title: Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen.
abstract: BACKGROUND
  Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data.
  
  
  OBJECTIVE
  The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity.
  
  
  METHODS
  Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations).
  
  
  RESULTS
  Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1.
  
  
  CONCLUSIONS
  Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.
authors.affiliation: Forschungszentrum Borstel, Biochemical and Molecular Allergology, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Petersen
authors.forename: G
authors.initials: G
authors.lastname: Schramm
authors.forename: M
authors.initials: M
authors.lastname: Schlaak
authors.forename: W M
authors.initials: WM
authors.lastname: Becker
mesh_terms: D000485:Allergens; D000937:Antigen-Antibody Reactions; D004220:Disulfides / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006031:Glycosylation; D006801:Humans; D006909:Hydroxyproline / Q000276:immunology; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D000485:Allergens; D004220:Disulfides; D010940:Plant Proteins; C090396:PHLPI protein, Phleum pratense; D007073:Immunoglobulin E; D006909:Hydroxyproline