple proportion between glycation degree and immunogenicity. Extensive glycation at 60°C minimized interactions between protein and antibodies. It suggests, that hindrance of such interactions by modified residues (Gupta et al., 2016) was dominant phenomenon at high glycation degree.
 
Glycation is considered as a method for reduction of food protein allergenicity,but is not strongly recommended for this purpose (Gupta et al., 2016). Influence of extensive glycation on ovomucoid immunogenicity was studied by Jiménez-Saiz et al., (2011) and observed possibility to reduce immunogenicity via extensive glycation. Drastic reaction conditions may lead to occurrence of unpredicted and undesired products. There was necessary to include experiment with moderately glycated protein and compare its immunogenicity with this extensively glycated.  Our results show, that moderate glycation (performed at relatively low temperature 37°C) does not provide reduction of ovomucoid immunoreactivity. Significant reduction of this property requires high temperature and/or long time leading to extensive glycation. Davis et al., (2016) state that advanced glycation end products are a diverse group of compounds, which can form endogenously and glycation of molecules may negatively affect their function. The American Diabetes Association makes no recommendation regarding avoidance of advanced glycation end products, but many researchers are concerned that they may be pro-inflammatory and way worsen cardiac function and kidney function and also contribute to obesity.

CONCLUSION

Glycation may be recommended as a way to reduce or inhibit immunoreactivity, however the protein’s nutritional value may decrease. In case of allergies, such action is necessary and purposeful. It is essential to choose such reaction conditions which do not cause products harmful to human’s health. It is needed to research the toxicity of proteins, in this case ovomucoid.

COMPETING INTEREST

The authors declare that they have no competing interests.

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