Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN308 and ASN409) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation.

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk.

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY.

Monitoring glycation-induced structural and biofunctional changes in chicken immunoglobulin Y by different monosaccharides.

Chicken IgY has been applied in the food industry as an important supplement. Glycation of IgY is of special interest due to its possible influence on the structure and functionality of IgY. IgY was subjected to in vitro glycation with 4 different monosaccharides, including glucose (Glu), mannose (Man), fucose (Fuc), and fructose (Fru). The objective of this paper was to characterize the formation of IgY-sugar conjugates using ultraviolet spectra, fluorescence spectra, circular dichroism spectra, Fourier transform infrared spectra, and SDS-PAGE and differential scanning calorimetry analysis. The antigen epitopes of native and glycated IgY was determined by enzyme-linked immunosorbent assay (ELISA). The existence of broad bands in stacking gel and sample well demonstrated that reducing monosaccharides covalently bound to IgY. The secondary structure of IgY was altered from a well-defined ß-sheet structure to a random coil structure. Fluorescence spectra showed that IgY was more hydrophilic after glycation. Thermal stability of glycated IgY was remarkably increased over that of native IgY. However, ELISA results would suggest that the antigen epitopes recognized by the polyclonal antibody and overall conformation changed in the IgY molecule due to a decrease in polyclonal antibody binding to glycated IgY. Data suggested that glycation induced by reducing sugars significantly affects the structure and antigen-binding ability of IgY, which could reduce the utility of IgY in certain applications.

A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate.

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale.