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Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1
Author(s) -
Ball Tanja,
Edstrom William,
Mauch Ludwig,
Schmitt Jacky,
Leistler Bernd,
Fiebig Helmut,
Sperr Wolfgang R.,
Hauswirth Alexander W.,
Valent Peter,
Kraft Dietrich,
Almo Steven C.,
Valenta Rudolf
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1432-1033.2004.04403.x
Subject(s) - epitope , immunoglobulin e , allergen , recombinant dna , basophil activation , sf9 , biology , glycoprotein , chemistry , immunology , microbiology and biotechnology , spodoptera , basophil , biochemistry , antibody , allergy , gene
Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross‐reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus‐infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C‐terminal 40% of Phl p 1, a major IgE‐reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain‐like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell‐expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 Å resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect‐cell expressed Phl p 1 compared to Escherichia coli ‐expressed Phl p 1. Moreover, insect cell‐expressed Phl p 1 induces potent histamine release and leads to strong up‐regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.