Mapping of IgE and IgG4 Antibody-Binding Epitopes in Cyn d 1, the Major Allergen of Bermuda Grass Pollen

Abstract

Background: Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG₄ antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. Methods: Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG_1–4 antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. Results: Four major IgE-binding epitopes (peptides 15^–1, 21, 33^–2 and 35⁺¹, corresponding to amino acids 70–79, 101–110, 159–167 and 172–181) and 5 major IgG₄-binding epitopes (peptides 15^–1, 30^–2, 33^–2, 35⁺¹ and 39, corresponding to amino acids 70–79, 144–153, 159–167, 172–181 and 192–200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG₄. Their critical amino acids were all characterized. Major epitopes for human IgG₁ to IgG₄ are almost identical. Conclusions: This is the first study to map the sequential epitopes for human IgE and IgG₄ subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis.