Lysine as a critical amino acid for IgE binding in Phl p 5b C terminus Journal Article
| Authors: | Gehlhar, K.; Rajashankar, K. R.; Hofmann, E.; Betzel, C.; Weber, W.; Werner, S.; Bufe, A. |
| Article Title: | Lysine as a critical amino acid for IgE binding in Phl p 5b C terminus |
| Abstract: | Background: Allergens induce the formation of specific immunoglobulin (Ig)E and harbor at least two IgE-binding regions (epitopes) to facilitate crosslinking of basophilic or mast-cell-bound specific IgE antibodies. Studies mapping linear epitopes have shown that these regions often contain charged or hydrophobic amino acids. Nevertheless, these studies are hampered by limited significance due to the often conformational nature of IgE epitopes. This prompted us to study the role of lysines in the context of an intact 3-dimensional model. Methods: Major allergen Phl p 5b from timothy grass bears 12 lysines in its C-terminal half. Using site-directed mutagenesis, we substituted all 10 surface-exposed lysines by alanines. Results: Although structural integrity of the lysine-deficient mutant was not altered, IgE-binding capacity measured by ELISA inhibition tests and crosslinking activity in ex vivo basophil stimulation and in vivo skin prick tests were significantly diminished. Interestingly, binding of specific IgG antibodies was considerably less reduced by loss of lysines. Conclusion: Lysine is an important amino acid for IgE binding in more than one epitope of major grass pollen allergen Phl p 5b C terminus. Allergenicity, but not IgG binding of the molecule, is substantially diminished by single amino acid substitutions without structural integrity being hampered. Copyright © 2006 S. Karger AG. |
| Keywords: | clinical article; controlled study; unclassified drug; human cell; binding affinity; amino acid substitution; carboxy terminal sequence; in vivo study; enzyme linked immunosorbent assay; amino acid sequence; molecular sequence data; immunoglobulin g; three dimensional imaging; recombinant proteins; epitope; mutagenesis, site-directed; binding sites; alanine; protein structure; allergens; antigen binding; cell stimulation; ex vivo study; site directed mutagenesis; immunoglobulin g antibody; lysine; ribonucleases; immunoglobulin e; epitopes; circular dichroism; antigen-antibody reactions; binding, competitive; cross linking; phosphoric diester hydrolases; plant proteins; site-directed mutagenesis; immunoglobulin-e-binding region; phl p 5b; pollen allergen; immunoglobulin e antibody; phl p 5b allergen; pollen antigen; allergenicity; basophil; phleum pratense; prick test; basophils; pyrophosphatases; skin tests |
| Journal Title: | International Archives of Allergy and Immunology |
| Volume: | 140 |
| Issue: | 4 |
| ISSN: | 1018-2438 |
| Publisher: | S. Karger AG |
| Date Published: | 2006-07-01 |
| Start Page: | 285 |
| End Page: | 294 |
| Language: | English |
| DOI: | 10.1159/000093706 |
| PUBMED: | 16735798 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 9" - "Export Date: 4 June 2012" - "CODEN: IAAIE" - "Source: Scopus" |
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