Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1
| Autor: | Helmut Fiebig, Steven C. Almo, Rudolf Valenta, Tanja Ball, Jacky Schmitt, Alexander W. Hauswirth, Dietrich Kraft, Bernd Leistler, Peter Valent, Ludwig Mauch, William Edstrom, Wolfgang R. Sperr |
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| Rok vydání: | 2004 |
| Předmět: |
Timothy-grass
Allergy biology Cell Biology Immunoglobulin E medicine.disease_cause biology.organism_classification medicine.disease Biochemistry Epitope chemistry.chemical_compound Allergen chemistry Escherichia Immunology otorhinolaryngologic diseases biology.protein medicine Antibody Molecular Biology Histamine |
| Zdroj: | FEBS Journal. 272:217-227 |
| ISSN: | 1742-4658 1742-464X |
| DOI: | 10.1111/j.1432-1033.2004.04403.x |
| Popis: | 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 A 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. |
| Databáze: | OpenAIRE |
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