title: Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4.
abstract: BACKGROUND
  Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens.
  
  
  OBJECTIVE
  We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4.
  
  
  METHODS
  Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays.
  
  
  RESULTS
  The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein.
  
  
  CONCLUSIONS
  The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs.
authors.affiliation: Institute for Molecular Biosciences (IMB), University of Graz, Graz, Austria.
authors.forename: Domen
authors.initials: D
authors.lastname: Zafred
authors.affiliation: Research and Development Division, Allergopharma Joachim Ganzer KG, Reinbek, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nandy
authors.affiliation: Research and Development Division, Allergopharma Joachim Ganzer KG, Reinbek, Germany.
authors.forename: Linda
authors.initials: L
authors.lastname: Pump
authors.affiliation: Research and Development Division, Allergopharma Joachim Ganzer KG, Reinbek, Germany.
authors.forename: Helga
authors.initials: H
authors.lastname: Kahlert
authors.affiliation: Institute for Molecular Biosciences (IMB), University of Graz, Graz, Austria. Electronic address: walter.keller@uni-graz.at.
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000485:Allergens / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D001491:Basophils / Q000276:immunology; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D031784:Phleum / Q000276:immunology*; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D011058:Pollen / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000485:Allergens; C100980:Phl p 4 protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D015086:2,6-Dichloroindophenol; D042843:Glucose 1-Dehydrogenase
keywords: BBE; CCD; CD; Circular dichroism; Cross-reactive carbohydrate determinant; FAD; FSAS; Flavin adenine dinucleotide; Fractional solvent-accessible surfaces; GPA; Grass pollen allergen; Grass pollen allergy; Phl p 4; RMSD; Reticuline oxidase (informally known as the berberine bridge enzyme); Root-mean-square deviation; Sec c 4; basophil release assay; bicovalently attached flavin adenine dinucleotide; cross-reactive carbohydrate determinant; dehydrogenase; recombinant allergen