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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Experimental Pneumology, Ruhr University Bochum, Bochum, Germany. kirsten.gehlhar@rub.de
authors.forename: Kirsten
authors.initials: K
authors.lastname: Gehlhar
authors.forename: Kanagalaghata R
authors.initials: KR
authors.lastname: Rajashankar
authors.forename: Eckhard
authors.initials: E
authors.lastname: Hofmann
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Weber
authors.forename: Sandra
authors.initials: S
authors.lastname: Werner
authors.forename: Albrecht
authors.initials: A
authors.lastname: Bufe
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions / Q000235:genetics / Q000276:immunology*; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002942:Circular Dichroism; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D008239:Lysine / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011755:Pyrophosphatases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D012882:Skin Tests
chemical_list: D000485:Allergens; C427944:ENPP3 protein, human; D007074:Immunoglobulin G; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C102306:Phl p Vb protein, Phleum pratense; D012260:Ribonucleases; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D008239:Lysine