title: Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen.
abstract: Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.
authors.affiliation: Institute of General and Experimental Pathology, Vienna General Hospital, University of Vienna, Austria. Susanne.Vrtala@akh-wien.ac.at
authors.forename: S
authors.initials: S
authors.lastname: Vrtala
authors.forename: S
authors.initials: S
authors.lastname: Fischer
authors.forename: M
authors.initials: M
authors.lastname: Grote
authors.forename: L
authors.initials: L
authors.lastname: Vangelista
authors.forename: A
authors.initials: A
authors.lastname: Pastore
authors.forename: W R
authors.initials: WR
authors.lastname: Sperr
authors.forename: P
authors.initials: P
authors.lastname: Valent
authors.forename: R
authors.initials: R
authors.lastname: Reichelt
authors.forename: D
authors.initials: D
authors.lastname: Kraft
authors.forename: R
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000393:Air Pollutants / Q000737:chemistry* / Q000276:immunology; D000485:Allergens / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000276:immunology; D006636:Histamine Release; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification* / Q000648:ultrastructure; D006109:Poaceae; D011058:Pollen / Q000737:chemistry* / Q000276:immunology* / Q000648:ultrastructure; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000393:Air Pollutants; D000485:Allergens; D018076:DNA, Complementary; D000939:Epitopes; C095238:PHLPVI protein, Phleum pratense; D010446:Peptide Fragments; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007073:Immunoglobulin E