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Keywords:

  • food allergy;
  • IgE;
  • N-linked glycosylation;
  • ovomucoid;
  • Pichia pastoris;
  • Th1/Th2

Summary

Background Food allergies are on the rise and it is estimated that in North America, 8% of the children and 4% of the adults have food allergies. Food allergies tend to occur more often in children than in adults due to their immature digestive and immune systems. Hen's egg is among the most common cause of food-induced allergic reactions in North America.

Objective The present study was undertaken to investigate the role of N-glycans of the third domain of ovomucoid in IgE binding and modulation of allergen-specific immune response in BALB/c mice.

Methods The cDNA encoding the third domain of ovomucoid was inserted into the yeast genome and expressed in Pichia pastoris X-33 cells, under the control of the glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for constitutive expression to obtain a post-translationally modified and functionally active ovomucoid third domain. Upon expression, the protein was secreted into the extracellular medium and was purified by size exclusion chromatography. The recombinant protein was produced at 10 mg/L of the culture supernatant. BALB/c mice were sensitized with the recombinant and native forms of glycosylated ovomucoid third domain antigen. The allergic response of the native and the recombinant glycosylated forms of ovomucoid third domain antigens were compared using antibody and cytokine measurements.

Results ELISA tests indicated a significant decrease in specific IgE antibodies to the recombinant N-linked glycosylated form (P-Gly), when compared with the native glycosylated form (DIII+) using mice sera. Immunization with P-Gly induced the production of IFN-γ [T-helper type 1 (Th1) response] and lowered the production of IL-4 (Th2 response), and a skewed balance towards the Th1 cytokine demonstrated that P-Gly has a modulating ability on Th1/Th2 balance to down-regulate Th2 response. Furthermore, N-linked glycan (N28) in the third domain of ovomucoid was shown to be associated with suppression of the allergic response.

Conclusion Therefore, we can conclude that P-Gly facilitates and contributes to the discovery of new molecular target for the development of a safe and specific therapeutic vaccine for the treatment of egg allergy, and oligosaccharides do seem to play a major role in the suppression of IgE-binding activity.