Elsevier

Molecular Immunology

Volume 71, March 2016, Pages 152-160
Molecular Immunology

Molecular and immunological analysis of hen’s egg yolk allergens with a focus on YGP42 (Gal d 6)

https://doi.org/10.1016/j.molimm.2016.02.005Get rights and content

Highlights

  • Sera of 25 egg white allergy patients demonstrated IgE binding for egg yolk proteins.

  • Mass spec analysis revealed Gal d 6 as the important allergen of egg yolk.

  • Recombinant Gal d 6 (rGal d 6) inhibited IgE binding to crude egg yolk by up to 30%.

  • This is the first report of concomitant sensitization to egg white and yolk proteins.

  • rGal d 6 has the potential to be used in diagnosis and immunotherapy of egg allergy.

Abstract

Allergy to hen’s (Gallus domesticus) egg white is one of the most common forms of food allergy. Allergy to hen’s yolk also exists however, to a lesser extent when compared to egg white allergy. Two minor allergens from the hen’s egg yolk known as α-livetin (Gal d 5) and YGP42 (Gal d 6) were discovered recently. In this study, we investigated whether sensitization to egg white is associated with reactivity to egg yolk as well. Sera obtained from 25 patients with allergy to egg white were tested for specific IgE binding for egg yolk proteins through western immunoblotting. 36% of patients were found with true IgE-sensitization against egg yolk proteins. It was found that most of the IgE reactive yolk proteins were fragments of major precursor proteins of hen; vitellogenin-1 (VTG-1), vitellogenin-2 (VTG-2) and apolipoprotein B (Apo B). The egg yolk allergen Gal d 6 is the C-terminal part of VTG-1 and was found to be allergenic in significant percentage of egg white allergy patients. These results highlight the significance of Gal d 6 as an important allergen of egg yolk. Therefore, the secondary aim of this study involved developing a recombinant version of YGP42 in an Escherichia coli expression system. Recombinant Gal d 6 (rGal d6) was expressed as a fusion peptide with a 6 × His tag and purified using metal chelating resin. The inhibition ELISA results showed that rYGP42 was IgE reactive and was able to inhibit IgE binding to crude egg yolk (CEY) by up to 30%. Traditionally, it was thought that allergy to egg yolk occurred independently from sensitization to egg white. This study underlies the importance of concomitant sensitization to egg yolk proteins in patients allergic to egg white. Evidence reported in this study strongly suggests that egg yolk has potentially undiscovered allergens and therefore warrants further investigation. Furthermore, IgE reactive Gal d 6 presented in this study has the potential to be used in diagnosis and immunotherapy to treat egg allergy.

Introduction

Allergy to hen’s egg is one of the most common forms of food allergy in childhood, second only to cow’s milk allergy (Caubet et al., 2011). In developed countries, the prevalence of egg allergy is estimated to range from 0.5 to 2.5% among infants and young children (Rona et al., 2007). Albumen of the chicken egg contains the well-known dominant allergens namely, ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3) and lysozyme (Gal d 4). The egg yolk, on the other hand, also contains two minor allergens known as α-livetin (Gal d 5) and YGP42 (Gal d 6). The allergenic properties of the hen’s egg white proteome have been extensively studied. By contrast, egg yolk proteins and its involvement in food allergy have not come under the limelight of many studies over the years.

The knowledge regarding the association between allergy to egg white and allergy to egg yolk is very limited in the literature. Few studies in the literature support evidence about a significant positive correlation between the allergenicities of egg white and yolk. It has been speculated that this may be due to the common allergenic epitopes that some egg white and yolk proteins share (Anet et al., 1985, Walsh et al., 1987). Furthermore, Walsh et al. reported two egg yolk proteins namely; apovitellenin I and apovitellenin V to be IgE reactive indicating that there could be potentially undiscovered allergens in the egg yolk. Also, the same authors demonstrated a good correlation between IgE binding to egg yolk proteins and the clinical incidence in most egg allergy patients (Walsh et al., 1988).

The objective of this study was threefold. The first objective was to investigate if patients sensitised to hen’s egg white also would be allergic to egg yolk through immunological methods. We found that majority of egg white allergy patients to be seropositive to various egg yolk proteins. The second aim was to establish the identity of IgE reactive proteins as shown from the results of the first objective. The third objective involved production of recombinant version of Gal d 6 (rGal d 6) since it was found to be IgE reactive in a significant number of patients with egg white allergy indicating it is an important allergen. Here, the rGal d 6 was produced and purified as a soluble fraction and showed that it is allergenic using inhibition ELISA immunoassay.

Section snippets

Human patients’ sera

Sera from 25 patients allergic to hen’s egg white were obtained from the Royal Children’s Hospital (Melbourne, VIC, Australia). The egg white specific IgE levels of patients are shown in Table 1. Experimentation involving patients’ sera was conducted in compliance with the National Statement on Ethical Conduct in Human Research (2007) with approval from Deakin University Faculty of Science, Engineering and Built Environment Human Ethics Advisory Group (HEAG), with a project approval number of

Immunological analysis of CEY and mass spectrometry analysis of IgE reactive hen’s egg yolk proteins

The complexity of the CEY is evident from the protein profile generated from SDS-PAGE (Fig. 1). The resulting protein profile of the CEY shows a large number of protein bands. Although there are some overlapping bands, around 13 different proteins ranging from 10 to 250 kDa could be clearly distinguished (Fig. 1). Furthermore, already known egg yolk allergens Gal d 5 and Gal d 6 were also clearly detectable on the SDS-PAGE of CEY approximately at 65 kDa and 42 kDa, respectively.

Western immunoassay

Discussion

Hen’s egg is one of the most well-known sources of allergens with several allergens present within the egg white and the yolk. The allergens of the egg white have much stronger allergenic potential than the yolk proteins. The role of egg white allergens in regard to food allergy has been well investigated compared to egg yolk allergy. Also, majority of studies reported in literature have investigated allergy to egg white and allergy to egg yolk separately and therefore, overlooking the aspect

Conflict of interest

There is no conflict of interest.

Author contributions

CS conceived the idea, contributed to the study design and supervised the study. CDS and PD performed experiments, collected data and prepared manuscript. TD provided chicken egg and tissue samples and MLKT provided reagents essential for the immunological analysis. All authors reviewed and edited the manuscript.

Acknowledgements

We would like to thank Poultry Cooperative Research Centre (CRC) (established and supported under the Australian Government’s Cooperative Research Centres Program) and Deakin University’s Centre for Molecular and Medical Research (MMR) for providing this study with the required research funding, Australian Animal Health Laboratory (AAHL) of Commonwealth Scientific and Industrial Research Organisation (CSIRO) for supplying animal tissues required for the study, and the Murdoch Children’s

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